InteractiveFly: GeneBrief

similar: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References


Gene name - similar

Synonyms -

Cytological map position - 99D3--7

Function - transcription factor

Keywords - oxygen homeostasis, response to hypoxia, trachea

Symbol - sima

FlyBase ID: FBgn0266411

Genetic map position - 3R

Classification - PAS domain, Helix-loop-helix dimerization domain

Cellular location - nuclear and cytoplasmic



NCBI link: Precomputed BLAST | Entrez Gene

Recent literature
Zarndt, R., Piloto, S., Powell, F.L., Haddad, G.G., Bodmer, R. and Ocorr, K. (2015). Cardiac responses to hypoxia and reoxygenation in Drosophila. Am J Physiol Regul Integr Comp Physiol [Epub ahead of print]. PubMed ID: 26377557
Summary:
An adequate supply of oxygen is important for the survival of all tissues, but is especially critical for tissues with high energy demands such as the heart. This study used the genetic model system Drosophila to investigate cardiac responses to acute (30 minutes), sustained (18 hours), and chronic (3 weeks) hypoxia with reoxygenation. Whereas hearts from wild type flies recover quickly after acute hypoxia, exposure to sustained or chronic hypoxia significantly compromises heart function upon reoxygenation. Hearts from flies with mutations in sima, Drosophila homolog of the Hypoxia Inducible Factor alpha subunit (HIFα), exhibit exaggerated reductions in cardiac output in response to hypoxia. Heart function in hypoxia-selected flies, selected over many generations for survival in a low oxygen environment, reveals reduced cardiac output in terms of decreased heart rate and fractional shortening compared to their normoxia controls. Hypoxia-selected flies also have smaller hearts, myofibrillar disorganization and increased extracellular collagen deposition, consistent with the observed reductions in contractility. This study indicates that longer duration hypoxic insults exert deleterious effects on heart function that are mediated in part by sima and advances Drosophila models for the genetic analysis of cardiac-specific responses to hypoxia and reoxygenation.

Cagin, U., Duncan, O. F., Gatt, A. P., Dionne, M. S., Sweeney, S. T. and Bateman, J. M. (2015). Mitochondrial retrograde signaling regulates neuronal function. Proc Natl Acad Sci U S A 112: E6000-6009. PubMed ID: 26489648
Summary:
Mitochondria are key regulators of cellular homeostasis, and mitochondrial dysfunction is strongly linked to neurodegenerative diseases, including Alzheimer's and Parkinson's. Mitochondria communicate their bioenergetic status to the cell via mitochondrial retrograde signaling. To investigate the role of mitochondrial retrograde signaling in neurons, we induced mitochondrial dysfunction in the Drosophila nervous system. Neuronal mitochondrial dysfunction causes reduced viability, defects in neuronal function, decreased redox potential, and reduced numbers of presynaptic mitochondria and active zones. Neuronal mitochondrial dysfunction stimulates a retrograde signaling response that controls the expression of several hundred nuclear genes. Drosophila hypoxia inducible factor alpha (HIFalpha) ortholog Similar (Sima) regulates the expression of several of these retrograde genes, suggesting that Sima mediates mitochondrial retrograde signaling. Remarkably, knockdown of Sima restores neuronal function without affecting the primary mitochondrial defect, demonstrating that mitochondrial retrograde signaling is partly responsible for neuronal dysfunction. Sima knockdown also restores function in a Drosophila model of the mitochondrial disease Leigh syndrome and in a Drosophila model of familial Parkinson's disease. Thus, mitochondrial retrograde signaling regulates neuronal activity and can be manipulated to enhance neuronal function, despite mitochondrial impairment.

Kamps-Hughes, N., Preston, J. L., Randel, M. A. and Johnson, E. A. (2015). Genome-wide identification of hypoxia-induced enhancer regions. PeerJ 3: e1527. PubMed ID: 26713262
Summary:
This study presents a genome-wide method for de novo identification of enhancer regions. This approach enables massively parallel empirical investigation of DNA sequences that mediate transcriptional activation and provides a platform for discovery of regulatory modules capable of driving context-specific gene expression. The method links fragmented genomic DNA to the transcription of randomer molecule identifiers and measures the functional enhancer activity of the library by massively parallel sequencing. A Drosophila melanogaster library was transfected into S2 cells in normoxia and hypoxia, and 4,599,881 genomic DNA fragments were assayed in parallel. The locations of the enhancer regions strongly correlate with genes up-regulated after hypoxia and previously described enhancers. Novel enhancer regions were identified and integrated with RNAseq data and transcription factor motifs to describe the hypoxic response on a genome-wide basis as a complex regulatory network involving multiple stress-response pathways. Of particular interest, an intronic enhancer in Sima was identified that contains both HIF-1 and NF-&kapppa;B binding sites, suggesting HIF-1 autoregulation and integration of NF-&kapppa;B signaling at a basal level in the hypoxic response. The enhancer region, while intronic to the full-length Sima transcript isoforms, is upstream of an alternative transcriptional start site that produces a transcript isoform that is up-regulated after hypoxia,
Perez-Perri, J.I., Dengler, V.L., Audetat, K.A., Pandey, A., Bonner, E.A., Urh, M., Mendez, J., Daniels, D.L., Wappner, P., Galbraith, M.D. and Espinosa, J.M. (2016). The TIP60 complex is a conserved coactivator of HIF1A. Cell Rep [Epub ahead of print]. PubMed ID: 27320910
Summary:
Hypoxia-inducible factors (HIFs) are critical regulators of the cellular response to hypoxia. Despite their established roles in normal physiology and numerous pathologies, the molecular mechanisms by which they control gene expression remain poorly understood. This study reports a conserved role for the TIP60 complex as a HIF1 transcriptional cofactor in Drosophila and human cells. TIP60 (KAT5) is required for HIF1-dependent gene expression in fly cells and embryos and colorectal cancer cells. HIF1A interacts with and recruits TIP60 to chromatin. TIP60 is dispensable for HIF1A association with its target genes but is required for HIF1A-dependent chromatin modification and RNA polymerase II activation in hypoxia. In human cells, global analysis of HIF1A-dependent gene activity reveals that most HIF1A targets require either TIP60, the CDK8-Mediator complex, or both as coactivators for full expression in hypoxia. Thus, HIF1A employs functionally diverse cofactors to regulate different subsets of genes within its transcriptional program.

Lin, X. W., Tang, L., Yang, J. and Xu, W. H. (2016). HIF-1 regulates insect lifespan extension by inhibiting c-Myc-TFAM signaling and mitochondrial biogenesis. Biochim Biophys Acta [Epub ahead of print]. PubMed ID: 27469241
Summary:
Diapause (developmental arrest) is characterized by dramatic depression of metabolic activity and profoundly extends insect lifespan, similar to the Caenorhabditis elegans dauer stage and Drosophila longevity; however, the molecular mechanism of low metabolism in insect diapause is unclear. This study showed that HIF-1α expression is significantly increased in diapause-destined pupal brains compared to nondiapause-destined pupal brains and that HIF-1α negatively regulates mitochondrial biogenesis. HIF-1α mediates this effect by inhibiting c-Myc activity via proteasome-dependent degradation of c-Myc. The mitochondrial transcription factor A (TFAM), which encodes a key factor involved in mitochondrial transcription and mitochondrial DNA replication, is activated by the binding of c-Myc to the TFAM promoter, thereby inducing transcription. Loss of TFAM expression is a major factor contributing to reducing the mitochondrial activity. Thus, the HIF-1alpha-c-Myc-TFAM signaling pathway participates in the regulation of mitochondrial activity for insect diapause or lifespan extension.
Wang, C. W., Purkayastha, A., Jones, K. T., Thaker, S. K. and Banerjee, U. (2016). In vivo genetic dissection of tumor growth and the Warburg effect. Elife 5 [Epub ahead of print]. PubMed ID: 27585295
Summary:
A well-characterized metabolic landmark for aggressive cancers is the reprogramming from oxidative phosphorylation to aerobic glycolysis, referred to as the Warburg effect. Models mimicking this process are often incomplete due to genetic complexities of tumors and cell lines containing unmapped collaborating mutations. In order to establish a system where individual components of oncogenic signals and metabolic pathways can be readily elucidated, this study induced a glycolytic tumor in the Drosophila wing imaginal disc by activating the oncogene PDGF/VEGF-receptor (Pvr). This causes activation of multiple oncogenic pathways including Ras, PI3K/Akt, Raf/ERK, Src and JNK. Together this network of genes stabilizes Hifα (Sima) that in turn, transcriptionally up-regulates many genes encoding glycolytic enzymes. Collectively, this network of genes also causes inhibition of pyruvate dehydrogenase (PDH) activity resulting in diminished ox-phos levels. The high ROS produced during this process functions as a feedback signal to consolidate this metabolic reprogramming.
Misra, T., Baccino-Calace, M., Meyenhofer, F., Rodriguez-Crespo, D., Akarsu, H., Armenta-Calderon, R., Gorr, T. A., Frei, C., Cantera, R., Egger, B. and Luschnig, S. (2016). A genetically encoded biosensor for visualizing hypoxia responses in vivo. Biol Open. PubMed ID: 28011628
Summary:
.Cells experience different oxygen concentrations depending on location, organismal developmental stage, and physiological or pathological conditions. Responses to reduced oxygen levels (hypoxia) rely on the conserved Hypoxia-Inducible Factor 1 (HIF-1). Understanding the developmental and tissue-specific responses to changing oxygen levels has been limited by the lack of adequate tools for monitoring HIF-1 in vivo. To visualise and analyse HIF-1 dynamics in Drosophila, this study used a hypoxia biosensor consisting of GFP fused to the oxygen-dependent degradation domain (ODD) of the HIF-1 homologue Sima. GFP-ODD responds to changing oxygen levels and to genetic manipulations of the hypoxia pathway, reflecting oxygen-dependent regulation of HIF-1 at the single-cell level. Ratiometric imaging of GFP-ODD and a red-fluorescent reference protein reveals tissue-specific differences in the cellular hypoxic status at ambient normoxia. Strikingly, cells in the larval brain show distinct hypoxic states that correlate with the distribution and relative densities of respiratory tubes. A set of genetic and image analysis tools is presented that enable new approaches to map hypoxic microenvironments, to probe effects of perturbations on hypoxic signalling, and to identify new regulators of the hypoxia response.
Batie, M., Druker, J., D'Ignazio, L. and Rocha, S. (2017). KDM2 family members are regulated by HIF-1 in hypoxia. Cells 6(1) [Epub ahead of print]. PubMed ID: 28304334
Summary:
Hypoxia is not only a developmental cue but also a stress and pathological stimulus in many human diseases. The response to hypoxia at the cellular level relies on the activity of the transcription factor family, hypoxia inducible factor (HIF). HIF-1 is responsible for the acute response and transactivates a variety of genes involved in cellular metabolism, cell death, and cell growth. This study shows that hypoxia results in increased mRNA levels for human lysine (K)-specific demethylase 2 (KDM2) family members, KDM2A and KDM2B, and also for Drosophila melanogaster KDM2, a histone and protein demethylase. In human cells, KDM2 family member's mRNA levels are regulated by HIF-1 but not HIF-2 in hypoxia. Interestingly, only KDM2A protein levels are significantly induced in a HIF-1-dependent manner, while KDM2B protein changes in a cell type-dependent manner. Importantly, it was demonstrated that in human cells, KDM2A regulation by hypoxia and HIF-1 occurs at the level of promoter, with HIF-1 binding to the KDM2A promoter being required for RNA polymerase II recruitment. Taken together, these results demonstrate that KDM2 is a novel HIF target that can help coordinate the cellular response to hypoxia. In addition, these results might explain why KDM2 levels are often deregulated in human cancers.
BIOLOGICAL OVERVIEW

In mammalian systems, the heterodimeric basic helix-loop-helix (bHLH)-PAS transcription hypoxia-inducible factor (HIF) has emerged as the key regulator of responses to decreased oxygen concentrations (reviewed by Bruick, 2001: Semenza, 2001; see also Kaelin, 2002; Bruick, 2003; Bracken, 2003). A homologous system is present in Drosophila, and its activity has been characterized in vivo during development. By using transcriptional reporters in developing transgenic flies, it has been shown that hypoxia-inducible activity rises to a peak in late embryogenesis and is most pronounced in tracheal cells. The bHLH-PAS proteins Similar (Sima) and Tango (Tgo) function as HIF-alpha and HIF-ß homologs, respectively; a conserved mode of regulation for Sima by oxygen has been demonstrated. Sima protein, but not its mRNA, is upregulated in hypoxia. Time course experiments following pulsed ectopic expression demonstrate that Sima is stabilized in hypoxia and that degradation relies on a central domain encompassing amino acids 692 to 863. Continuous ectopic expression overrode Sima degradation, which remains cytoplasmic in normoxia, and translocates to the nucleus only in hypoxia, revealing a second oxygen-regulated activation step. Abrogation of the Drosophila Egl-9 prolyl hydroxylase homolog, HIF prolyl hydroxylase, causes both stabilization and nuclear localization of Sima, indicating a central involvement in both processes. Tight conservation of the HIF/prolyl hydroxylase system in Drosophila provides a new focus for understanding oxygen homeostasis in intact multicellular organisms (Lavista-Llanos, 2002).

In multicellular organisms, oxygen homeostasis requires precise developmental coordination between the growth of metabolizing tissues and that of systems that supply oxygen. Recent advances have provided new insights into how this complex task is achieved. For instance, in mammals it has long been recognized that local hypoxia is a major stimulus for angiogenesis. More recently, the recognition that specific angiogenic growth factors (such as the vascular endothelial growth factor) are powerfully induced by hypoxia through the action of a DNA binding complex termed hypoxia-inducible factor (HIF) has provided mechanistic insights into the process (Lavista-Llanos, 2002 and references therein).

The HIF DNA-binding complex consists of a heterodimer of basic-helix-loop-helix-PAS (bHLH-PAS) proteins that binds a core element A/(G)CGTG within hypoxia response elements (HREs) (Wang, 1995a and b). Regulation by oxygen involves stabilization of the alpha-subunit in hypoxia, whereas the ß-subunit, a common partner for several other bHLH-PAS proteins, is constitutively expressed regardless of oxygen tension. Normoxic degradation of HIF-alpha is mediated via ubiquitination and subsequent proteolysis, which requires oxygen-dependent interaction with the Von Hippel-Lindau (VHL) tumor suppressor protein (Maxwell, 1999). This interaction is regulated by hydroxylation of specific prolyl residues within the HIF-alpha polypeptides (Ivan, 2001; Jaakkola, 2001, Masson, 2001; reviewed by Masson, 2003), and recent work has identified a series of {alpha}-ketoglutarate-dependent non-heme iron-dependent dioxygenases that catalyze HIF-alpha prolyl hydroxylation and thus regulate stability of the polypeptide in accordance with oxygen availability (Bruick, 2001; Epstein, 2001). Other studies of HIF induction in mammalian tissue culture systems have defined additional regulatory steps that involve oxygen-dependent subcellular localization (Berra, 2001; Kallio, 1998) and coactivator recruitment (Carrero, 2000; Ema, 1999), although these processes are so far less well understood (Lavista-Llanos, 2002 and references therein).

In addition to angiogenic growth factors, HIF-1 drives the expression of genes involved in a broad array of systemic and cellular adaptive responses to hypoxia, suggesting a central role for HIF-1 as a regulator of oxygen homeostasis. However, although targeted inactivation of different HIF-alpha and HIF-ß subunits in the mouse is associated with several severe or lethal phenotypes involving defective vascular development (Carmeliet, 1998; Iyer, 1998; Kozak, 1997; Maltepe, 1997; Ryan, 1998), few mechanistic studies of the HIF-1 system have been conducted in vivo, and the critical interfaces between developmental processes and HIF activation remain largely undefined (Lavista-Llanos, 2002 and references therein).

To better understand HIF regulation in vivo and the role of hypoxia in developmental processes, the system has been characterized in Drosophila. In insects, air reaches the tissues by passive diffusion through a specialized tubular network termed the tracheal system. Moreover, development of tracheal terminal branches is oxygen dependent and shares many features with mammalian oxygen-dependent angiogenesis. For instance, oxygen-regulated expression of Drosophila Branchless/FGF guides tracheal migration during development and also drives extension of plastic terminal branches in a manner similar to the function of vascular endothelial growth factor in mammalian angiogenesis. The existence of a Drosophila HIF homolog has been inferred from DNA-binding assays with nuclear extracts from normoxic or hypoxic SL2 cells (Nagao, 1996), and transfection studies in mammalian cells have suggested that the Drosophila bHLH-PAS protein Similar (Sima) (Nambu, 1996) might function as an HIF-alpha homolog (Bacon, 1998). However, the system has not yet been fully defined or characterized in vivo in the fly (Lavista-Llanos, 2002).

Transgenic flies have been used to demonstrate and characterize in vivo the operation of a hypoxia inducible transcription response homologous to mammalian HIF. The work confirms the candidacy of Sima and Tgo as the Drosophila homologs of mammalian HIF-alpha and HIF-ß, respectively, defines a conserved multistep mode of regulation for Sima and provides new insights into the mechanisms regulating HIF proteins, as well as into the spatial and temporal operation of the hypoxia-responsive system during Drosophila development (Lavista-Llanos, 2002).

By tracking reporter gene activation in developing flies, the oxygen concentration dependence, developmental regulation, and spatial distribution of the transcriptional response were studied. Serial studies of the hypoxia response during development indicate that induction by hypoxia is modest in early embryogenesis and mid-embryogenesis and then rises sharply to peak levels at the end of embryogenesis, thereafter remaining relatively high throughout the larval stages. This developmentally restricted capacity fits well with the adaptive requirements of Drosophila larvae. After eclosion larvae usually dig into the substrate, while feeding actively, and are probably subjected to major variations in environmental oxygen tension so that enhanced activity of the HIF system is likely to be of critical importance at this stage (Lavista-Llanos, 2002).

Interestingly and somewhat unexpectedly, analysis of reporter expression patterns in developing flies shows enhanced hypoxia-inducible activity in the cells of the tracheal system. Although experiments using severe hypoxia and genetic inactivation of Sima proteolysis demonstrate a widespread potential for transcriptional activation by this system, exposure to more moderate hypoxia clearly demonstrates enhanced activity in tracheal cells. This was reflected both in higher expression levels of Sima and in higher activity of different HRE-linked reporter genes and, moreover, was shown to be a cell autonomous function that was preserved in cells of tracheal fate even in the face of mutations that disrupt tracheal architecture (Lavista-Llanos, 2002).

The existence of enhanced responses to hypoxia in cells composing the organ of oxygen delivery is clearly of interest and raises questions as to its function, particularly since current models indicate that the regulation of tracheal development by oxygen is guided by signals arising in the metabolizing tissues outside the tracheae. Interestingly, some of the branches of the tracheal system run alongside the Drosophila nervous system, and one possibility is that the tracheae function as sensory organs for hypoxia, as does the carotid body in mammals. A hypoxia pathway affecting behavioral responses has been described in flies, and it will be interesting to determine whether hypoxia-induced behavioral responses share a regulatory mechanism with the HIF system (Lavista-Llanos, 2002 and references therein).

In the current work, the hypoxia response element (HRE) transgenic reporter system (binding of the Sima/Tango heterodimer to a cognate DNA binding site that triggers a reporter system) was used to define upstream control mechanisms operating on the Drosophila HIF system. These studies identify Sima as the regulatory Drosophila HIF subunit and demonstrate a major mode of regulation through oxygen-dependent proteolysis that involves a central oxygen-dependent degradation domain (ODDD). Interestingly, both of the sites of prolyl hydroxylation that operate in mammalian HIF-alpha subunit ODDD (Masson, 2001) appear to be conserved in Sima. Furthermore, genetic ablation of the Drosophila HIF prolyl hydroxylase results in striking upregulation of both Sima and reporter gene activity in vivo. This strongly supports a conserved mode of proteolytic regulation of Sima following prolyl hydroxylation at one or both of these sites (Lavista-Llanos, 2002).

In contrast with the mammalian system, where HIF prolyl hydroxylase activity is represented by the three PHD isoforms (Bruick, 2001, Epstein, 2001), survey of the Drosophila genome revealed only one homolog (Taylor, 2001), raising questions about the potential of this activity to regulate precisely tuned physiological responses. Interestingly, however, the CG1114 gene is itself a Sima target, demonstrating the operation of a conserved feedback control with the potential to contribute to the complex demands of physiological oxygen homeostasis (Lavista-Llanos, 2002).

Studies of Sima regulation also demonstrate an additional regulatory step. Transgenic overexpression of Sima in normoxic embryos resulted in cytoplasmic accumulation of the protein and little transcriptional activity. In contrast, similar levels of overexpression in hypoxia resulted in nuclear accumulation and a strong transcriptional response, demonstrating the presence of a second oxygen-regulated mechanism controlling Sima subcellular localization. An oxygen-regulated nuclear localization step has previously been demonstrated for mammalian HIF-alpha (Berra, 2001, Kallio, 1998, Luo, 2001), although not in every study. However, demonstration of conservation of this mode of regulation in Drosophila Sima provides strong support for the physiological relevance of this process. These findings suggest that Sima subcellular localization is controlled by an active mechanism that maintains the protein in the cytoplasm in normoxia as opposed to an hypoxia-dependent machinery that mediates nuclear import. Although the strong transcriptional activity of mammalian HIF-alpha that is observed after deletion of the ODDD (Elson, 2001, Huang, 1998), mutation of the VHL binding sites (Masson, 2001), or inactivation of VHL (Maxwell, 1999) is consistent with a role for this domain in cytoplasmic localization in normoxia, this has not been tested in studies of mammalian HIF-alpha that have examined subcellular localization directly. Moreover, although induction of nuclear localization by iron chelators and cobaltous ions (Kallio, 1998) suggests a similar mode of regulation to proteolytic regulation, neither the source of the oxygen-sensitive signal nor the mechanism of transduction have been defined. In vivo studies in flies show induction of nuclear Sima after inactivation of CG1114 either by RNAi or by mutation, thus clearly implicating this gene product in the process of cytoplasmic localization in normoxia. Moreover, Sima nuclear localization was also observed in flies bearing the Delta692-863 transgene, indicating that this sequence is absolutely required for cytoplasmic localization (Lavista-Llanos, 2002).

Very recently nonproteolytic regulation of mammalian HIF-alpha subunits involving the C-terminal transactivation domains has been shown to be regulated by hydroxylation of a specific asparaginyl residue by an enzymatic activity that, like the prolyl hydroxylases involved in HIF proteolysis, demonstrates the properties of an alpha-ketoglutarate-dependent dioxygenase (Lando, 2002b). Thus, regulatory hydroxylation of HIF-alpha residues by this class of enzyme appears to extend to both specific asparaginyl and prolyl residues. Currently, the precise substrate requirements of the CG1114 gene product are not defined, and it is not clear whether effects on nuclear localization are mediated through the conserved prolyl residues, possibly reflecting additional functions of the VHL ubiquitylation complex, or whether other sequences within the Sima ODDD mediate this process. Further biochemical and genetic studies should clarify these new insights into the HIF system (Lavista-Llanos, 2002).

Overall, the high degree of conservation in the Drosophila system indicates that genetic studies in this organism should be highly informative in analyses of both the upstream pathways regulating the HIF system, and the downstream physiological effects in an intact organism (Lavista-Llanos, 2002).

Mitochondrial retrograde signaling regulates neuronal function

Mitochondria are key regulators of cellular homeostasis, and mitochondrial dysfunction is strongly linked to neurodegenerative diseases, including Alzheimer's and Parkinson's. Mitochondria communicate their bioenergetic status to the cell via mitochondrial retrograde signaling. To investigate the role of mitochondrial retrograde signaling in neurons, mitochondrial dysfunction was induced in the Drosophila nervous system. Neuronal mitochondrial dysfunction causes reduced viability, defects in neuronal function, decreased redox potential, and reduced numbers of presynaptic mitochondria and active zones. Neuronal mitochondrial dysfunction stimulates a retrograde signaling response that controls the expression of several hundred nuclear genes. Drosophila hypoxia inducible factor alpha (HIFalpha) ortholog Similar (Sima) regulates the expression of several of these retrograde genes, suggesting that Sima mediates mitochondrial retrograde signaling. Remarkably, knockdown of Sima restores neuronal function without affecting the primary mitochondrial defect, demonstrating that mitochondrial retrograde signaling is partly responsible for neuronal dysfunction. Sima knockdown also restores function in a Drosophila model of the mitochondrial disease Leigh syndrome and in a Drosophila model of familial Parkinson's disease. Thus, mitochondrial retrograde signaling regulates neuronal activity and can be manipulated to enhance neuronal function, despite mitochondrial impairment (Cagin, 2015).

The human brain constitutes approximately 2% of body weight but consumes 20% of available oxygen because of its high energy demand. Mitochondria are abundant in neurons and generate the majority of cellular ATP through the action of the mitochondrial ATP synthase complex. Mitochondrial disorders are one of the most common inherited disorders of metabolism and have diverse symptoms, but tissues with a high metabolic demand, such as the nervous system, are frequently affected. The primary insult in all mitochondrial diseases is to mitochondrial function, but the etiology of these diseases is highly pleiotropic. This phenomenon is poorly understood, but suggests that the cellular response to mitochondrial dysfunction may be complex and vary between cell types and tissues (Cagin, 2015).

Mitochondrial retrograde signaling is defined as the cellular response to changes in the functional state of mitochondria. Mitochondrial retrograde signaling enables communication of information about changes in processes such as mitochondrial bioenergetic state and redox potential to the rest of the cell and is thus a key mechanism in cellular homeostasis. The best characterized retrograde responses involve mitochondrial dysfunction eliciting changes in nuclear gene transcription. In yeast, mitochondrial dysfunction causes changes in the expression of genes involved in supplying mitochondria with oxaloacetate and acetyl CoA, the precursors of α-ketoglutarate and glutamate, to compensate for failure of the tricarboxylic acid (TCA) cycle (Cagin, 2015).

In proliferating mammalian cell models, mitochondrial retrograde signaling is more diverse and involves increases in cytosolic-free Ca2+, leading to activation of Ca2+-responsive calcineurin, causing the up-regulation of genes controlling Ca2+ storage and transport. In addition to mitochondrial diseases, alterations in mitochondrial function are also associated with late onset neurodegenerative diseases such as Alzheimer's and Parkinson's. Thus, the neuronal response to mitochondrial function may be altered in these diseases and contribute to disease progression. However, neuronal-specific mitochondrial retrograde signaling is poorly understood and its role in neuronal homeostasis is completely unknown (Cagin, 2015).

This study has developed a neuronal-specific model of mitochondrial dysfunction in Drosophila and used this to characterize mitochondrial retrograde signaling in vivo. Retrograde signaling is shown to regulate neuronal function and can be manipulated to alleviate the effects of mitochondrial dysfunction in neurons (Cagin, 2015).

This study shows that the Drosophila HIFα ortholog Sima is potentially a key regulator of the mitochondrial retrograde response in the nervous system and that knockdown of Sima dramatically improves neuronal function in this and other models of mitochondrial dysfunction. Surprisingly, Sima activity in part causes the dysfunction of neurons containing defective mitochondria. Previous studies of Drosophila mutants in the regulatory and catalytic subunits of the mitochondrial DNA polymerase Polγ have demonstrated that loss of mtDNA replication in Drosophila causes mtDNA loss, reduced neuronal stem cell proliferation, and developmental lethality. To avoid the pleiotropic effects of using homozygous mutant animals, this study developed a neuronal-specific model of mitochondrial dysfunction. The phenotypes resulting from TFAM overexpression and expression of a mitochondrially targeted restriction enzyme were characterized, and both of these tools were used to model neuronal-specific mitochondrial dysfunction (Cagin, 2015).

Overexpression of mitochondrial transcription factor A (TFAM) results in mitochondrial dysfunction caused by inhibition of mitochondrial gene expression, rather than an alteration in mtDNA copy number. Overexpression of TFAM has been shown to have different effects depending on the cell type, model system, or ratio of TFAM protein to mtDNA copy number. The current results are consistent with in vitro studies and overexpression of human TFAM in mice and human cells, which have shown that excess TFAM results in the suppression of mitochondrial gene transcription. Ubiquitous expression of mitoXhoI causes early developmental lethality and that, although there was no significant mtDNA loss, the majority of mtDNA was linearized. Given that mtDNA is transcribed as two polycistronic mRNAs, a double-stranded break in coxI would block the transcription of the majority of mitochondrially encoded genes, resulting in severe mitochondrial dysfunction (Cagin, 2015).

Using a Drosophila motor neuron model, mitochondrial dysfunction was found to cause a reduction in the number of active zones, loss of synaptic mitochondria, and locomotor defects. Mitochondrial dysfunction caused by overexpression of PINK1 or Parkin decreases the rate of mitochondrial transport in vitro and in vivo. Furthermore, a recent study using KillerRed demonstrated that local mitochondrial damage results in mitophagy in axons. Therefore, the acute loss of synaptic mitochondria in the current model may result from defects in mitochondrial transport and/or mitophagy (Cagin, 2015).

Previous studies in mice have examined the effects of neuronal mitochondrial dysfunction by using mitoPstI expression, or targeted knockout of TFAM. Knockout of TFAM specifically in mouse dopaminergic neurons (the 'MitoPark' mouse model) causes progressive loss of motor function, intraneuronal inclusions, and eventual neuronal cell death. Interestingly, cell body mitochondria are enlarged and fragmented and striatal mitochondria are reduced in number and size in MitoPark dopaminergic neurons, suggesting that the effects of neuronal mitochondrial dysfunction are conserved in Drosophila and mammals. Larvae mutant for the mitochondrial fission gene drp1 have fused axonal mitochondria and almost completely lack mitochondria at the NMJ, similar to motor neurons overexpressing TFAM or expressing mitoXhoI (Cagin, 2015).

Adult drp1 mutant flies also have severe behavioral defects. Synaptic reserve pool vesicle mobilization is inhibited in drp1 mutant larvae because of the lack of ATP to power the myosin ATPase required for reserve pool tethering and release. Reserve pool vesicle mobilization is likely to be similarly affected in TFAM overexpressing or mitoXhoI-expressing motor neurons, which would result in locomotor defects in these animals (Cagin, 2015).

Interestingly, expression of the Arctic form of β-amyloid1-42 (Aβ) in Drosophila giant fiber neurons also leads to the depletion of synaptic mitochondria and decreased synaptic vesicles. Synaptic loss and alterations in neuronal mitochondrial morphology have also been observed in postmortem tissue from Alzheimer's disease patients. The parallels between these phenotypes and those in the current model suggest a common underlying mechanism (Cagin, 2015).

Using microarray analysis, this study found that mitochondrial dysfunction in neurons regulates the expression of hundreds of nuclear genes. The Drosophila CNS contains different neuronal subtypes, and glial cells, so the results of the microarray are heterogeneous, representing the pooled response to mitochondrial dysfunction throughout the CNS. Mitochondrial dysfunction was phenotypically characterized in motor neurons, but not all of the genes identified from the microarrays are expressed in motor neurons, e.g., Ilp3. The specific genes that are regulated differ depending on whether mitochondrial dysfunction results from TFAM overexpression or knockdown of ATPsynCF6. However, a core group of approximately 140 genes are similarly regulated in both conditions (Cagin, 2015).

Yeast mutants in different components of the TCA cycle result in differing retrograde responses and comparison of somatic cell hybrids (cybrids) carrying the A3243G mtDNA mutation with cybrids completely lacking mtDNA (ρ0 cells) showed overlapping but distinct gene expression profiles. Moreover, another study comparing cybrids with increasing levels of the A3243G mtDNA mutation showed markedly different alterations in nuclear gene expression, depending on the severity of mitochondrial dysfunction (Cagin, 2015).

Taken together, these data suggest that the cellular response to mitochondrial dysfunction is not uniform and adapts to the specific defect and severity of the phenotype. Therapeutic strategies targeting mitochondrial dysfunction in human disease may therefore need to be tailored to the specific mitochondrial insult. Concomitant with the current findings, previous studies have shown that in yeast, Drosophila, and mammalian-proliferating cells, retrograde signaling activates the expression of hypoxic/glycolytic genes and the insulin-like growth factor-1 receptor pathway to compensate for mitochondrial dysfunction. Rtg1 and Rtg3, the transcription factors that coordinate the mitochondrial retrograde response in yeast, are not conserved in metazoans. In mammalian proliferating cellular models, the retrograde response activates the transcription factors nuclear factor of activated T cells (NFAT), CAAT/enhancer binding protein δ (C/EBPδ), cAMP-responsive element binding protein (CREB), and an IκBβ-dependent nuclear factor κB (NFκB) c-Rel/p50. Whether these transcription factors regulate mitochondrial retrograde signaling in the mammalian nervous system is not known (Cagin, 2015).

HIFα/Sima is a direct regulator of LDH expression in flies and mammals, and this study found that Sima also regulates the expression of two other retrograde response genes, Thor and Ilp3, in the Drosophila nervous system. Importantly, Sima is required for the increase in Thor expression in response to mitochondrial dysfunction. Sima has been strongly implicated as a key regulator of mitochondrial retrograde signaling in Drosophila S2 cells knocked down for the gene encoding subunit Va of complex IV. sima, Impl3, and Thor expression were all increased in this model, and there is a significant overlap with the genes regulated in the current model (Cagin, 2015).

These data support the possibility that the Drosophila HIFα ortholog Sima is a key transcriptional regulator of neuronal mitochondrial retrograde signaling. HIFα is stabilized in hypoxia through the action of prolyl hydroxylases and this mechanism was thought to require ROS, but HIFα stabilization may in fact be ROS independent. In mammalian cells carrying the mtDNA A1555G mutation in the 12S rRNA gene, mitochondrial retrograde signaling has been shown to be activated by increased ROS, acting through AMPK and the transcription factor E2F1 to regulate nuclear gene expression. In the Drosophila eye, loss of the complex IV subunit cytochrome c oxidase Va (CoVa) causes decreased ROS. However, retrograde signaling upon loss of CoVa was not mediated by decreased ROS, but by increased AMP activating AMPK. Similarly, the small decrease in redox potential in neurons in response to mitochondrial dysfunction in the current model makes it unlikely that ROS are the mediator of the retrograde signal. Moreover, HIFα physically interacts with several transcriptional regulators including the Drosophila and mammalian estrogen-related receptor and Smad3, as well as its heterodimeric binding partner HIFβ, to regulate gene expression. Mitochondrial retrograde signaling may modulate these or other unidentified HIFα interactors and, thus, control HIF target gene expression without directly regulating HIFα (Cagin, 2015).

In cancer cell models, mitochondrial dysfunction promotes cell proliferation, increased tumourigenicity, invasiveness, and the epithelial-to-mesenchymal transition via retrograde signaling. In these models, inhibition of retrograde signaling prevents these tumourigenic phenotypes. Neuronal mitochondrial dysfunction in the current model causes a cellular response, resulting in a severe deficit in neuronal function. This response may have evolved to protect neurons, through decreased translation and increased glycolysis, from the short-term loss of mitochondrial function. Over longer periods, however, this response may be counterproductive because it results in decreased neuronal activity and locomotor function. Inhibition of neuronal mitochondrial retrograde signaling, through knockdown of Sima, dramatically improves neuronal function. Thus, mitochondrial retrograde signaling contributes to neuronal pathology and can be modified to improve the functional state of the neuron (Cagin, 2015).

Importantly, this intervention works without altering the primary mitochondrial defect. Knockdown of Sima not only abrogates the acute defects in neuronal function, but also suppresses the reduced lifespan caused by neuronal mitochondrial damage. The benefits of reduced Sima expression therefore extend throughout life. In addition to TFAM overexpression, this study also shows that Sima knockdown in neurons rescues a Drosophila model of the mitochondrial disease Leigh syndrome. However, Sima knockdown does not rescue the lethality caused by a temperature-sensitive mutation in coxI (Cagin, 2015).

Mitochondrial diseases are complex, and mutations in different COX assembly factors cause varying levels of COX deficiency in different tissues. The increasing number of Drosophila models of mitochondrial dysfunction will help to unravel the mechanisms underlying the varied pathology of mitochondrial diseases. Ubiquitous knockdown of Sima also partially restores the climbing ability of parkin mutant flies. The ability of reduced Sima expression to rescue both mitochondrial dysfunction and Parkinson's disease models reinforces the link between mitochondrial deficiency and Parkinson's and suggests that retrograde signaling may be a therapeutic target in Parkinson's disease. HIF1α inhibitors are in clinical trials for lymphoma and so, if the current findings can be replicated in mammalian models, HIF1α inhibitors may be candidates for repurposing to treat mitochondrial diseases and neurodegenerative diseases associated with mitochondrial dysfunction, such as Parkinson's disease (Cagin, 2015).


REGULATION

Regulation of Sima by hypoxia: Functional evidence for homology with mammalian HIF-1alpha

Hypoxia inducible factor-1 (HIF-1) is a heterodimeric complex of two basic-helix-loop-helix proteins of the PAS family that is critical for oxygen-dependent expression of many mammalian genes. Regulation is mediated by the alpha subunit (HIF-1 alpha) and sequences from HIF-1 alpha can confer hypoxia-inducible activity on a Ga14 fusion protein (as evidenced by Gal4 activation of a luciferase reporter construct). To analyze conservation of this system of gene regulation between Drosophila and mammalian cells, Ga14 fusions were constructed with a series of Drosophila basic-helix-loop-helix PAS (bHLH-PAS) proteins and hypoxia inducibility was tested for in transfected Hep3B cells. Ga14 functions with Similar (Sima) but not other Drosophila bHLH-PAS proteins, showed inducible activity following exposure to stimuli that classically activate mammalian HIF-1:hypoxia, cobaltous ions, and desferrioxamine. Sima protein accumulates in Drosophila SL2 cells following hypoxia. Together these findings indicate the existence of functional homologies between Sima and HIF-1 alpha, and that conservation enables Sima to interact with the hypoxia signal transduction system in mammalian cells (Bacon, 1998).

To analyze the mechanisms underlying activation of human HIF-1, portions of individual HIF-1 genes (HIF-1a and ARNT) were fused to a Gal4 DNA binding domain (amino acids 1-147) and assayed for oxygen regulated activity in mammalian cells co-transfected with a Gal4 responsive reporter plasmid. These experiments have demonstrated that fusion of HIF-1alpha to Gal4 confers oxygen regulated activity on the chimeric gene, and have led to the definition of oxygen dependent regulatory domains within the HIF-1alpha molecules. To determine whether particular Drosophila bHLH-PAS proteins could function in an analogous manner similar Gal4 fusions were created with four Drosophila bHLH-PAS genes; sim, sima, trh and darnt/tgo (Bacon, 1998).

Hep3B cells were co-transfected with plasmids expressing these fusion genes and the reporter pUASAsc1 tk-luc, and split for parallel incubations in normoxia and conditions known to induce HIF-1. Aliquots of transfected cells were thus incubated in normoxia or exposed to hypoxia (1% oxygen), cobaltous ions, or the iron chelator desferrioxamine (DFO) in normoxia. Whereas a low constitutive level of transactivation was observed with Gal/Sim, no transactivation was observed with Gal/Trh. In contrast, the Gal/Sima fusion showed transactivation which was clearly increased by hypoxia, cobaltous ions and desferrioxamine and the Gal/DARNT fusion showed strong transactivation in normoxic cells which was not increased by hypoxia or cobalt and or only modestly increased by DFO. This suggested that Sima possessed at least one region that was capable of interaction with an oxygen regulated signal in mammalian cells. To pursue the inducible response conveyed by Sima further, the activity of the Gal/Sima fusion was compared with an analogous Gal/ HIF-1a fusion in Hep3B cells. Though the inducible response was somewhat greater for the HIF-1a fusion, a similar pattern of responses was observed for each fusion gene. In each case activity was increased by hypoxia and DFO, but not by azide. Inhibitors of the respiratory chain like azide do not mimic hypoxia in HIF activation. Activation of the Gal/Sima fusion therefore reflects the characteristics of HIF-1 activation, suggesting that the Gal4/Sima fusion interacts with the same or a closely similar transduction system in mammalian cells (Bacon, 1998).

Comparison of HIF-1a and Sima amino acid sequence reveals the highest level of conservation in the N-terminal basic-helix-loop-helix and PAS domains involved in DNA binding and dimerization. Much lower though significant conservation is observed between the rest of the HIF-1a molecule and sequences in an internal domain of Sima, whereas the most C-terminal domain of approximately 500 amino acids in Sima has no significant homology with HIF-1a (Bacon, 1998).

Studies of Gal/HIF-1alpha fusions have demonstrated regulatory function for several domains lying 3' to the bHLH PAS domains in HIF-1alpha. To determine if the oxygen inducible activity conferred by Sima was consistent with this homology in its central region, a set of N-terminal deletions was made and tested for activity as Gal4 fusions. Deletions to amino acids 365, 527, 665 and 816, all of which removed the bHLH and PAS domains of Sima, but retained differing extents of sequence homologous to HIF-1a in the internal domain, all retained inducible activity. In contrast, two further deletions to amino acids 1043 and 1135, that removed all of this internal domain, but retained the whole or part of the non-conserved C-terminal domain showed no evidence of inducible activity. These experiments indicate that the nonconserved glutamine-rich C-terminal sequence of Sima has significant transactivation capacity, but cannot confer inducibility, whereas inclusion of sequences homologous to the distal portion of HIF-1a is sufficient to confer an inducible response. Consideration of the overall levels of activity of the fusions also indicates that sequences C-terminal to amino acid 665 are sufficient for powerful transactivation with sequences N-terminal to this region being functionally repressive (Bacon, 1998).

A hypoxia-inducible transcriptional response in Drosophila mediated by Sima and Tango

By tracking reporter gene activation in developing flies, the oxygen concentration dependence, developmental regulation, and spatial distribution of the transcriptional response were studied. Based on the observation that an HIF homolog present in extracts of Drosophila SL2 cells can bind a mammalian HRE, the transcriptional system predicted by these experiments was characterized by the generation of transgenic flies bearing mammalian HREs linked to a LacZ reporter gene. A transgenic line bearing a pentamer of an 18-bp sequence from the murine erythropoietin HRE (Epo-LacZ), that is known to be induced by both Trachealess and Single minded, was tested. However, no significant induction of ß-Gal expression was observed when these embryos were exposed to hypoxia. Mutational analysis of the erythropoietin HRE in mammalian cells has indicated that, while this 18-bp sequence is sufficient for binding to HIF-1, an adjacent site that binds an as-yet-unknown factor is also critical for the transcription activity. The erythropoietin gene is not conserved in flies, and it was reasoned that the additional factor(s) binding to this adjacent site might be missing in flies. Therefore, for the design of a new HRE-LacZ reporter, the mammalian lactate dehydrogenase A (LDH-A) gene, which is hypoxia responsive and well conserved in flies, was tested. The mammalian LDH-A hypoxic enhancer includes two HREs separated by an 8-bp spacer and a cyclic AMP responsive element (CRE) located 16 bp further downstream. Both HREs and the CRE consensus have been shown to be necessary for strong hypoxic induction in mammalian cells (Firth, 1995). Transgenic lines were generated with an LDH-LacZ reporter based on a 233-bp fragment from the murine LDH-A enhancer bearing the three relevant boxes described above. In two independent lines carrying this element, approximately 10-fold ß-galactosidase induction was observed in hypoxic embryos. These findings demonstrate the operation in Drosophila embryos of a conserved HRE-dependent transcriptional response to hypoxia and provided an opportunity to characterize the transcriptional response to hypoxia in developing flies (Lavista-Llanos, 2002).

To study how the overall response to hypoxia is influenced by development, synchronized populations of insects were used that were subjected to 5% oxygen for 8 h at different stages of development and tested their ability to induce the reporter was tested. Hypoxia-inducible ß-Gal activity rises dramatically at late embryogenesis, although a relatively high induction of the reporter was recorded at all larval stages. Thus, the response is strongly modulated by developmental parameters, reaching peak levels at late embryogenesis (Lavista-Llanos, 2002).

The range of ambient oxygen concentrations over which the reporter is induced in the whole animal was examined. The response to different oxygen concentrations was studied in stage 16 to 17 embryos. Hypoxic induction was found to be maximal between 3% and 5% oxygen, decreasing gradually as the oxygen concentration rises. This result indicates that activation by low oxygen is strikingly concentration dependent although, presumably because of oxygen gradients within the developing flies, the point of maximal activation is shifted relative to the concentrations of 0.1% to 1.0% oxygen that are maximally active in tissue culture cells. Remarkably, at 1% oxygen, expression of the reporter was below that of the normoxic controls, most probably reflecting general arrest of cell metabolism in very severe hypoxia (Lavista-Llanos, 2002).

Although the first experiments with X-Gal staining suggested ubiquitous reporter expression, this result was obtained by using long incubation times on stage 17 embryos, where the cuticle is beginning to be secreted, and it was surmised that very high levels of reporter product may have obscured spatial localization. By using X-Gal staining for 30 min on stage 15 to 16 embryos, a distinct expression pattern that appeared to correspond to tracheal branches was observed. Indeed, double staining of hypoxic embryos with an antitracheal lumen antibody revealed hypoxia-inducible ß-Gal reporter in the tracheal system, with stronger expression in the lateral trunks and dorsal branches (Lavista-Llanos, 2002).

To study the expression of the reporter in more detail and to assess postembryonic stages, an additional reporter construct was developed based on the binary Gal4/UAS system that would allow more sensitive detection in vivo particularly in the presence of the developing cuticle. A 51-bp fragment was engineered from the core of the LDH-A enhancer as a dimer controlling expression of Gal4 (LDH-Gal4) and several transgenic fly lines were generated with single insertions in all of the three chromosomes. These lines were crossed with different UAS-green fluorescent protein (GFP) or UAS-GFPn.LacZ lines, and expression of the reporter was monitored in normoxia and hypoxia (5% oxygen for 4 h). Unlike the LDH-LacZ lines, these lines showed constitutive reporter expression in salivary glands. However, hypoxia-inducible expression was remarkably similar between the different reporter lines. Double immunofluorescence experiments with antibodies to ß-Gal and the tracheal marker Trachealess demonstrated that hypoxia-dependent transcription is indeed elicited mostly in tracheal cells but also in scattered patches on the ectoderm. More detailed studies of developmental regulation showed that, whereas stage 11 embryos showed no induction of the reporter, scattered cells of the tracheal system began to express the reporter during stages 12 and 13, with the number of cells expressing the reporter increasing gradually from this stage onward. By the end of embryogenesis and throughout the larval stages all of the tracheal cells showed a strong response to hypoxia. To answer the question of whether enhanced hypoxia responsiveness in the tracheal system reflects some microenvironmental signal connected with the position or integrity of the developing tracheal system, hypoxic induction of the reporter was studied in embryos homozygous for a mutation in the gene breathless (btl), which encodes a Drosophila FGF receptor that is required for tracheal cell migration. Tracheal cells fail to migrate in btlMZ13 mutants, and the tracheal system is not developed. In these mutants, however, the nonmigrating tracheal cells express the hypoxic reporter normally, clearly indicating that tracheal integrity is not required for the hypoxic response (Lavista-Llanos, 2002).

Enhanced hypoxia-inducible transcription in tracheal cells is of interest since the tracheal system is directly responsible for oxygen delivery in the fly. Nevertheless, current models of oxygen-dependent tracheal plasticity involve a receptor-mediated chemotactic outgrowth of terminal branches that is generated by hypoxia-inducible expression of the Drosophila FGF homolog Branchless in extratracheal metabolizing tissues. Therefore, it would be predicted that the hypoxic machinery should operate in nontracheal tissues as well, although perhaps with different sensitivity. To pursue this, more severe hypoxic conditions (4% oxygen for 16 h) were applied to embryos bearing the hypoxic reporter and the GFP expression was recorded. Under these conditions, hypoxia-inducible expression was clearly observed outside the tracheae, with approximately one-quarter of late embryos or larvae manifesting widespread reporter expression across the ectoderm, esophagus, gut, fat body, muscles, and gonads, as well as the trachea. However, tracheal expression remained dominant, and responses in extratracheal cells were more patchy in the majority of embryos. When embryos were placed at an oxygen concentration of 3% or less, arrest of embryonic development occurred, and tissue-specific expression of the reporter could not be recorded. Taken together with the analysis of responses to graded hypoxia in LDH-LacZ flies, this suggests that most if not all tissues in the developing fly can respond to this system, although in many nontracheal tissues this appears to be at a threshold that is close to that which produces metabolic and developmental arrest. In contrast, the tracheal system appears to respond with greater sensitivity. Tracking inducible expression precisely in adult flies was more difficult because of cuticle development. Nevertheless, strong hypoxia-inducible expression was evident in the legs (Lavista-Llanos, 2002).

The experiments described above established that the HIF response is conserved and strongly active in developing flies, suggesting that genetic studies in Drosophila may be used to explore the upstream and downstream connections of the pathway in an in vivo system (Lavista-Llanos, 2002).

To test this a proposed role for Tgo in the hypoxic response, embryos that were homozygous for a strong tgo mutant allele (tgo5) were examined. These embryos failed to induce the reporter in hypoxia, strongly supporting the role of Tgo as the HIF-ß subunit and indicating that, as in mammalian cells, this protein is absolutely required for the hypoxia response (Lavista-Llanos, 2002).

The best candidate HIF-alpha homolog is Similar (Sima) since it shows the highest amino acid identity, and protein levels are increased at 1% O2 in Drosophila SL2 cells (Bacon, 1998). To investigate the role of Sima in the transcriptional response to hypoxia, immunofluorescent detection of Sima was performed in embryos subjected to hypoxia (5% oxygen for 14 h). By performing the fixation immediately (0.5 to 1 min) after opening the hypoxic chamber, strong Sima staining could be detected in stage 15 to 16 embryos subjected to hypoxia, but not in normoxic controls, in a pattern of expression corresponding to the tracheae that strikingly mimics expression of the reporter in hypoxia. To pursue this further, synchronized stage 8 wild-type embryos bearing the LDH-Gal4/UAS-GFP.LacZ reporter were kept in hypoxia, and protein extracts were analyzed by Western blotting with anti-Sima and anti-ß-Gal antisera (Bacon, 1998). Sima protein levels are increased in hypoxia, paralleling induction of the ß-Gal reporter. However, Sima mRNA increased only slightly in hypoxia (1.3- to 1.5-fold), whereas ß-Gal mRNA was upregulated by 9.7- to 10.4-fold. These results indicate that Sima is controlled posttranscriptionally, most probably at the level of protein degradation. To test this, the protein was overexpressed in hs-Gal4/UAS-Sima embryos by giving a 37°C heat shock for 20 min and the decay of Sima protein levels was compared in hypoxia and normoxia. Sima levels 4 h after heat shock are much higher in hypoxia than in normoxia. At 16 h after heat shock, expression of Sima can still be visualized in patches of cells in hypoxic embryos but is undetectable in embryos kept in normoxia, thus confirming that Sima is stabilized in hypoxia (Lavista-Llanos, 2002).

Proteolytic control of mammalian HIF-alpha is dependent on a central oxygen-dependent degradation domain (ODDD) (Huang, 1998; Pugh, 1997) that contains two sites of oxygen-dependent prolyl hydroxylation (Masson, 2001). Although overall conservation between HIF-1alpha and Sima outside the basic and HLH domains is low (Bacon, 1998; Nambu, 1996), sequence comparison indicates that these prolyl residues are both conserved (Sima Pro747 and Pro850), suggesting that they might define a Sima ODDD. To test this, a deletion of Sima was generated between amino acids 692 and 863 (Delta692-863) and the deleted Sima was expressed in UAS transgenic lines through a heat shock-Gal4 driver as described above for full-length Sima. This deletion stabilizes Sima in normoxia with no further changes in stability when heat-shocked embryos are incubated in hypoxia. These findings demonstrate that Sima is regulated by oxygen-dependent proteolysis in a manner similar to mammalian HIF-alpha (Lavista-Llanos, 2002).

To override the rate of Sima degradation and shed light on an additional mode of regulation, the protein was overexpressed by using an engrailed-Gal4 (en-Gal4) driver. The en-Gal4 element was recombined in a chromosome bearing a UAS-nGFP-LacZ element (with a nuclear localization signal) that was used to mark the nuclei. Sima protein, expressed in the characteristic striped engrailed pattern, was easily detected by immunofluorescence in stages 11 to 15 normoxic embryos. Surprisingly, however, the protein was almost exclusively localized in the cytoplasm. In another experiment, Sima was expressed in embryos bearing the direct LDH-LacZ hypoxic reporter (lacking the UAS-nGFP-LacZ element). In normoxia, the reporter was induced only at very low levels. In contrast, when the same experiments were performed with embryos exposed to 5% oxygen, Sima was detected predominantly in the nucleus, and embryos exhibited strong expression of the reporter in the expected engrailed pattern. In contrast, expression of Trachealess or a chimeric protein exhibiting specificity for Single minded target genes under en-Gal4 did not activate the reporter in either normoxia or hypoxia (Lavista-Llanos, 2002).

This confirms that Sima mediates activity of the HRE reporter but also indicates the existence of an additional oxygen-regulated nuclear localization mechanism, as has been proposed in mammalian cells (Kallio, 1998; Luo, 2001). Studies of mammalian HIF-alpha have demonstrated that stabilization by deletion of the ODDD is associated with constitutive upregulation of transcriptional activity (Elson, 2001; Huang, 1998). Therefore, it was reasoned that the deletion might also affect subcellular localization. The Drosophila system provided an opportunity to compare the localization of Delta692-863 with full-length Sima under similar expression conditions. Accordingly, Delta692-863 was expressed similarly in embryos through the en-Gal4 driver. Deletion of the ODDD caused constitutive localization of Sima in the nucleus irrespective of oxygen levels. Consistent with this, the LDH-LacZ transcriptional reporter showed strong constitutive expression in the expected engrailed pattern after crossing into embryos expressing Delta692-863. These results indicate that the Sima ODDD also mediates signals critically required for cytoplasmic localization of Sima in normoxia (Lavista-Llanos, 2002).

A Drosophila sequence homologous to the HIF prolyl hydroxylases was recently reported (HIF prolyl hydroxylase) (Bruick, 2001; Taylor, 2001). These enzymes are known to promote proteasomal destruction of HIF-alpha through the hydroxylation of key prolyl residues in the ODDD. To assess whether CG1114 regulates Sima levels and the transcriptional response to hypoxia in vivo, CG1114 expression was abrogated by injecting double-stranded RNA into early embryos bearing the LDH-Gal4/UAS-nGFP.LacZ reporter gene. Sima protein was strongly upregulated in normoxic embryos injected with CG1114 RNAi but not in individuals injected with an unrelated double-stranded RNA. Sima upregulation induced expression of the LDH-Gal4/UAS-nGFP.LacZ nuclear hypoxic reporter. Furthermore, complete overlap between Sima protein and the nuclear reporter product demonstrated that Sima was exclusively localized in the nucleus. To further assess the role of CG1114 in the regulation of Sima, flies were examined that bore mutations predicted to inactivate this gene completely. A lethal P element insertional mutation [l(3)02255], mapping 336 nucleotides upstream of the initiation codon of the CG1114 gene, is available from Drosophila public stock collections. By crossing l(3)02255 heterozygous mutant flies with a strain carrying the Df(3R)3-4 chromosomal deficiency that covers CG1114 gene, it was verified that the insertion was indeed causing the lethal phenotype. Sima levels and induction of the transcriptional response to hypoxia in l(3)02255 embryos bearing the LDH-Gal4/UAS-nGFP.LacZ reporter were analyzed. Sima is ubiquitously upregulated in homozygous mutant embryos in normoxia, and high levels of Sima result in concomitant strong induction of the LDH-Gal4/UAS-nGFP.LacZ reporter in all embryonic tissues (Lavista-Llanos, 2002).

Mammalian HIF prolyl hydroxylase genes are themselves induced by hypoxia (Elson, 2001), suggesting the existence of a feedback response that limits HIF induction in hypoxia. To test whether this aspect of regulation is also conserved in Drosophila and does indeed represent a feedback response dependent on activity in the transcriptional pathway itself, tests were performed for induction by both hypoxia and Sima overexpression. CG1114 gene expression is strongly upregulated in hypoxia and is strongly induced in the typical engrailed pattern by overexpression of Sima by using an en-Gal4 driver (Lavista-Llanos, 2002).

The hypoxia-induced paralogs Scylla and Charybdis inhibit growth by down-regulating S6K activity upstream of TSC in Drosophila

Diverse extrinsic and intrinsic cues must be integrated within a developing organism to ensure appropriate growth at the cellular and organismal level. In Drosopohila, the insulin receptor/TOR/S6K signaling network plays a fundamental role in the control of metabolism and cell growth. scylla and charybdis (a. k. a. charybde), two homologous genes identified as growth suppressors in an EP (enhancer/promoter) overexpression screen, act as negative regulators of growth. The genes are named after mythological monsters that lived in the Strait of Messina between Sicily and Italy, posing a threat to the passage of ships. The simultaneous loss of both genes generates flies that are more susceptible to reduced oxygen concentrations (hypoxia) and that show mild overgrowth phenotypes. Conversely, either scylla or charybdis overactivation reduces growth. Growth inhibition is associated with a reduction in S6K but not PKB/Akt activity. Together, genetic and biochemical analysis places Scylla/Charybdis downstream of PKB and upstream of TSC1. Furthermore, scylla and charybdis are induced under hypoxic conditions and scylla is a target of Drosopohila HIF-1 (hypoxia-inducible factor-1: Similar) like its mammalian counterpart RTP801/REDD1, thus establishing a potential cross-talk between growth and oxygen sensing (Reiling, 2004).

RTP801/REDD1 was shown to be induced by hypoxia (Shoshani, 2002). This prompted an investigation of the effects of hypoxia on the scylla charybdis double mutants. scylla charybdis mutant larvae were raised on normal food at room temperature in a hypoxia chamber containing 9% oxygen during their entire development. In general, control flies as well as scylla charybdis homozygotes were 3-4 d delayed in development under these hypoxic conditions. However, whereas adult homozygous scylla charybdis double mutants could readily be recovered under standard culture conditions in normoxia, homozygous mutants of three independent scylla charybdis allelic combinations (char180 in combination with scy31, scy113, or scyEP9.85) were strongly underrepresented compared to normoxic conditions. The appearance of an increased number of dead pupae in the vials was observed. The scylla113 char180 double-mutant combination had the strongest effect, and only two escapers (out of 326 scored flies) hatched, whereas under normoxia flies with this genotype were recovered with nearly the expected Mendelian ratio. The eclosed homozygotes raised under hypoxia did not show obvious morphological defects. Thus, whereas simultaneous loss of Scylla and Charybdis under normoxic conditions results in a slight increase in growth, their absence under reduced oxygen concentrations severely compromises larval development. This indicates that Scylla and Charybdis have a critical function for survival under hypoxic conditions (Reiling, 2004).

The transcription factor HIF-1 is the key regulator of changes in gene expression in response to hypoxia. It consists of two bHLH-PAS domain protein subunits (HIF-1alpha and HIF-1beta). Under conditions of low oxygen, the HIF-1 protein complex is stabilized and binds to Hypoxia Response Elements (HRE), short regulatory DNA sequences (core recognition sequence 5'-TACGTG-3') located in the genomic region of target genes. Both the scylla and charybdis loci possess several HREs. Since RTP801/REDD1, a mammalian homolog of scylla and charybdis, is a direct target gene of HIF-1 and is induced under hypoxic conditions, it was enquired whether this function is evolutionarily conserved. Wild-type larvae were subjected to hypoxia (between 2% and 5% O2) and checked for the induction of scylla and charybdis expression. It is mainly the endoreplicative tissue such as fat body, gut, salivary glands, and tracheae that respond to changes in oxygen concentrations. scylla mRNA expression was up-regulated in the larval fat body and in the gut after hypoxia. charybdis, in contrast, is mildly induced in the midgut but not in the fat body. Whether hypoxia had an effect on Scylla protein levels and distribution was also tested. To detect the endogenous Scylla protein, advantage was taken of a transgenic Scylla-reporter line (a so-called protein trap line). This protein trap line bears a promoter-less green fluorescent protein (GFP)-reporter transgene in the scylla locus generating a Scylla-GFP fusion protein. Scylla-GFP is expressed in most larval tissues. Under normoxic conditions, nuclear accumulation of Scylla protein is observed in some cells of the endoreplicative tissue. Consistent with the mRNA data, upon exposure of third instar larvae to various hypoxia conditions, an up-regulation and nuclear localization of Scylla protein was observed in the fat body and in the gut (Reiling, 2004).

In Drosopohila, the bHLH-PAS family comprises Period (Per), Trachealess (Trh), Single-minded (Sim), Spineless (Ss), Dysfusion (Dys), Tango (Tgo), and Similar (Sima). Tgo is the ubiquitously expressed HIF-1beta ortholog, which dimerizes with any of the alpha-subunits. Sima has been shown to fulfill analogous functions to its mammalian homolog HIF-1 (Reiling, 2004).

To test whether scylla/charybdis transcription is regulated by bHLH-PAS proteins that recognize the same DNA stretch, sim, trh or sima were overexpressed together with tgo using the Lsp2-Gal4-driver that is active specifically in the fat body during the third larval stage. For Sima, a form lacking the oxygen-dependent degradation domain (ODD) was used, rendering it refractory to proteolytic destruction under normoxic conditions. Only the coexpression of tgo and sima induced scylla but not charybdis expression as assessed by mRNA in situ hybridization. This does not preclude, however, the possibility that charybdis is a target of Tgo-Sima, since its endogenous induction was observed upon hypoxia, but only in the gut and not in the fat body. Since neither expression of trh with tgo nor sim induced scylla or charybdis expression, the regulation of scylla by the Tgo-Sima heterodimer is specific. Thus, scylla and charybdis, like their mammalian homolog RTP801/REDD1, are induced by hypoxia, and at least scylla appears to be a direct target gene of the HIF-1 homolog Tgo-Sima (Reiling, 2004).

Alternative splicing of Sima

Oxygen homeostasis depends upon sensing variations in oxygen tension (pO2) and signal transduction leading to physiologically appropriate changes in gene expression. Virtually all aerobic prokaryotes and eukaryotes have evolved various mechanisms for regulating genes during times of oxygen deprivation. Particularly in hypoxia-tolerant species, i.e., those capable of surviving and, even thriving, in environments with little (hypoxia) or no (anoxia) oxygen present, these changes in gene expression, together with a drastically reduced steady-state of ATP levels, are critical determinants of the organism’s resilience to low oxygen. In contrast to most endothermic vertebrates (birds and mammals), invertebrates are often hypoxia-tolerant. Embryos and adult stages of the fruitfly Drosophila melanogaster, for example, possess a rich and varied repertoire of survival strategies endowing them to withstand, and fully recover from, even hour-long exposure to anoxia (N2 atmosphere). These responses vary once oxygen levels have declined to 1.6%-3%, the critical pO2 of the species, below which, generally, insect respiration ceases to be regulated. The following strategies are employed by fruitfly embryos and adults when faced with hypoxic/anoxic challenges: (1) ability to sense falling pO2 within seconds via a nitric oxide signaling pathway [Wingrove, 1999); (2) anoxic stupor ranging from loss of coordination to complete immobility throughout the entire period of O2 deprivation; (3) greatly reduced O2 consumption down to 20% of normoxic values; (4) glycogen-fueled anaerobiosis with lactate as major endproduct; (5) general and reversible chromatin condensation; (6) cell cycle arrest. Another important oxygen-sensitive response in insects is the long known hypoxic stimulation of outgrowth and proliferation of tracheal termini for improved supply of limiting amounts of O2 to tissues. This hypoxia-induced ramification of insect breathing tubes poses intriguing parallels to the O2 response in mammalian angiogenesis (Gorr, 2004 and references therein).

A wide range of animals, from mammals to fruitflies to nematodes, share a common pathway that links sensing of changes in pO2 to transcriptional regulation. Central to hypoxia-mediated gene expression are hypoxia-inducible (transcription) factors, or HIFs (Drosophila homolog: Sima), which belong to the family of basic-helix-loop-helix (bHLH)/PAS transcription factors. In mammals, C. elegans and Drosophila, HIF is a heterodimer of a- and b-subunits that specifically recognizes short cis-regulatory E-box motifs called hypoxia-response elements (HREs) in the promoter and/or enhancer regions of a number of genes. Functional HREs, i.e., those capable of oxygen-dependent binding of HIF proteins in vitro and transactivating reporter genes in vivo, can be summarized in the following consensus: 5’ B(A/G)CGTGVBBB 3’ (with B = all bases except A, V = all bases except T). The central four-base core (underlined) is critical for the binding of any HIF complex, be it mammalian, insect or crustacean. Oxygen-dependent activation of HIF is, in cell culture, controlled primarily at the protein level through specific oxidative modifications of the alpha-subunit. The HIF oxygen sensor is a novel prolyl hydroxylase that catalyzes the O2-dependent hydroxylation of proline residues within the oxygen-dependent degradation domain (ODD) of HIF-1a (Huang, 1998). Once hydroxylated, HIF-1a rapidly binds to the von Hippel-Lindau (VHL) tumor suppressor product, enabling ubiquitination and rapid degradation by the proteasome (Salceda, 1997; Maxwell, 1999; Cockman, 2000; Tanimoto, 2000). Conversely, hypoxia, and hypoxia-mimicking agents such as transition metals (e.g., Co2+) and iron chelators (e.g., desferrioxamine), inhibit proline hydroxylation of HIF-1a, thus enabling the protein to escape proteolytic degradation and to dimerize with HIF-1b (ARNT: Drosophila homolog Tango). The HIF dimer then translocates into the nucleus where it activates target genes containing hypoxia response element (HRE) binding sites. This scheme for activation of HIF extends from mammals to invertebrates. In contrast, little is known about the means utilized by hypoxic cells to regulate HIF function (Gorr, 2004 and references therein).

In Drosophila melanogaster, hypoxia-specific HIF-like DNA binding activity was first demonstrated in so-called SL2 cells cultured from late embryos of the fruit fly (Nagao, 1996). Subsequently, structural homologs of HIF subunits were cloned in the form of the bHLH/PAS proteins Similar (Sima) [HIF-1a homolog] (Ohshiro, 1997) and Tango (dARNT) [HIF-1b homolog]. As expected for HIF proteins, Sima and Tango transcripts are ubiquitously expressed throughout embryogenesis and in nearly all tissues of Drosophila. Moreover, as in mammals, Sima's stability and Drosophila HIF activity seem to be regulated in an O2-dependent fashion. Functionality of homologous amino acid motifs in Sima, predicted to confer normoxic instability (Srinivas, 1999), was confirmed by hypoxic inductions of luciferase reporter plasmids through Sima/Gal4 fusion constructs (Bacon, 1998). The fact that this transactivation was observed in mammalian cells, indicates that Sima is able to functionally substitute for human HIF-1alpha, and provides evidence for the close conservation of these signaling pathways between mammals and insects. Recent work on transgenic flies by Lavista-Llanos (2002) showed that, in vivo, HIF activity is most pronounced in developing tracheal cells, wherein proliferation is hypoxia-stimulated. Both Sima and Tango are absolutely required for regulating the transcription of HRE-reporter constructs within tracheae of hypoxic fly embryos. Other alpha-like bHLH/PAS proteins, known to also heterodimerize with Tango in a tissue-specific manner are unable to elicit this hypoxic response. These include the neurogenic factor Single-minded (Sim), and most notably, Trachealess (Trh), the master regulator that drives early tracheal development. Spineless (Ss), Drosophila’s aryl hydrocarbon receptor (Ahr) homolog, and Dysfusion (Dys), another tracheogenetic bHLH/PAS factor, are two additional partner proteins for Tango. Therefore, Tango can form functional heterodimers with five partners: Sima, Sim, Trh, Ss, Dys. This growing list of Tango heterodimers with close, or even overlapping, DNA specificities begs the question as to how, in the face of potential competition for Tango and/or binding site interference by coexpressed Tango partners, the signaling pathways mediated by each alpha-like bHLH/PAS protein are maintained and controlled (Gorr, 2004 and references therein).

To address the control and maintenance of Drosophila HIF signaling, SL2 cells (Schneider cells, line 2) were used. These cells were originally cultured from late, 20-24 h old Drosophila embryos, a time of maximal HIF activity during Drosophila’s development. The fact that, Sim and Trh, potential Tango competitors, are not expressed in this cell line, makes the investigations less complicated. Thus, SL2 cells are well suited as a model for investigating function and control of Drosophila HIF/Sima. In hypoxic cultures of SL2 cells Sima is expressed in the form of full-length (fl) and splice-variant (sv) isoforms. The following evidence supports the role of full-length Sima as functional HIFapha and the role of SL2 HIF as a transcriptional activator or suppressor. The pO2-dependence of Sima abundance matches that of HIF activity. HIF-dependent changes of candidate target gene expression were detected through variously effective stimuli: hypoxia (strong) > iron chelation (e.g., desferrioxamine) (moderate) >> transition metals (e.g., cobalt) ~= normoxia (ineffective). Sima overexpression augments hypoxic induction or suppression of different targets. In addition to the full-length exon 1-12 transcript yielding the 1510 amino acid HIFalpha homolog, the sima gene also expresses, specifically under hypoxia, an exon 1-7+12 splice variant, which translates into a 426 amino acid Sima truncation termed svSima. svSima contains bHLH and PAS sequences identical with Sima, but, due to deletion of exons 8-11, lacks the oxygen-dependent degradation domain and nuclear localization signals. Overexpressed svSima fails to transactivate reporter genes. However, it attenuates HIF (flSima:Tango) stimulated reporter expression in a dose-dependent manner. Thus svSima has the potential for regulating Drosophila HIF-function under steady and hypoxic pO2 by creating a cytosolic sink for Sima’s partner protein Tango (Gorr, 2004).

Drosophila Cyclin D/Cdk4 requires Hif-1 prolyl hydroxylase, potentially independently of Sima/Hif, to drive cell growth

The Drosophila cyclin-dependent protein kinase complex Cyclin D/Cdk4 induces cell growth (accumulation of mass) as well as proliferation (cell cycle progression). To understand how CycD/Cdk4 promotes growth, a screen was performed for modifiers of CycD/Cdk4-driven overgrowth in the eye. Loss-of-function mutations in Hif-1 prolyl hydroxylase (Hph), an enzyme involved in the cellular response to hypoxic stress, dominantly suppress the growth but not the proliferation function of CycD/Cdk4. hph mutant cells are defective for growth, and, remarkably, ectopic expression of Hph is sufficient to increase cellular growth. Epistasis analysis places Hph downstream of CycD/Cdk4. Overexpressed CycD/Cdk4 causes an increase in Hph protein in tissues where Hph induces growth, suggesting a mechanism whereby Hph levels are regulated posttranscriptionally in response to CycD/Cdk4. These data suggest that Hph, in addition to its function in hypoxic response, is a regulator of cellular growth and that it is a key mediator for CycD/Cdk4. Nevertheless, observations weigh against an important role for Hif-1 in Hph-driven growth, suggesting a potential Hif-1-independent function of Hph (Frei, 2004).

The synthetic promoter element Glass multiple reporter (GMR) is expressed in the eye imaginal disc posterior to the morphogenetic furrow, where only cells in the second mitotic wave undergo one synchronized cell division. GMR can be used to drive expression of the yeast transcription factor Gal4. Gal4 can be used to direct transcription of transgenes placed next to the UAS binding site of Gal4. Therefore, UAS transgenes driven by Glass-activated GMR-Gal4 are expressed predominantly in postmitotic cells. Under these circumstances, expression of CycD/Cdk4 leads to an enlargement of the adult eye, bigger ommatidia and bristles, and a general rough appearance (Datar, 2000). Although some ommatidia have additional cells, the main cause of the enlargement is an increase in cell size leading to 61% larger ommatidia. In order to identify loss-of-function mutants that modify this phenotype, CycD/Cdk4 was expressed in a deficiency collection background and screened under the light microscope for modifiers. Out of 162 deficiencies that cover 60%-70% of the genome, four deficiencies were isolated that dominantly suppress CycD/Cdk4. Df(3R)6-7, which deletes polytene segments 82D3/8-F3/6, led to a decrease in the enlargement of the eye and bristle size. Most strikingly, under these conditions, CycD/Cdk4 led to an increase in ommatidia size of only 17. Subsequently, partial overlapping deficiencies were tested: Df(3R)3-4 showed the same suppression phenotype but not a third deficiency, Df(3R)110, demonstrating that the gene of interest is between 82F3 and F7. All available mutants in this region were tested, and two lethal P element insertions were identified that showed an identical suppression phenotype compared to the deficiencies: l(3)02255 is inserted 104 bp, and l(3)S030304 is inserted 1111 bp upstream of the putative transcription start site of Hph/dmHph/CG1114 (Frei, 2004).

Whether l(3)02255 and l(3)S030304 are alleles of hph and whether their loss of function causes the suppression of CycD/Cdk4 were addressed. Northern blot experiments revealed that both heterozygous mutants l(3)02255 and l(3)S030304 have reduced expression of hph compared to the endogenous levels and that l(3)02255/l(3)S030304 transheterozygotes lack all detectable hph expression. In addition to these loss-of-function mutants, the EP3200 line has an EP insertion 382 bp upstream of Hph. Expression of hph using this EP element, by the hs-Flp Act>CD2>Gal4 system, led to a weak expression of hph in the absence of a heat shock, due to leakage of the system. A further increase in hph expression occured upon heat shock (Frei, 2004).

A full-length Hph cDNA was cloned under the control of a UAS promoter and injected into flies. These Hph transgenes suppress at least partially the cell growth phenotype of homozygous hph mutants and completely suppress the reduced viability of l(3)02255/l(3)S030304 transheterozygotes. Furthermore, when UAS-Hph transgenes were coexpressed with CycD/Cdk4 using the GMR-Gal4 driver, the dominant suppression of CycD/Cdk4-driven overgrowth by l(3)02255 was inhibited and the overgrowth phenotype was restored. It is concluded that l(3)02255 and l(3)S030304 are alleles of Hph and that the reduction of Hph can suppress CycD/Cdk4-induced overgrowth (Frei, 2004).

Whether growth or proliferation would be suppressed by hph in the eye imaginal disc was tested. GFP was expressed either alone or together with CycD/Cdk4 in wild-type, hph02255/+, or Df(3R)3-4/+ backgrounds using the GMR-Gal4 driver. Imaginal eye discs from wandering third instar larvae were dissected, trypsinized to single cells, and analyzed by FACS for their cell size by using the forward scatter (FCS). Expression of CycD/Cdk4 in a wild-type background led to an increase in the forward scatter of 20%-30%, which was reduced to 10%-15% in an hph02255/+ or Df(3R)3-4/+ background. Acridine orange staining showed that the suppression phenotype was not due to an increase in cell death. Furthermore, the cell cycle distribution was analyzed of eye imaginal discs from wandering larvae or pupae 48 hr after prepupae formation. At both time points, the increase in cells entering S and G2/M phases of the cell cycle due to ectopic expression of CycD/Cdk4 was not altered in an hph02255/+ background. Taken together, these data demonstrate that the cell size and proliferation functions of CycD/Cdk4 can be separated. Furthermore, Hph is required for the increase in cell size but not required for proliferation, suggesting that Hph functions downstream of CycD/Cdk4 in a growth-specific manner (Frei, 2004).

In the experiments described above, CycD/Cdk4 was induced in mostly postmitotic cells of the eye imaginal disc. To test suppression by hph in mitotically dividing cells, CycD/Cdk4 was induced during larval development, and wing discs cells were analyzed. Ectopic expression of CycD/Cdk4 shows a distinctive induction of growth: cells divide at a faster rate but are otherwise indistinguishable from control cells from the same disc. Therefore, when single clones are measured, the clone area is increased, and the clone consists of more cells with no change in cell size or cell cycle phasing. Since columnar cells of wing discs form a single cell layer, measuring the clone area gives an accurate estimation of the amount of mass that was accumulated during the growth of the clone. CycD/Cdk4 was overexpressed together with GFP in random clones using the hs-Flp Act>CD2>Gal4 system and analyzed after a 48 hr growth period. Compared to external control clones expressing only GFP, expression of CycD/Cdk4 caused a 75% increase in the median clone size. This phenotype depends on Hph, since the median clone size was reduced to control level in a heterozygous hph02255 mutant background. The suppression did not correlate with an increase in apoptosis, since coexpression of the cell death inhibitor p35 gave identical phenotypes. When cell size and cell cycle phasing were analyzed by FACS, there was no difference between cells expressing CycD/Cdk4 and internal control cells in either wild-type or hph/+ mutant backgrounds. These results demonstrate that the induction of growth by CycD/Cdk4 depends on normal levels of Hph. Furthermore, since hph suppressed growth but not proliferation in the eye imaginal discs, expression of CycD/Cdk4 in the wing should lead to a change in cell size if only growth but not proliferation were suppressed. However, no difference in cell size was detected, suggesting that the increase in proliferation caused by CycD/Cdk4 is secondary to the induction of growth (Frei, 2004).

Whether hph function was required for normal rates of cell growth was tested. Most hph0225/hph02255 or hph02255/Df(3R)3-4 animals die during embryogenesis, and only a few larvae hatch. These mutant larvae have severe growth defects and die within 2 to 3 days. Transheterozygotic hphS030304/hph02255 mutants develop normally until pupariation, but very few escaper adults eclose. These escapers are smaller than their heterozygous siblings but have normal body proportions. Weight measurements showed that hphS030304/hph02255 mutant flies are 18% lighter than heterozygotes. Therefore, hph mutant animals show a phenotype similar to homozygous cdk43 flies (Meyer, 2000) or wild-type flies reared at low oxygen (Palos, 1979; Frazier, 2001; Frei, 2004).

To test whether hph mutant cells are autonomously defective for growth, homozygous mutant clones were induced in the fat body using ionizing radiation. hph02255 was crossed to flies expressing GFP under the control of a constitutively active promoter inserted on the same arm of the chromosome as Hph (3R). The progeny were irradiated during embryogenesis, emerging larvae were grown in regular food, wandering third instar larvae were dissected, and their fat bodies were fixed and mounted. Homozygous hph02255 mutant cells lacked GFP, whereas heterozygous mutant cells expressed GFP. hph02255/hph02255 cells were smaller and contain less DNA than heterozygous neighboring cells. Importantly, the presence of a UAS-Hph transgene partially suppresses this phenotype, indicating that loss of Hph is the cause of the growth defect (Frei, 2004).

Whether ectopic expression of Hph is sufficient to stimulate growth was tested. EP3200 or UAS-Hph transgenes were used to induce Hph expression. Cell clones expressing Hph were induced in wing imaginal discs, and the median clone size was measured. Expression of Hph led to an increase in clone area very similar to CycD/Cdk4. Surprisingly, expression of Hph together with CycD/Cdk4 stimulated clonal growth to the same extent as Hph alone. However, in the presence of the apoptosis inhibitor p35, an additive phenotype was detected when both growth drivers were coexpressed. As for CycD/Cdk4, overexpressed Hph did not change cell size or cell cycle phasing, as assayed by FACS (Frei, 2004).

To test whether Hph functions downstream of CycD/Cdk4 also in this tissue, clones expressing Hph were induced in a homozygous cdk43 mutant background, and the median clone size was measured. Under these conditions, Hph led to a very similar induction of growth as in a wild-type background. FACS analysis indicates that there are no detectable changes in cell size or cell cycle phasing. These data show that Hph is sufficient to stimulate growth, and the finding that this stimulation is independent of Cdk4 suggests that Hph functions downstream of CycD/Cdk4 (Frei, 2004).

Ectopic expression of CycD/Cdk4 in the posterior compartment of the wing imaginal disc using the en-Gal4 promoter leads to an enlargement of the posterior compartment in adult wings with no change in trichome (hair) density. Since the trichome density is proportional to the number of cells per area, the increase in compartment size is due to more cells of the same size. When Hph was expressed under en-Gal4 control using EP3200 or UAS-Hph transgenes, a similar result was obtained: posterior compartments were bigger and contained more cells of the same size. Thus, Hph induces growth in a similar manner to CycD/Cdk4 in wing imaginal discs. However, when Hph was expressed in the eye imaginal disc using the GMR-Gal4 driver, no increase in cell size, as assayed by FACS, was observed. Furthermore, adult eyes were not enlarged. Further experiments are required to understand why Hph expression is not sufficient to increase growth in the eye imaginal disc (Frei, 2004).

Little is known about how Hph RNA or protein levels are regulated. In Drosophila embryos, hph is expressed uniformly and does not seem to be subject to patterning. Vertebrate cells have three Hph orthologs (Taylor, 2001): HPH-3 protein localizes to the nucleus, HPH-2 exclusively to the cytoplasm, and HPH-1 mainly in the cytoplasm with a little staining in the nucleus (Huang, 2002; Metzen, 2003; Frei, 2004).

To test whether the subcellular localization or levels of Hph are altered in response to CycD/Cdk4, polyclonal antibodies were raised to full-length Hph. To test the specificity of the antiserum, Hph was overexpressed in the posterior compartment of the wing using the en-Gal4 driver. Hph staining increased in posterior regions, both in the peripodial and columnar epithelium. Homozygous hph mutant cells, marked by the absence of GFP, lacked detectable Hph staining. Therefore, the serum is specific for Hph and is able to detect endogenous levels of Hph. Furthermore, in third instar imaginal wing discs, Hph staining was uniform throughout the disc and specific for the nucleoplasm of the cells. Very little staining was detectable in the cytoplasm or the nucleolus (Frei, 2004).

When Hph staining was analyzed in cells expressing ectopic CycD/Cdk4, increased Hph levels were observed. Antiserum staining in the peripodial epithelium shows that these cells had an increase in nucleoplasmic as well as cytoplasmic Hph. In contrast, when homozygous cdk43 mutant cells, marked by the absence of GFP, were analyzed, only background levels were detected. Therefore, Hph protein levels are regulated in response to CycD/Cdk4 gain and loss of function. When hph expression was analyzed by RT-PCR from wing discs expressing ectopic CycD/Cdk4, no effect on Hph RNA levels was detected. Furthermore, when whole third instar larvae were analyzed by RT-PCR or microarray analysis, no change in Hph expression was observed. Taken together, these observations suggest that CycD/Cdk4 affects Hph levels posttranscriptionally (Frei, 2004).

The hydroxylation activity of HPHs depends of Fe2+ bound to the active site (Epstein, 2001). Therefore, iron chelators like deferoxamine mesylate (DFO) are commonly used to experimentally mimic hypoxic conditions. When Drosophila larvae are raised on regular food supplemented with 2 mM DFO, they show an induction in Hif-1α/β, as assayed with a reporter construct (Lavista-Llanos, 2002), that is very similar to that seen under hypoxic conditions. When CycD/Cdl4 or Hph are expressed in the posterior compartment of the wing imaginal disc, the increase in compartment areas is suppressed by DFO. Moreover, when DFO is added to flies expressing CycD/Cdk4 in the postmitotic eye using the GMR-Gal4 driver, the enlargement of the adult eye as well as the rough appearance is suppressed, however, not to the same extent as in the heterozygous hph mutant backgrounds. This suggests that the hydroxylation activity of Hph is required for its growth function and that Hph is a major growth effector of CycD/Cdk4 (Frei, 2004).

The finding that Drosophila Hph functions downstream of CycD/Cdk4 and is sufficient to increase growth when overexpressed suggests that CycD/Cdk4 and Hph work in a common pathway. Consistent with this, heterozygous hph mutants do not suppress the extra growth induced by components of the insulin pathway or dMyc. Moreover, increases in cell size and changes in cell cycle phasing induced by the insulin signaling pathway, dMyc, or Ras in wing imaginal disc cells, do not depend on Cdk4. Taken together, these results suggest that the CycD/Cdk4-Hph pathway functions separately from these other growth regulatory pathways (Frei, 2004).

Since the kinase activity of Cdk4 is required for the induction of growth and proliferation (Meyer, 2000), Hph could be a phosphorylation target of Cdk4. The consensus sequence of vertebrate pocket proteins, the only known targets of cyclin D1/Cdk4, can be different from the classical CDK sequence. In Drosophila Rbf1, two potential sites have been found that disrupt its regulation by CycD/Cdk4 and CycE/Cdk2: T356PLTR and S728PHPK (Xin, 2002). Both sites are different from the vertebrate consensus sequence. Therefore, a search for putative consensus sequences on Hph is difficult. However, there are three sites that have the minimal requirement of a serine or threonine residue followed by a proline: T91PDAP, T204PGTT, and T285PPAA. None of these resemble the consensus sequences recognized by either the vertebrate or Drosophila complex. Nevertheless, future experiments should address whether CycD/Cdk4 phosphorylates Hph on these or other sites and how this affects Hph function (Frei, 2004).

It is proposed that in wing discs, Hph protein levels are regulated in response to CycD/Cdk4. Although it cannot be excluded that growth is also induced in an Hph-independent manner, the findings that overgrowth driven by CycD/Cdk4 and Hph is suppressed nearly completely by the iron chelator DFO or by heterozygosity for hph suggest that this is a major mechanism. Moreover, it is proposed that the small size of flies reared at low oxygen concentrations is caused at least partially by a decrease in Hph activity due to the absence of oxygen (Frei, 2004).

How does Hph induce growth? Since the hydroxylation inhibitor DFO suppressed the increases in growth caused by CycD/Cdk4 or Hph, Hph's hydroxylation activity is probably required. The only characterized hydroxylation target of Hph is Hif-1α, a mediator of the transcriptional response to hypoxia (Semenza, 2001; Kaelin, 2002; Bruick, 2003). Although mutant alleles of the Drosophila Hif-1α ortholog sima are not available, a partial loss-of-function allele of the Hif-1β ortholog, tango1, was available. To test the potential role of Hif1 in growth control, the ey-Flp/FRT method was used to generate flies in which the eyes were >80% homozygous mutant for tango1. If Hph stimulates growth by hydroxylating Hif-1α and targeting it for degradation, then loss of Hif-1 activity might be expected to result in overgrowth phenotypes. Contrary to this expectation, overgrowth was not observed in tango1/tango1 eyes. Moreover, GMR-driven expression of CycD/Cdk4 led to the same degree of overgrowth in tango1/tango1 eyes as in wild-type controls. Although these observations weigh against an important role for Hif-1 in Hph-driven growth, it is important to note that tango1 is not a null allele and that Tango is thought to be expressed in excess over its binding partner, Sima. Thus, further analysis using sima mutants and overexpression will be required to definitively test whether Hph drives cell growth via a Hif-1-dependent mechanism or through hydroxylation of novel targets. The finding that only one of the three vertebrate Hph orthologs is required for regulation of Hif-1α levels in vivo (Berra, 2003) further suggests that additional targets may be important (Frei, 2004).

There is little data that suggest a growth function for vertebrate HPH. Rat HPH-1/SM-20 was identified first as a gene upregulated by growth factors or serum. The induction is very fast and peaks at 60 min after stimulation. Remarkably, this induction does not require de novo protein synthesis, as it is not blocked by the translation inhibitor cyclohexamide. The effect on growth upon deregulation of mouse Falkor/HPH-3 is controversial: whereas expression of a C-terminal fragment induced cells to grow faster and to a higher density, expression of a wild-type construct had no effect. An antisense oligonucleotide specific for Falkor induced cells to grow faster. Thus, the function of vertebrate HPH family member in growth control is still ambiguous (Frei, 2004).

Drosophila Hph has at least two functions: response to hypoxia and regulation of growth. How are they linked? In response to hypoxia, Sima/Tango activity is strongly induced in endoreplicative tissues like trachea, gut and fat body, and to a much lesser extent, in imaginal discs (Lavista-Llanos, 2002). Although endopreplicative cells lacking Hph are impaired for growth, ectopic overexpression of Hph in these cells does not increase their size. In contrast, in imaginal discs, Hph can increase growth when overexpressed. It is speculated that in endoreplicative tissues, Hph's main function is to regulate the hypoxic response and, to a minor extent, growth, whereas in imaginal tissues, Hph's main function is to regulate growth. Taken to the environment of wild Drosophila, this suggests that hypoxic conditions, which are often found in fermenting fruit, may induce a strong hypoxic response in endoreplicative tissues. Since these tissues are metabolically highly active, this response may be required for the generation of sufficient ATP by the induction of glycolysis. In imaginal discs, cell cycle progression is not controlled primarily by extrinsic factors but by disc intrinsic growth cues. Therefore, even under hypoxic stress, growth and development of imaginal discs continues but may be slowed down, presumably by inactivation of Hph activity, in order to ensure the formation of adult animals (Frei, 2004).

In fat body cells, Hph is a nuclear protein, and homozygous Cdk4 mutant cells lack detectable Hph levels. Moreover, ectopic expression of CycD/Cdk4 leads to more Hph protein in the cytoplasm and/or the nucleus. Surprisingly, a reporter line showed an increase, rather than a decrease, in Sima activity upon expression of CycD/Cdk4. It is proposed that in the fat body, Hph induced by CycD/Cdk4 is not sufficient to hydroxylate Hif-1α. In addition to the cofactors oxygen and iron, hydroxylation activity requires the binding of 2-oxoglutarate to the active site of HPH (Epstein, 2001; Bruick, 2001). 2-oxoglutarate is an intermediate of the citrate cycle, and its levels might correlate with the metabolic activity of the cell. Therefore, Hph protein may be induced by CycD/Cdk4 but may require 2-oxoglutarate and oxygen for catalytic activity in the fat body. In this model, Hph would integrate the regulation of growth by CycD/Cdk4 and its upstream regulators, with the regulation of growth by the metabolic activity, mediated by oxygen and 2-oxoglutarate (Frei, 2004).

insulin-PI3K/TOR pathway induces a HIF-dependent transcriptional response in Drosophila by promoting nuclear localization of HIF-alpha/Sima

The hypoxia-inducible factor (HIF) is a heterodimeric transcription factor composed of a constitutively expressed HIF-ß subunit and an oxygen-regulated HIF-alpha subunit. A hypoxia-inducible transcriptional response has been defined in Drosophila that is homologous to the mammalian HIF-dependent response. In Drosophila, the bHLH-PAS proteins Similar (Sima) and Tango (Tgo) are the functional homologues of the mammalian HIF-alpha and HIF-ß subunits, respectively. HIF-alpha/Sima is regulated by oxygen at several different levels that include protein stability and subcellular localization. Insulin can activate HIF-dependent transcription, both in Drosophila S2 cells and in living Drosophila embryos. Using a pharmacological approach as well as RNA interference, it has been determined that the effect of insulin on HIF-dependent transcriptional induction is mediated by PI3K-AKT and TOR pathways. Stimulation of the transcriptional response involves upregulation of Sima protein but not sima mRNA. Finally, the effect of the activation of the PI3K-AKT pathway on the subcellular localization of Sima protein was analyzed in vivo. Overexpression of dAKT and dPDK1 in normoxic embryos provokes a major increase in Sima nuclear localization, mimicking the effect of a hypoxic treatment. A similar increase in Sima nuclear localization was observed in dPTEN homozygous mutant embryos, confirming that activation of the PI3K-AKT pathway promotes nuclear accumulation of Sima protein. It is concluded that regulation of HIF-alpha/Sima by the PI3K-AKT-TOR pathway is a major conserved mode of regulation of the HIF-dependent transcriptional response in Drosophila (Dekanty, 2005).

Insulin stimulation or exposure to hypoxia can induce common target genes; such transcriptional response depends on the Drosophila HIFalpha and HIFß homologues Sima and Tango. Evidence is provided that insulin-stimulated HRE response is transduced by the PI3K-AKT pathway and, furthermore, that the effect depends on TOR and involves an increase in Sima protein levels, whereas mRNA levels are not affected. These results are in good agreement with the reported effect of the PI3K-AKT/TOR pathway on mammalian HIF, because in several cell lines activation of this pathway led to an increase in HIF protein levels or stabilization of the protein (Dekanty, 2005).

In addition, the subcellular localization of Sima depends on oxygen tension in a dose-dependent manner, and activation of the PI3K-AKT pathway also causes a major increase in Sima nuclear localization. This regulatory mechanism might represent another conserved aspect of HIF regulation, because one recent report suggests that HIF-alpha accumulates in the nucleus of retinal epithelial cells upon IGF-1alpha treatment. The molecular bases of HIFalpha nuclear accumulation upon hypoxia or PI3K activation are so far unclear. Nucleo-cytoplasmic localization of many transcription factors results from a steady-state equilibrium between nuclear import and nuclear export, and accumulation in one or the other compartment depends on the relative rate of import versus export. Whether HIF nuclear accumulation upon hypoxia or growth factor stimulation depends on regulated nuclear import or regulated nuclear export remains to be determined (Dekanty, 2005).

All major cellular features of oxygen-dependent regulation of HIF proteins are conserved in Drosophila and, thus, activation of the HRE response in Drosophila by the PI3K-AKT and TOR pathways extends even further the notion of a conserved HIF system in evolution. The functional significance of the regulation exerted by PI3K-AKT and TOR pathways over the HRE response was discussed in the context of cancer biology, because the loss of PTEN or the tuberous sclerosis complex 1 (TSC1) and TSC2 proteins is frequently associated with human tumors. What is the functional meaning of PI3K-AKT pathway regulation of the HRE response in normal cells? The PI3K-AKT and TOR pathways are regulated in part by growth factors and other endocrine signals and thus, endocrine control of the HRE response is conserved among animal species that have diverged 700 million years ago. It seems reasonable to postulate that the main physiological role of HRE induction by the PI3K-AKT pathway is the stimulation of glycolysis, but a function in the regulation of animal body size and growth control is another interesting possibility (Dekanty, 2005).

A cardinal function of the PI3K-AKT and TOR pathways throughout evolution is to regulate growth, and to determine the final size of developing organs and whole organisms. Genetic studies in Drosophila have shown that a reduction of the activity of the PI3K-AKT pathway results in flies with a reduced body size, bearing smaller cells. Likewise, a reduction in TOR signaling provokes growth decrease and, conversely, over-activation of TOR signaling due to loss-of-function of its negative regulators TSC1 and/or TSC2, leads to an increase in cell and body size. The effect of TOR on cell growth was reported to be mediated at least in part by S6K, a kinase that phosphorylates the ribosomal protein S6, leading to translational activation (Dekanty, 2005).

Besides its role in growth control, the insulin-PI3K-AKT pathway has been traditionally implicated in the regulation of circulating glucose levels and anabolic metabolism. It has been demonstrated that the cellular bases of glucose sensing and regulation of serum glucose are conserved between mammals and Drosophila, and it has been proposed that PI3K-AKT signaling in conjunction with the TOR pathway coordinates growth according to environmental conditions and the nutritional status of the organism. The mechanism involved in this coordination is still unclear. Oxygen tension is one environmental factor that has been shown to modulate growth in Drosophila, because hypoxic flies have a reduced body size (Frazier, 2001). A mechanistic explanation to this phenomenon has been provided by showing that overexpression of Sima protein causes a reduction in cell size in an autonomous manner (Centanin, 2005). Consistent with this, it has been shown that hypoxia provokes a reduction in Drosophila TOR pathway activity and that such reduction results from hyperactivation of the TSC1-TSC2 tumor suppressor complex. Similar results have been reported in mammalian cells, implying that TOR is regulated by hypoxia. Furthermore, hypoxia-dependent TSC1-TSC2 stimulation and growth inhibition are mediated by the product of a HIF/Sima-inducible gene called scylla in Drosophila and RTP801/REDD1 in mammals (Dekanty, 2005).

The results establish a direct link between pathways largely implicated in growth regulation (PI3K-AKT and TOR) and the hypoxia-responsive machinery (HIF/Sima). It is suggested that in hypoxia, HIF prolyl hydroxylase (Hph)/Fatiga activity is reduced, resulting in HIF/Sima stabilization and induction of an HRE response. One of the genes induced by hypoxia is scylla/RTP801/REDD1, which in turn activates TSC1-TSC2. Then, stimulation of the TSC complex provokes reduction of TOR activity and decreases S6K phosphorylation, resulting in growth inhibition. According to this model, PI3K-TOR activation of HIF-alpha/Sima might generate a negative feedback loop to limit or downregulate growth; in this scenario, low oxygen levels are expected to enhance Sima-dependent inhibition of growth (Dekanty, 2005).

It has been reported that mitochondrial dysfunction inhibits Hph/Fatiga activity, thereby triggering the transcriptional response to hypoxia, and also that it concomitantly provokes growth defects. It was proposed that Hph/Fatiga operates as an integration node between oxygen levels and growth regulation. The current results have shown that the effect of Hph/Fatiga on growth regulation is conveyed at least in part by Sima. Further studies will reveal whether Hph/Fatiga also plays Sima-independent roles in cell and organ size determination (Dekanty, 2005).

Regulation of Drosophila embryonic tracheogenesis by dVHL and hypoxia

The tracheal system of Drosophila is an interconnected network of gas-filled epithelial tubes that develops during embryogenesis and functions as the main gas-exchange organ in the larva. Larval tracheal cells respond to hypoxia by activating a program of branching and growth driven by HIF-1α/sima-dependent expression of the breathless (btl) FGF receptor. By contrast, the ability of the developing embryonic tracheal system to respond to hypoxia and integrate hard-wired branching programs with sima-driven tracheal remodeling is not well understood. This study shows that embryonic tracheal cells utilize the conserved ubiquitin ligase (von Hippel-Lindau) (dVHL) to control the HIF-1 α/sima hypoxia response pathway, and two distinct phases of tracheal development with differing hypoxia sensitivities and outcomes were identified: a relatively hypoxia-resistant 'early' phase during which Sima activity conflicts with normal branching and stunts migration, and a relatively hypoxia-sensitive 'late' phase during which the tracheal system uses the dVHL/sima/btl pathway to drive increased branching and growth. Mutations in the archipelago (ago) gene, which antagonizes btl transcription, re-sensitize early embryos to hypoxia, indicating that their relative resistance can be reversed by elevating activity of the btl promoter. These findings reveal a second type of tracheal hypoxic response in which Sima activation conflicts with developmental tracheogenesis, and identify the dVHL and ago ubiquitin ligases as key determinants of hypoxia sensitivity in tracheal cells. The identification of an early stage of tracheal development that is vulnerable to hypoxia is an important addition to models of the invertebrate hypoxic response (Mortimer, 2008).

The development and survival of an organism are dependent on its ability to adapt to changing environmental conditions. Responses to some environmental changes, for example in nutrient availability, temperature, or oxygen concentration, involve alterations in patterns of gene expression that allow the organism to survive periods of environmental stress. In metazoan cells, the cellular response to reduced oxygen is mediated primarily by the HIF (hypoxia inducible factor) family of transcription factors, which are heterodimers composed of α and β subunits belonging to the bHLH Per-ARNT-Sim (bHLH-PAS) protein family. The HIF-1 αβ heterodimer is the primary oxygen-responsive HIF in mammalian cells and binds to a specific DNA sequence termed hypoxia response element (HRE) present in the promoters of target genes involved in energy metabolism, angiogenesis, erythropoiesis, and autophagy. HIF-1 activity is inhibited under normoxic conditions by two hydroxylase enzymes that use dioxygen as a substrate for catalysis to hydroxylate specific proline or aspartate residues in the HIF-1α subunit. These modifications limit HIF-1 activity by either reducing HIF-1α levels or inhibiting its ability to activate HRE-containing target promoters. One of these inhibitory mechanisms involves the 2-oxoglutarate/Fe(II)-dependent HIF-1 prolyl hydroxylase (HPH), which attaches a hydroxyl group onto each of two conserved proline residues in the oxygen-dependent degradation domain (ODD) of mammalian HIF-1α. These modifications create a binding site in the HIF-1α ODD for the Von Hippel-Lindau (VHL) protein, the substrate adaptor component of a ubiquitin ligase that subsequently polyubiquitinates HIF-1α and targets it for degradation by the proteasome. This degradation mechanism operates constitutively in normoxia and is epistatic to otherwise wide spread expression of HIF-1α mRNA. HIF-1α protein is also modified by a second oxygen-dependent hydroxylase termed Factor Inhibiting HIF (FIH) that hydroxylates an asparagine residue in the HIF-1α C-terminal activation domain. This blocks interaction with the CBP/p300 transcriptional co-factor and thus further restricts expression of HIF-1 responsive genes. These parallel O2-dependent hydroxylation mechanisms by HPH and FIH ensure that HIF-1α levels and activity remain low in normoxic conditions. However as oxygen levels become limiting in the cellular environment, rates of hydroxylation decline and HIF-1α is rapidly stabilized in a form that dimerizes with HIF-1β, translocates to the nucleus, and promotes transcription of HRE-containing target genes (Mortimer, 2008).

Evidence suggests that invertebrate homologs of HIF-1 are also regulated in response to changes in oxygen availability. In the fruit fly Drosophila melanogaster, the HPH homolog fatiga (fga) has been shown to genetically antagonize the HIF-1α homolog similar (sima) during development. The Drosophila VHL homolog dVHL has also been shown to be capable of binding to human HIF-1α and stimulating its proteasomal turnover in vitro. In addition, the Drosophila genome encodes a well-characterized HIF-1β homolog tango (tgo), and two potential FIH homologs (CG13902 and CG10133; Berkeley Drosophila Genome Project) that have yet to be analyzed functionally. Spatiotemporal analysis of sima activation using sima-dependent hypoxia-reporter transgenes has shown that exposure to an acute hypoxic stress induces Sima most strongly in cells of the larval and embryonic tracheal system, while induction of reporter activity in other tissues requires more chronic exposure to low oxygen. The larval tracheal system is composed of an interconnected network of polarized, epithelial tubes that duct gases through the organism. As the trachea acts as the primary gas-exchange organ in the larva, it is thus a logical site of hypoxia sensitivity. During larval stages, specific cells within the tracheal system called 'terminal cells' respond to hypoxia by initiating new branching and growth that results in the extension of fine, unicellular, gas-filled tubes toward hypoxic tissues in a manner somewhat analogous to mammalian angiogenesis . Studies have shown that sima and its upstream antagonist fga function within terminal cells to regulate this process. sima is necessary for terminal cell branching in hypoxia and its ectopic activation, by either transgenic overexpression or loss of fga, is sufficient to induce excess branching even in normoxia. These phenotypes have been linked to the ability of sima to promote expression of the breathless (btl) gene, which encodes an FGF receptor that is activated by the branchless (bnl) FGF ligand. This receptor/ligand pair is known to act via a downstream MAP-kinase signaling cascade to promote cell motility and tubular morphogenesis in a variety of systems. Excessive activation of this pathway within tracheal cells by transgenic expression of btl is sufficient to drive excess branching. Reciprocally, misexpression of the bnl ligand in certain peripheral tissues is sufficient to attract excess terminal cell branching. Indeed production of secreted factors such as Bnl may be a significant part of the physiologic mechanism by which hypoxic cells attract new tracheal growth. Sima-driven induction of btl in conditions of hypoxia thus allows larval terminal cells to enter what has been termed an 'active searching' mode in which they are hyper-sensitized to signals emanating from nearby hypoxic non-tracheal cells (Mortimer, 2008 and references therein).

The role of the btl/bnl pathway in tracheal development is not restricted to hypoxia-induced branching of larval terminal cells. It also plays a critical, earlier role in the initial development of the embryonic tracheal system from the tracheal placodes, groups of post-mitotic ectodermal cells distributed along either side of the embryo that undergo a process of invagination, polarization, directed migration, and fusion to create a network of primary and secondary tracheal branches . btl and bnl are each required for this process via a mechanism in which restricted expression of bnl in cells outside the tracheal placode represents a directional cue for the migration of btl-expressing cells within the placode. Accordingly, btl expression is normally highest in pre-migratory and migratory embryonic fusion cells. In contrast to the larval hypoxic response, sima does not appear to be required for morphogenesis of the embryonic tracheal system. Rather, developmentally programmed signals in the embryo dictate a stereotyped pattern of btl and bnl expression that leads to a similarly stereotyped pattern of primary and secondary tracheal branches. The btl/bnl pathway thus responds to developmental signals to drive a fixed pattern of branching in the embryo, while in the subsequent larval stage it responds to hypoxia-dependent sima activity to facilitate the homeostatic growth of larval terminal cells and tracheal remodeling (Mortimer, 2008 and references therein).

Under normal circumstances, developing Drosophila tissues do not begin to experience hypoxia until the first larval stage, when organismal growth and movement begin to consume more oxygen than can be provided by passive diffusion alone. As a consequence, the first hypoxic challenge normally occurs after the btl/bnl-dependent elaboration of the primary and secondary embryonic branches is complete. Thus, the ability of the larval tracheal system to drive new branching and remodeling via sima and btl represents the response of a developed 'mature' tracheal system to reduced oxygen availability. By contrast the effect of hypoxia on embryonic tracheal development, which requires tight spatiotemporal control of Btl signaling to pattern the tracheal network, is not as well understood. Given that the trachea does not function as a gas-exchange organ until after fluid is cleared from the tubes at embryonic stage 17, it may be that the transcriptional response of embryonic tracheal cells to hypoxia leads to mainly metabolic changes rather than to a btl-driven program of tubulogenesis and remodeling. However, if the embryonic tracheal system does utilize the sima pathway to induce hypoxia-dependent changes in btl gene transcription, then hypoxic exposure of embryos might be predicted to produce a situation of competing developmental and homeostatic inputs that converge on the btl/bnl pathway. The ability of tracheal cells to integrate such signals may then determine whether or not the embryonic tracheal system is able to adapt to oxygen stress, or whether embryonic tracheal development represents a sensitive period during which the organism's ability to respond to changes in oxygen levels is inherently limited by a pre-programmed pattern of developmental gene expression (Mortimer, 2008).

This study shows that the embryonic tracheal system utilizes the dVHL/sima pathway to respond to hypoxia, but that the type and severity of resulting phenotypes depend on the developmental stage of exposure. Hypoxic challenge while embryonic tracheal cells are responding to developmentally programmed btl/bnl migration signals disrupts tracheal development and results in fragmented and unfused tracheal metameres. In contrast, hypoxic challenge at a somewhat later embryonic stage after fusion is complete results in overgrowth of the primary tracheal branches and the production of extra secondary branches. Interestingly, it was found that the threshold of hypoxia required to induce tracheal phenotypes in the early embryo is higher than that required to induce excess branching phenotypes in later embryonic stages, indicating that tracheal patterning events in the embryo are relatively resistant to hypoxia. Genetic analysis indicates that both types of hypoxic tracheal phenotypes -- stunting and overgrowth -- require sima and can be phenocopied in normoxia by reducing expression of the HIF-1α ubiquitin ligase gene dVHL specifically within tracheal cells. Moreover, it was found that reduced dVHL expression in the larval trachea leads to excess terminal cell branching in a manner quite similar to that observed in fga mutants. Molecular and genetic data indicate that excess btl transcription is a major cause of hypoxia-induced tracheal phenotypes. Consistent with this, mutations in the archipelago (ago) gene, which antagonizes btl transcription in tracheal fusion cells, synergize strongly with dVHL inactivation to disrupt tracheal migration and branching. Interestingly, ago mutations also lower the threshold of hypoxia required to elicit tracheal phenotypes in the 'early' embryo, suggesting that the relative activity of the btl promoter can affect hypoxic sensitivity. These findings show that the dVHL/sima pathway plays an important role in tracheal development, and identify two distinct phases of embryonic development that show different phenotypic outcomes of activating this pathway: an early phase during which sima activity conflicts with developmental control of tracheal branching and migration, and a later phase during which the tracheal system uses the dVHL/sima/btl pathway to adapt to hypoxia by increasing its future capacity to deliver oxygen to target tissues (Mortimer, 2008).

Hypoxia-induced remodeling of tracheal terminal cells represents the response of a developed larval tracheal system to reduced levels of O2 in the environment. By contrast, the response of the developing embryonic tracheal system to systemic hypoxia has not been as well characterized. In light of the observation that embryonic tracheal cells display hypoxia-induced activation of a Sima-reporter) and that sima promotes btl expression in larval tracheal cells, embryonic exposure to hypoxia may thus produce a situation in which hard-wired btl/bnl patterning signals in the embryo come into conflict with the type of sima/btl-driven plasticity of tracheal cell branching seen in the larva. This study examined the effect of hypoxia on embryonic tracheal branching and migration. It was found that hypoxia has dramatic effects on the patterns of morphogenesis of the primary and secondary tracheal branches. Surprisingly, varying the timing and severity of hypoxic challenge is able to shift the outcome from severely stunted tracheal branching to excess branch number and enhanced branch growth. Genetic and molecular data indicate that both classes of phenotypes, stunting and overgrowth, involve regulation of sima activity and btl transcription by dVHL, and that the effects of hypoxia on tracheal development can be mimicked in normoxia by tracheal-specific knockdown of dVHL. This observation confirms a central role for dVHL in restricting the hypoxic response in vivo, and identifies a role for dVHL as a required inhibitor of sima and btl during normal tracheogenesis (Mortimer, 2008).

Since Trh and Sima/HIF-1α share a similar consensus DNA binding site, it is likely that the tracheal phenotypes elicited by either hypoxia or dVHL knockdown are to some degree the product of a combined 'Trh/Sima-like' transcriptional activity in tracheal cells. This conclusion is supported both by the general phenotypic similarity (i.e. migration and overgrowth defects) between hypoxia/dVHL knockdown and trh overexpression, by the modest ability of trh alleles to suppress dVHLi phenotypes, and by the previously demonstrated overlap of transcriptional activity between Trh and human HIF-1α. Indeed, Trh is well-established as a required activator of developmental btl expression. However, because the excess Btl activity that occurs in hypoxia or in the absence of dVHL occurs independently of a change in Trh expression, it thus appears to be mediated largely by increased sima activity (Mortimer, 2008).

This analysis suggests that there are two distinct developmental 'windows' of embryogenesis during which hypoxia has opposite effects on tracheal branching. The first corresponds to a period immediately before and during primary branch migration that is relatively insensitive to hypoxia. Embryos in this stage show a minimal response to 1% O2, but show a nearly complete arrest of migration in 0.5% O2. Interestingly, a prior study found that similarly staged embryos (stage 11) respond to complete anoxia by prolonged developmental arrest, from which they can emerge and resume normal development. These somewhat paradoxical results -- that acute hypoxia is more detrimental to development than chronic anoxia -- might be explained by the observation that chronic exposure to low O2 induces Sima activity throughout the embryo while acute exposure activates Sima only in tracheal cells. The former scenario may result in coordinated developmental and metabolic arrest throughout the organism, while in the latter scenario developmental patterns of gene expression in non-tracheal cells may proceed such that tracheal cells emerging from an 'early' hypoxic response find an embryonic environment in which developmentally hard-wired migratory signals emanating from non-tracheal cells have ceased (Mortimer, 2008).

The second type of tracheal response occurs during a later 'window' of embryogenesis after btl/bnl-driven primary and secondary branch migration and fusion are largely complete. It involves sinuous overgrowth of the primary and secondary branches, and duplication of secondary branches. As in the 'early' response, 'late' hypoxic phenotypes are controlled by the dVHL/sima pathway, yet unlike the 'early' response, these phenotypes occur at high penetrance even at 1% O2. Thus the 'late' embryonic tracheal system is relatively sensitized to hypoxia and responds with increased branching in a manner similar to larval terminal cells. Indeed, much as larval branching increases with decreasing O2 levels, it was observed that dorsal trunk growth in the late embryo is graded to the degree of hypoxia. The mechanism underlying the differential sensitivity of the 'early' and 'late' tracheal system may be quite complex. However, it was found that tracheogenesis can be sensitized to hypoxia by reducing activity of ago, a ubiquitin ligase component that restricts btl transcription in tracheal cells via its role in degrading the Trh transcription factor. Increasing transcriptional input on the btl promoter thus appears to sensitize 'early' tracheal cells to hypoxia. As Sima also controls btl transcription, one explanation of the difference in sensitivity between different embryonic stages may thus lie in differences in the activation state of the btl promoter. If so then the activity of the endogenous btl regulatory network may be an important determinant of the threshold of hypoxia required to elicit changes in tracheal architecture (Mortimer, 2008).

An organism can have its hypoxic response triggered in two ways, either by systemic exposure of the whole organism to a reduced O2 environment or by localized hypoxia produced by increased O2 consumption in metabolically active tissues. Data from this study and others suggests there may be distinctions between these two triggers. Exposing larvae or embryos to a systemic pulse of hypoxia results in a 'btl-centric' response specifically in tracheal cells. Outside of an 'early' vulnerable period which corresponds to embryonic branch migration and fusion, elevated Btl activity in embryonic tracheal cells promotes branch duplications and overgrowth similar to that seen in larvae. By contrast, tracheal growth induced by localized hypoxia in the larva has been suggested to involve a 'bnl-centric' model in which the hypoxic tissue secretes Bnl and recruits new tracheal branching. Whether this type of mechanism operates in embryos, or whether embryos ever experience localized hypoxia in non-tracheal cells, has not been established (Mortimer, 2008).

tHE data indicate that dVHL is a central player in the hypoxic response pathway in embryonic and larval tracheal cells. A prior study found that injection of dVHL dsRNA into syncytial embryos disrupted normal tracheogenesis, but was technically limited in its ability to conduct a detailed analysis of dVHL function in development and homeostasis. The current study found that dVHL knockdown specifically in tracheal cells mimics the effect of systemic hypoxia on embryonic tracheal architecture and larval terminal cell branching. dVHL knockdown thus phenocopies loss of the HPH gene fga, which normally functions to target Sima to the dVHL ubiquitin ligase in normoxia. Moreover, all phenotypes that result from reduced dVHL expression can be rescued by reducing sima activity, suggesting that Sima is the major target of dVHL in the tracheal system. These data support a model in which dVHL, fga, and sima function as part of a conserved VHL/HPH/HIF-1α pathway to control tracheal morphogenesis in embryos and larvae. The btl receptor appears to be an important target of this pathway in embryonic (this study) and larval tracheal cells. Knockdown of dVHL elevates btl transcription in embryonic placodes and tracheal branches, and removal of a copy of the gene effectively suppresses dVHL tracheal phenotypes. Reciprocally, overexpression of wild type btl in embryonic tracheal cells can produce migration defects and sinuous overgrowth, while expression of a constitutively active btl chimera (btlλ) also leads to primary branch stunting and duplication of secondary branches. Interestingly, pupal lethality associated with tracheal-specific knockdown of dVHL is not sensitive to the dose of btl, but is dependent on sima. Thus the dVHL/sima pathway may have btl independent effects on tracheal cells in later stages of development (Mortimer, 2008).

Cell autonomy of HIF effects in Drosophila: tracheal cells sense hypoxia and induce terminal branch sprouting

Drosophila tracheal terminal branches are plastic and have the capacity to sprout out projections toward oxygen-starved areas, in a process analogous to mammalian angiogenesis. This response involves the upregulation of FGF/Branchless in hypoxic tissues, which binds its receptor Breathless on tracheal cells. This study show that extra sprouting depends on the Hypoxia-Inducible Factor (HIF)-α homolog Sima and on the HIF-prolyl hydroxylase Fatiga that operates as an oxygen sensor. In mild hypoxia, Sima accumulates in tracheal cells, where it induces breathless, and this induction is sufficient to provoke tracheal extra sprouting. In nontracheal cells, Sima contributes to branchless induction, whereas overexpression of Sima fails to attract terminal branch outgrowth, suggesting that HIF-independent components are also required for full induction of the ligand. It is proposed that the autonomous response to hypoxia that occurs in tracheal cells enhances tracheal sensitivity to increasing Branchless levels, and that this mechanism is a cardinal step in hypoxia-dependent tracheal sprouting (Centanin, 2008).

This study has analyzed the role of the Drosophila HIF-α homolog Sima and the oxygen-sensing prolyl-4-hydroxylase Fga in tracheal terminal branching. It is assumed that during embryonic stages, tracheal development depends on hard-wired developmental cues, and, later, in larval stages, tracheal terminal branching is driven by local hypoxia in the target tissues. The observations carried out in this study indicate that the tracheal system of sima mutant third-instar larvae is indistinguishable from that of wild-type individuals, including the pattern of terminal branches. Thus, the results imply that if terminal branching during normal development was mediated by tissue hypoxia, the mechanism involved in such a local response should be Sima independent. This is a remarkable difference between Drosophila tracheogenesis and the development of the mammalian vascular system, in which HIF proteins are critically required for both vasculogenesis and developmental angiogenesis (Centanin, 2008).

It was also shown that Sima does play a cardinal role in hypoxia-dependent tracheal terminal branch sprouting, as well as in the formation of terminal branches that compensate for poor oxygenation in exceptional situations in which a neighboring branch is missing. Sima-dependent extra sprouting is negatively regulated by the oxygen-sensing prolyl-4-hydroxylase Fga, since fga mutants displayed an extra sprouting phenotype that was even stronger than that observed in wild-type individuals exposed to hypoxia. This extra sprouting phenotype is the first demonstration that loss of function of a HIF-prolyl hydroxylase can provoke an angiogenic-like phenotype. Thus, it seems reasonable to expect that conditional knockdown of mammalian PHDs in an appropriate cell type will promote angiogenesis (Centanin, 2008).

The long-standing paradigm for mammalian angiogenesis is that low oxygen levels trigger HIF accumulation in target tissues, which, in turn, mediates VEGF induction that, upon binding to VEGF receptors on endothelial cells, attracts the outgrowth of newly formed blood capillaries. Nevertheless, this apparently passive role of endothelial cells has recently been challenged. It has been demonstrated that in endothelial cell-specific HIF-α knockout mice the angiogenic response is impaired, highlighting a central role of the oxygen-sensing machinery in endothelial cells (Centanin, 2008).

This study has shown that the specialized Drosophila tracheal cells that respond to hypoxia by projecting angiogenic-like subcellular processes -- i.e., the terminal branches -- are apparently more sensitive to hypoxia than any other cell type in the larva. The sensory threshold to induce Sima-driven gene activation in these cells is shifted to near-normoxic oxygen tension. An alternative interpretation of the data is that tracheal terminal cells are similarly sensitive but more hypoxic than other cells, thereby inducing hypoxia-dependent transcription with higher sensitivity. In either case, the results suggest that Sima-dependent transcription within the tracheal terminal cells is part of the mechanism of oxygen sensing and tracheal extra sprouting (Centanin, 2008).

To test this hypothesis directly, EGFP-labeled sima homozygous mutant terminal cells were generated, and it was found that the ability of these cells to ramify upon a hypoxic stimulus is largely impaired. Furthermore, whether overexpression of Sima in the tracheae can provoke the angiogenic-like response was examined, and it was found that, indeed, expression of Sima restricted to the tracheal system is sufficient to induce extra sprouting. In contrast, overexpression of Sima -- or of a nondegradable variant of Sima -- in flip-out random clones outside the tracheae failed to provoke a similar phenotype, suggesting that accumulation of Sima in these cells is not sufficient for extra sprouting. Interestingly, in these Sima flip-out clones, a cell-autonomous response was observed, in which long subcellular processes projected from the cells that overexpressed Sima. Thus, although it is clear that bnl is induced in hypoxia and attracts the extension of terminal branches, the data support the notion that Sima is necessary, but not sufficient, for bnl induction in hypoxia (Centanin, 2008).

This study investigated which Sima target genes might be responsible for tracheal extra sprouting in fga mutants or upon exposure of wild-type larvae to hypoxia. Northern blot analyses indicated that bnl and btl are both upregulated in mildly hypoxic larvae or fga mutants. However, bnl homozygous EGFP-labeled terminal cells of larvae exposed to hypoxia retained their branching capacity, suggesting that extra sprouting in hypoxia is not mediated by an autocrine effect of Bnl, upon Sima-dependent induction in tracheal cells. In contrast, btl is directly induced by Sima in tracheal cells, and, consistent with this, overexpression of Btl in tracheal cells is sufficient to mimic the phenotypes of larvae exposed to hypoxia. Thus the data suggest that Sima-dependent transcriptional induction of btl in tracheal terminal cells is a critical step of the angiogenic-like response of the tracheal system in hypoxic larvae (Centanin, 2008).

In summary, it is proposed that tracheal cells respond to hypoxia in an autonomous manner, by promoting the accumulation of Sima, which induces expression of the receptor Btl, thereby increasing sensitivity of these cells to the ligand Bnl. Concomitantly, Bnl is induced in hypoxic target tissues through a mechanism that also involves the participation of Sima, and serves to cue the outgrowth of terminal branches toward O2-starved areas (Centanin, 2008).

During angiogenesis, vertebrate VEGF receptors are upregulated in endothelial cells of blood vessels that invade hypoxic tissues, and, particularly, Flt-1 induction is HIF dependent. Endothelial-specific overexpression of VEGF receptors might reveal to what extent this induction is a cardinal step in the angiogenic response to hypoxia (Centanin, 2008).

Oxygen sensing in Drosophila: multiple isoforms of the prolyl hydroxylase fatiga have different capacity to regulate HIFalpha/Sima

The Hypoxia Inducible Factor (HIF) mediates cellular adaptations to low oxygen. Prolyl-4-hydroxylases are oxygen sensors that hydroxylate the HIF alpha-subunit (Similar in Drosophila), promoting its proteasomal degradation in normoxia. Three HIF-prolyl hydroxylases, encoded by independent genes, PHD1, PHD2, and PHD3, occur in mammals. PHD2, the longest PHD isoform includes a MYND domain, whose biochemical function is unclear. PHD2 and PHD3 genes are induced in hypoxia to shut down HIF dependent transcription upon reoxygenation, while expression of PHD1 is oxygen-independent. The physiologic significance of the diversity of the PHD oxygen sensors is intriguing. This study has analyzed the Drosophila PHD locus, fatiga, which encodes 3 isoforms, FgaA, FgaB and FgaC that are originated through a combination of alternative initiation of transcription and alternative splicing. FgaA includes a MYND domain and is homologous to PHD2, while FgaB and FgaC are shorter isoforms most similar to PHD3. Through a combination of genetic experiments in vivo and molecular analyses in cell culture, it was shown that that fgaB but not fgaA is induced in hypoxia, in a Sima-dependent manner, through a HIF-Responsive Element localized in the first intron of fgaA. The regulatory capacity of FgaB is stronger than that of FgaA, as complete reversion of fga loss-of-function phenotypes is observed upon transgenic expression of the former, and only partial rescue occurs after expression of the latter. It is concluded that diversity of PHD isoforms is a conserved feature in evolution. As in mammals, there are hypoxia-inducible and non-inducible Drosophila PHDs, and a fly isoform including a MYND domain co-exists with isoforms lacking this domain. These results suggest that the isoform devoid of a MYND domain has stronger regulatory capacity than that including this domain (Acevedo, 2010).

In response to oxygen deprivation cells, tissues and whole organisms induce the expression of a wide range of genes that tend to restore energy homeostasis. Hypoxic gene induction is mainly mediated by the Hypoxia Inducible Factor (HIF), a heterodimeric α/β transcription factor composed of two basic-Helix-Loop-Helix-PAS (bHLH-PAS) subunits. Whereas the HIFβ subunit is constitutive, HIFα is tightly regulated by oxygen levels through various mechanisms that include protein stability, transcription coactivator recruitment and subcellular localization. The molecular mechanism that controls HIFα protein stability has been characterized in detail: In normoxia, HIFα is ubiquitinated and degraded at the 26S proteasome, while in hypoxia the protein is stabilized. HIFα ubiquitination in nomoxia is mediated by the Von Hippel Lindau (VHL) tumor suppressor factor which is the substrate recognition subunit of a multimeric E3 ubiquitin ligase complex. Physical interaction between VHL and HIFα requires hydroxylation of 2 key prolyl residues in the HIFα sequence (P402 and P564 in human HIF-1α), which is catalyzed by specific prolyl-4-hydroxylases, named PHD1- PHD2 and PHD3. These enzymes are members of the Fe (II) and 2-oxoglutarate dependent dioxygenase superfamily that utilizes O2 as a co-substrate for catalysis. Under hypoxia, PHD hydroxylase activity is reduced, HIFα escapes hydroxylation and proteolysis, leading to HIF nuclear accumulation and transcriptional induction of target genes. HIF-dependent transcription involves direct binding to Hypoxia Response Elements (HREs) that are characterized by an invariant 5′CGTG 3′ core consensus. Interestingly, a negative feed back loop, limiting HIFα activity in chronic hypoxia or upon re-oxygenation has been reported: PHD2 and PHD3 mRNAs are induced by low oxygen in a HIF-dependent manner to shut-down HIF activity; PHD1 transcription is oxygen-independent (Acevedo, 2010 and references therein).

The occurrence of three mammalian PHD isoforms encoded by three independent genes (PHD 1, PHD2 and PHD3) has opened the question of how each of these enzymes contributes to HIF regulation. It has been shown that all three PHDs can hydroxylate HIFα in vitro, and that upon over-expression, they can all suppress HRE-reporter induction. Cell culture analysis revealed that, PHD2 has a dominant role in controlling HIF-1α in normoxia, while PHD3 is important for regulating HIF in hypoxia or upon re-oxygenation. Furthermore, in vivo studies showed that PHD2, but not PHD1 or PHD3 knockout mice, exhibit enhanced angiogenesis and erythropoiesis Fong, 2008; Takeda, 2008), whereas PHD1 knockout mice display metabolic differences under ischemic conditions (Aragones, 2008; Acevedo, 2010 and references therein).

Previous work has led to the identification of a hypoxia response system in Drosophila that is homologous to mammalian HIF, in which the bHLH-PAS protein Similar (Sima), and the prolyl-4-hydroxylase Fatiga (Fga) are the homologues of HIFα and PHDs, respectively (Lavista-Llanos, 2002). sima null mutant individuals are unable to carry out transcriptional responses to hypoxia, although they are fully viable in normoxia. fga loss-of-function alleles showed different levels of Sima accumulation in normoxia, as well as tracheal defects and lethality at different developmental stages. Interestingly, sima loss-of-function mutations rescued viability and tracheal defects of fatiga mutants, suggesting that Sima protein over-accumulation accounts for these phenotypes (Centanin, 2005; Acevedo, 2010 and references therein).

This study describes a characterization of the single fatiga locus. The locus encodes three Fatiga variants, FgaA, FgaB and FgaC that originate from a combination of alternative transcription initiation and alternative mRNA splicing. FgaA includes a MYND domain, so it is homologous to PHD2, while both FgaB and FgaC are shorter isoforms that lack the MYND domain, and are similar to PHD3. Expression pattern of FgaA and FgaB were analyzed, as well as their transcriptional induction in hypoxia. Whereas FgaA expression remains constant and relatively low throughout the life cycle, FgaB is strongly upregulated in adult stages. FgaB but not FgaA is induced in hypoxia in a Sima dependent manner, both in cell culture and in vivo. Cell culture studies revealed that an HRE lying 759 to 756 base pairs upstream of the FgaB transcription initiation site accounts for FgaB induction in hypoxia. Finally, the ability of FgaA and FgaB to shut down Sima-dependent gene expression was explored; although the two isoforms are active, the regulatory capacity of FgaB is clearly stronger than that of FgaA (Acevedo, 2010).

Three PHD variants occur in mammals, and one single PHD gene, named EGL9, has been reported in Caenorabditis elegans. In Drosophila, previous studies on Fatiga, the Drosophila PHD homologous gene, have focused on its role in the regulation of Sima protein abundance (Lavista-Llanos, 2002; Centanin, 2005) and CyclinD-dependent cellular growth (Frei, 2004). In these functional studies, however, the occurrence of diverse Fga isoforms has not been addressed. This study has analyzed the fatiga locus, revealing that three different PHD isoforms occur in the fruit fly, which are generated through a combination of alternative splicing and alternative initiation of transcription. One of the isoforms, FgaA, includes a MYND domain, so it is homologous to mammalian PHD2, and the other two isoforms, FgaB and FgaC, lack a MYND domain, and are similar to PHD3. Thus, the diversity of PHD isoforms, including or not a MYND domain, seems to be an ancestral condition in evolution maintained in phylogenetically distant phyla such as insects and mammals. The occurrence of a single PHD isoform including a MYND domain in C. elegans might be due to evolutionary loss of shorter PHD variants (Acevedo, 2010).

In mammals PHD2 and PHD3, but not PHD1 mRNAs, are HIF-inducible (Epstein, 2001). This work has shown that FgaB, but not FgaA, is hypoxia-inducible, and that this induction depends on Drosophila HIF/Sima. A HIF Responsive Element (HRE) that mediates hypoxic transcriptional activation of fgaB mRNAs is localized at the position –759 to –756 with respect to the transcription initiation site of fgaB. Most HREs of hypoxia inducible genes of various organisms localize at their 5' regulatory region no more than 1 Kb upstream to the transcription initiation site. The identified HRE upstream to the fgaB open reading frame adjusts to this general rule. Due to the structure of the fga locus, the 5' regulatory region of fgaB lies in the large (8630 bp) first intron of fgaA (Acevedo, 2010).

Sequence conservation of the HRE lying upstream of Drosophila fgaB transcription initiation site and the mammalian PHD3 HRE -- localized in its first intron -- is remarkable, and extends beyond CGTG HRE invariant core. Fourteen out of 17 nucleotides around the fgaB HRE (CTGGGCTACGTGAGCAT) are conserved in the PHD3 regulatory region. This observation supports the notion that oxygen-dependent induction of PHD isoforms is important for adaptation of organisms to changing oxygen conditions (Acevedo, 2010).

The fact that a single Drosophila PHD locus encodes different isoforms that parallel two of the mammalian PHD variants encoded by independent genes is remarkable, and argues in favor that a combination of PHDs including or not a MYND domain is functionally relevant. The role of the MYND domain in HIF prolyl-4-hydroxylases is intriguing. Although PHD2 is the most abundant mammalian isoform and hence, has a dominant role in controlling HIFα in normoxia, PHD3 has been reported to have stronger intrinsic hydroxylation capacity than PHD2, which includes the MYND domain. Consistent with this, the MYND domain has been proposed to mediate inhibition of PHD2 hydroxylase activity, as deletion of this domain led to increased activity of the enzyme. Supporting the notion of the MYND domain provoking reduction of PHD regulatory capacity, it has been shown that direct interaction of the peptidyl cis/trans isomerase FKBP38 with the MYND domain of PHD2 negatively regulates PHD2 protein stability. FKBP38 does not interact with the hydroxylase isoforms PHD1 or PHD3, which lack the MYND domain. Some reports, however, weigh in favor of a model of a MYND domain enhancing PHD negative regulation of HIF, as PHD2 but not PHD1 or PHD3 have the capacity to inhibit HIF transcriptional activity through a hydroxylation-independent mechanism. Consistent with this, proteins including a MYND domain have been reported to mediate transcriptional inhibition of other transcription factors, so it is conceivable that transcription inhibitory capacity is a general feature of this domain. Thus, it is still unclear as whether the MYND domain increases or decreases the regulatory capacity of PHDs. The results in Drosophila support the latter possibility, as the PHD isoform that lacks the MYND domain has stronger regulatory capacity than the isoform that includes this domain. Detailed biochemical and functional studies are required to define the precise role of this protein domain in transcriptional responses to hypoxia (Acevedo, 2010).

Computational and experimental characterization of dVHL establish a Drosophila model of VHL syndrome

The von Hippel-Lindau (VHL) cancer syndrome is associated with mutations in the VHL gene. The pVHL protein is involved in response to changes in oxygen availability as part of an E3-ligase that targets the Hypoxia-Inducible Factor for degradation. pVHL has a molten globule configuration with marginal thermodynamic stability. The cancer-associated mutations further destabilize it. The Drosophila homolog, dVHL, has relatively low sequence similarity to pVHL, and is also involved in regulating HIF1-α. Using in silico, in vitro and in vivo approaches this study demonstrates high similarity between the structure and function of dVHL and pVHL. These proteins have a similar fold, secondary and tertiary structures, as well as thermodynamic stability. Key functional residues in dVHL are evolutionary conserved. This structural homology underlies functional similarity of both proteins, evident by their ability to bind their reciprocal partner proteins, and by the observation that transgenic pVHL can fully maintain normal dVHL-HIF1-alpha downstream pathways in flies. This novel transgenic Drosophila model is thus useful for studying the VHL syndrome, and for testing drug candidates to treat it (Shmueli, 2014: PubMed).

HIF- and non-HIF-regulated hypoxic responses require the estrogen-related receptor in Drosophila melanogaster

Low-oxygen tolerance is supported by an adaptive response that includes a coordinate shift in metabolism and the activation of a transcriptional program that is driven by the hypoxia-inducible factor (HIF) pathway. The precise contribution of HIF-1a in the adaptive response, however, has not been determined. This study investigated how HIF influences hypoxic adaptation throughout Drosophila melanogaster development. Hypoxic-induced transcriptional changes were found to be comprised of HIF-dependent and HIF-independent pathways that are distinct and separable. Normoxic set-points of carbohydrate metabolites are significantly altered in sima mutants and these animals are unable to mobilize glycogen in hypoxia. Furthermore, it was found that the estrogen-related receptor (dERR), which is a global regulator of aerobic glycolysis in larvae, is required for a competent hypoxic response. dERR binds to dHIFa and participates in the HIF-dependent transcriptional program in hypoxia. In addition, dERR acts in the absence of dHIFa in hypoxia and a significant portion of HIF-independent transcriptional responses can be attributed to dERR actions, including upregulation of glycolytic transcripts. These results indicate that competent hypoxic responses arise from complex interactions between HIF-dependent and -independent mechanisms, and that dERR plays a central role in both of these programs (Li, 2013: 23382692).


DEVELOPMENTAL BIOLOGY

Embryonic

Whole-mount embryonic in situ hybridization experiments using different sima cDNA probes were performed to determine the sima expression pattern. Hybridization was observed in most or all cells from the blastoderm stage through the end of embryogenesis. The significant amounts of sima transcripts in syncitial blastoderm embryos suggest a maternal contribution of sima. The pattern of sima transcripts completely overlaps that of sim and trk (Nambu, 1996).


EFFECTS OF MUTATION

The Drosophila piwi gene is the founding member of the only known family of genes whose function in stem cell maintenance is highly conserved in both animal and plant kingdoms. piwi mutants fail to maintain germline stem cells in both male and female gonads. The identification of piwi-interacting genes is essential for understanding how stem cell divisions are regulated by piwi-mediated mechanisms. To search for such genes, the Drosophila third chromosome (~36% of the euchromatic genome) was screened for suppressor mutations of piwi2, and six strong and three weak piwi suppressor genes/sequences were identified. These genes/sequences interact negatively with piwi in a dosage-sensitive manner. Two of the strong suppressors represent known genes -- serendipity-delta and similar, both encoding transcription factors. These findings reveal that the genetic regulation of germline stem cell division involves dosage-sensitive mechanisms and that such mechanisms exist at the transcriptional level. In addition, three other types of piwi interactors were identified. The first type consists of deficiencies that dominantly interact with piwi2 to cause male sterility, implying that dosage-sensitive regulation also exists in the male germline. The other two types are deficiencies that cause lethality and female-specific lethality in a piwi2 mutant background, revealing the zygotic function of piwi in somatic development (Smulders-Srinivasan, 2003).

Like Sry-delta, Similar and Tango play a key role in the dosage-sensitive regulation of germline stem cell division. Similar is homologous to a large group of heterodimerizing transcriptional activators. It shows closest homology to the human hypoxia inducible factor-1alpha (HIF-1alpha) and has been shown to function in hypoxic response in Drosophila. HIF-1alpha binds to HIF-1ß to drive transcription of downstream genes. Since Tango is the only Drosophila homolog of HIF-1ß, it is likely to be a partner of Similar. Indeed, Tango interacts with Similar in the yeast two-hybrid system. However, Tango is also known to bind to two other Drosophila bHLH-PAS family proteins, Single-minded and Trachealess, to mediate the transcription of their downstream targets. By showing that tango suppresses the germline stem cell phenotype of piwi2, this study suggests that Tango heterodimerizes with Similar in the dosage-sensitive transcriptional activation of genes involved in germline stem cell division (Smulders-Srinivasan, 2003).

Reversion of lethality and growth defects in Fatiga oxygen-sensor mutant flies by loss of Hypoxia-Inducible Factor-alpha/Sima

Hypoxia-Inducible Factor (HIF) prolyl hydroxylase domains (PHDs) have been proposed to act as sensors that have an important role in oxygen homeostasis. In the presence of oxygen, they hydroxylate two specific prolyl residues in HIF-alpha polypeptides, thereby promoting their proteasomal degradation. So far, however, the developmental consequences of the inactivation of PHDs in higher metazoans have not been reported. This study describes novel loss-of-function mutants of fatiga (HIF prolyl hydroxylase), the gene encoding the Drosophila PHD oxygen sensor, that manifest growth defects and lethality. A null mutation in dHIF-alpha/similar (sima) is reported, that is unable to adapt to hypoxia but is fully viable in normoxic conditions. Strikingly, loss-of-function mutations of sima rescue the developmental defects observed in fatiga mutants and enable survival to adulthood. These results indicate that the main functions of Fatiga in development, including control of cell size, involve the regulation of dHIF/Sima (Centanin, 2005).

Recent work has led to the definition of widely operative signalling systems that control the transcriptional response to hypoxia through hypoxia-inducible factor (HIF). HIF proteins are a family of alpha/ß-heterodimers in which the common ß-subunit is constitutive and the alpha-subunits are oxygen-regulated by mechanisms that include transcriptional co-activator recruitment, subcellular localization and protein stabilization. The regulation of proteasomal degradation of alpha-subunits has been well characterized in cell culture and in in vitro systems. In the presence of oxygen, a series of 2-oxoglutarate and iron-dependent dioxygenases termed PHDs (prolyl hydroxylase domains) hydroxylate specific prolyl residues in the HIF-alpha oxygen-dependent degradation domain (ODDD), enabling its ubiquitination and proteasomal degradation. As molecular oxygen is absolutely required in the prolyl hydroxylation reaction and enzyme activity is sensitive to mild hypoxia, the PHDs have suitable characteristics that enable them to function as bona fide oxygen sensors that determine the half-life of HIF-alpha proteins, thereby controlling hypoxia-dependent transcription. Analyses of 'knockout' mouse strains have shown developmental roles of mammalian HIF proteins. They are required for the normal formation of the heart, brain, vasculature, cartilage and placenta, suggesting that fetal oxygen availability might have a role in these processes. However, this question remains open, and the developmental effects of genetic inactivation of the oxygen-sensitive PHD pathways have not yet been defined (Centanin, 2005).

The Drosophila bHLH-PAS proteins Similar (Sima) and Tango (Tgo) are, respectively, the functional homologues of HIF-alpha and HIF-ß in the fly. A lethal P-element insertional mutation (l(3)02255) is described in the Drosophila PHD gene (CG1114 in FlyBase) that fails to downregulate Sima protein in normoxia, thus driving constitutive activation of the transcriptional response to hypoxia. The aim of the present work was to investigate the developmental role of Drosophila PHD, which has been termed fatiga (fga; Spanish for 'fatigue') after its lack-of-oxygen phenotype (Centanin, 2005).

As a first step in the study of the functions of fga in development, new loss-of-function mutations were generated by mobilizing the l(3)02255 P-element, which is located between the second and third exons of the fga gene. Precise excisions of the transposon led to a reconstitution of the wild-type hypoxic response, as shown by hypoxia-inducible expression of transcriptional reporters that are based on the murine LDH-A enhancer. Imprecise excisions of the P-element resulted in three novel fga alleles (fga1, fga9 and fga64) that were characterized at the molecular level by Southern blot analysis and PCR experiments. fga9 conserved a 1.4 kb fragment of the original transposon; in fga64, a large genomic portion upstream of the insertion site was removed, and fga1 conserved a fragment of about 9 kb of the original P-element. In normoxic wild-type embryos, Sima protein and induction of the Ldh-Gal4 reporter are not detected; upon exposure to hypoxia (5% O2), both Sima protein and reporter expression are observed, mainly in the tracheal system. The molecular basis of this pattern of induction is now under investigation. Interestingly, fga loss-of-function alleles show different levels of accumulation of Sima protein in normoxia, which was widely expressed in fga1 and fga64, and shows some prevalence in the tracheal system in fga9, correlating with the constitutive induction of the hypoxic reporter. fga02255, fga1 and fga64 are lethal at the first larval instar and fga9 die in the pupal stage. Because overexpression of Sima through a ubiquitous Gal4 driver provokes lethality in the larval stages, it was reasoned that lethality in fga mutants could be due to overaccumulation of Sima protein in normoxia. To test this hypothesis and to determine if Fga is a dedicated regulator of Sima or whether, alternatively, it might modulate other molecular targets, attempts were made to analyse fga phenotypes in a sima-free genetic background. Loss-of-function mutations of sima have not been reported so far, but two different P-element insertions mapping within the sima locus were available from the Public Stock Centers. One of these insertion lines was able to respond to hypoxia and, thus, was indistinguishable from the wild type. In contrast, embryos homozygous for the other insertion, sima07607, did not express sima mRNA and failed to induce the Ldh-LacZ reporter in hypoxia. Introduction of a UAS-Sima transgenic element under the control of an hs-Gal4 driver was able to rescue induction of reporter expression, which was expressed in a wild-type pattern, indicating that the absence of Sima was indeed responsible for the lack of hypoxic response. Altogether, these results indicate that sima07607 is a sima loss-of-function allele (Centanin, 2005).

To explore whether the absence of Sima protein, and thus the inability to respond to hypoxia, affects developmental progression, phenotypes were analysed in sima07607 mutants. Homozygous mutant embryos developed without any obvious difference from the controls, and the first-instar larvae looked healthy and motile. Next, homozygous mutant or control larvae were placed in vials containing fresh food, which were then exposed to 21% or 5% O2 until individuals attained the pupal stage. sima07607 mutants were viable and fertile in normoxia, but virtually unable to develop in hypoxia. Precise excision of the P-element totally reverted hypoxia-dependent lethality, which indicated that the insertion was indeed responsible for this phenotype. Thus, it is concluded that, unlike Tango that participates as a common bHLH-PAS partner in several developmental processes in normoxia, Sima is necessary for developmental progression in hypoxia but not in normoxia (Centanin, 2005).

fga mutations caused a reduction in cell size, but it is unclear whether this effect depends on overaccumulation of Sima. The availability of sima07607 as a sima loss-of-function allele enabled this particular question to be answered and, more generally, the extent to which the developmental defects of fga loss-of-function mutations are due to the de-regulated accumulation of Sima protein to be addressed. As expected, in fga1sima07607 double homozygous mutants, Sima protein is undetectable and embryos do not show any expression of hypoxia-inducible reporters in normoxia. Consistent with previously described growth defects of fga mutants, fga9 pupae are smaller than their heterozygous siblings and, interestingly, they exhibited a delay in larval development, taking 2 additional days to reach the pupariation stage. Strikingly, fga1sima07607 double homozygous mutants are indistinguishable from the controls, both in their pupal weight and in the duration of larval development. Thus, the loss of Sima provokes the complete reversion of growth defects occurring in fga mutants. To answer whether overaccumulation of Sima is sufficient to account for the autonomous reduction in cell size reported for fga mutant cells, Sima protein was overexpressed in random clones using the flipase-induced recombination (FLP-OUT) technique, and the effect on cell size was analysed. Overexpression of Sima in isolated cells causes a marked autonomous reduction of cell size, which correlated with smaller nuclei. Taken together, these results indicate that Sima is a downstream effector of Fga as a regulator of cell growth. Further analyses were carried out on the tracheal system; once again defects (particularly, air-filling impairment) that are observed in fga mutants, are corrected in fga sima double mutants (Centanin, 2005).

Given the reversion of the analysed fga phenotypes in fga sima double mutants, it was of interest to test whether lethality that occurred following fga loss-of-function is also due to overaccumulation of Sima. This is indeed the case, since, in normoxia, fga1 sima07607 double homozygous mutants are viable to adulthood, even when many of these adults fail to complete emergence from the pupal case, and those that emerged looked weak and frequently die shortly afterwards. As expected, in hypoxia, this reversion of lethality does not occur, and fga or sima single mutants, as well as fga sima double mutant flies, die in the first larval stage. Overall, these results show that Drosophila development can proceed in the absence of PHD oxygen sensors, provided that the HIF-alpha subunit is absent and oxygen availability is not compromised. Thus, it is concluded that the most fundamental functions of Fatiga/PHD in development probably involve the downregulation of Sima protein levels. However, fga sima exarate adults show defects in wing and ovary development, which may imply that Fga is involved in patterning these organs in a Sima-independent manner. Detailed genetic and molecular analyses of fga sima double mutants should help to define Sima-independent developmental functions of the oxygen sensor in Drosophila (Centanin, 2005).

Cell autonomy of HIF effects in Drosophila: tracheal cells sense hypoxia and induce terminal branch sprouting

Drosophila tracheal terminal branches are plastic and have the capacity to sprout out projections toward oxygen-starved areas, in a process analogous to mammalian angiogenesis. This response involves the upregulation of FGF/Branchless in hypoxic tissues, which binds its receptor Breathless on tracheal cells. This study shows that extra sprouting depends on the Hypoxia-Inducible Factor (HIF)-alpha homolog Similar (Sima) and on the HIF-prolyl hydroxylase Fatiga that operates as an oxygen sensor. In mild hypoxia, Sima accumulates in tracheal cells, where it induces breathless, and this induction is sufficient to provoke tracheal extra sprouting. In nontracheal cells, Sima contributes to branchless induction, whereas overexpression of Sima fails to attract terminal branch outgrowth, suggesting that HIF-independent components are also required for full induction of the ligand. It is proposed that the autonomous response to hypoxia that occurs in tracheal cells enhances tracheal sensitivity to increasing Branchless levels, and that this mechanism is a cardinal step in hypoxia-dependent tracheal sprouting (Dekanty, 2008).

To address whether Sima and Fatiga participate in the regulation of tracheal terminal sprouting, phenotypic alterations of the tracheae of third-instar larvae exposed to hypoxia were examined in detail. For quantitative purposes, focus was placed on the dorsal branch of the third segment, whose terminal cell has a characteristic branching pattern, typically comprising of a main cellular process from which straight cellular extensions of about 1 μm diameter (hereafter, 'Thick Terminal Branches' [TTBs]) project and thinner extensions ramify thereafter. The average number of TTBs at the third dorsal branch of wild-type larvae maintained in normoxia was 5.65. In hyperoxia (60% O2), a slight but significant reduction of TTBs (4.82) was observed, whereas in hypoxia (5% O2), the average TTB number increased to 8.76. Of note, this increase in the number of TTBs was paralleled by a similar increase in the number of thinner terminal projections. Third-instar larvae displayed an average of 17.0 ± 2.7 thin projections at the third dorsal branch in normoxia and an average of 30.7 ± 3.4 thin projections at 5% O2, implying that, upon hypoxic exposure, the number of thin terminal projections increases to an extent similar to that of the TTBs. These results confirm the tight correlation between tracheal terminal branching and oxygen levels that has been known since Wigglesworths' pioneering studies in the 1950s, and they establish the number of TTBs as a good read out to analyze the extent of terminal branching. Next, it was tested whether fatiga mutant larvae, which are known to accumulate high levels of Sima protein in normoxia, have alterations in the number of ramifications. The fga1/fga9 allelic combination that can develop to the third-larval instar was used, and it was observed that, under normoxic conditions, these larvae displayed an average of 9.49 TTBs and 43.6 ± 5.3 thin terminal projections at the third dorsal branch, an overall number of ramifications even higher than that of wild-type larvae exposed to hypoxia. To test whether increased levels of Sima can account for the excess of TTBs in fga mutant larvae, fga sima double homozygous individuals, which are viable to adulthood, were uxamined. In fga sima double mutant larvae, the number of TTBs (5.14) and thin terminal projections (18.2 ± 3.3) reverted to wild-type levels, which, in turn, was very similar to those of sima homozygous mutants. These findings suggest that the extraterminal sprouting phenotype observed in fga mutants is due to increased levels of Sima protein (Dekanty, 2008).

To gather additional evidence of the participation of the Fatiga (Fga)-Sima system in tracheal hypoxia-dependent plasticity, focus was placed on a different oxygen-dependent modification that is typical of hypoxic larvae: upon exposure to 5% O2, most tracheal branches become tortuous and, in particular, ganglionic branches (the branches that reach the central nervous system) adopt a ringlet appearance. The proportion of larvae exhibiting at least one ringlet-shaped ganglionic branch (RSGB) was quantitated and it was found that, in normoxic larvae, all ganglionic branches were straight, whereas upon exposure to hypoxia, 36.4% of the larvae displayed at least one RSGB. Among fga1/fga9 larvae, the proportion of individuals exhibiting at least one RSGB dramatically increased, with 69.2% of them being RSGB positive. Strikingly, in fga sima double mutants, the incidence of RSGBs was reduced again to almost wild-type levels, suggesting that RSGBs in fga mutant larvae are also provoked by increased levels of Sima. These results paralleled those regarding the regulation of terminal sprouting, and they support the notion that oxygen-dependent tracheal plasticity of Drosophila larvae is controlled by the oxygen-sensing prolyl-4-hydroxylase Fga, through the regulation of Sima protein abundance (Dekanty, 2008).

This study has analyzed the role of the Drosophila HIF-α homolog Sima and the oxygen-sensing prolyl-4-hydroxylase Fga in tracheal terminal branching. It is assumed that during embryonic stages, tracheal development depends on hard-wired developmental cues, and, later, in larval stages, tracheal terminal branching is driven by local hypoxia in the target tissues. The observations carried out in this study indicate that the tracheal system of sima mutant third-instar larvae is indistinguishable from that of wild-type individuals, including the pattern of terminal branches. Thus, the results imply that if terminal branching during normal development is mediated by tissue hypoxia, the mechanism involved in such a local response should be Sima independent. This is a remarkable difference between Drosophila tracheogenesis and the development of the mammalian vascular system, in which HIF proteins are critically required for both vasculogenesis and developmental angiogenesis (Dekanty, 2008).

Sima does play a cardinal role in hypoxia-dependent tracheal terminal branch sprouting, as well as in the formation of terminal branches that compensate for poor oxygenation in exceptional situations in which a neighboring branch is missing. Sima-dependent extra sprouting is negatively regulated by the oxygen-sensing prolyl-4-hydroxylase Fga, since fga mutants displayed an extra sprouting phenotype that was even stronger than that observed in wild-type individuals exposed to hypoxia. This extra sprouting phenotype is the first demonstration that loss of function of a HIF-prolyl hydroxylase can provoke an angiogenic-like phenotype. Thus, it seems reasonable to expect that conditional knockdown of mammalian PHDs in an appropriate cell type will promote angiogenesis (Dekanty, 2008).

The long-standing paradigm for mammalian angiogenesis is that low oxygen levels trigger HIF accumulation in target tissues, which, in turn, mediates VEGF induction that, upon binding to VEGF receptors on endothelial cells, attracts the outgrowth of newly formed blood capillaries. Nevertheless, this apparently passive role of endothelial cells has recently been challenged. It has been demonstrated that in endothelial cell-specific HIF-α knockout mice the angiogenic response is impaired, highlighting a central role of the oxygen-sensing machinery in endothelial cells (Dekanty, 2008).

This study has shown that the specialized Drosophila tracheal cells that respond to hypoxia by projecting angiogenic-like subcellular processes (i.e., the terminal branches) are apparently more sensitive to hypoxia than any other cell type in the larva. The sensory threshold to induce Sima-driven gene activation in these cells is shifted to near-normoxic oxygen tension. An alternative interpretation of these data is that tracheal terminal cells are similarly sensitive but more hypoxic than other cells, thereby inducing hypoxia-dependent transcription with higher sensitivity. In either case, the results suggest that Sima-dependent transcription within the tracheal terminal cells is part of the mechanism of oxygen sensing and tracheal extra sprouting (Dekanty, 2008).

To test this hypothesis directly, EGFP-labeled sima homozygous mutant terminal cells were generated, and it was found that the ability of these cells to ramify upon a hypoxic stimulus is largely impaired. Furthermore, whether overexpression of Sima in the tracheae can provoke the angiogenic-like response was investigated, and it was found that, indeed, expression of Sima restricted to the tracheal system is sufficient to induce extra sprouting. In contrast, overexpression of Sima -- or of a nondegradable variant of Sima -- in flip-out random clones outside the tracheae failed to provoke a similar phenotype, suggesting that accumulation of Sima in these cells is not sufficient for extra sprouting. Interestingly, in these Sima flip-out clones, a cell-autonomous response was observed, in which long subcellular processes projected from the cells that overexpressed Sima. Thus, although it is clear that bnl is induced in hypoxia and attracts the extension of terminal branches, the data support the notion that Sima is necessary, but not sufficient, for bnl induction in hypoxia (Dekanty, 2008).

Which Sima target genes might be responsible for tracheal extra sprouting was investigated in fga mutants or upon exposure of wild-type larvae to hypoxia. Northern blot analyses indicated that bnl and btl are both upregulated in mildly hypoxic larvae or fga mutants. However, bnl homozygous EGFP-labeled terminal cells of larvae exposed to hypoxia retained their branching capacity, suggesting that extra sprouting in hypoxia is not mediated by an autocrine effect of Bnl, upon Sima-dependent induction in tracheal cells. In contrast, btl is directly induced by Sima in tracheal cells, and, consistent with this, overexpression of Btl in tracheal cells was sufficient to mimic the phenotypes of larvae exposed to hypoxia. Thus, the data suggest that Sima-dependent transcriptional induction of btl in tracheal terminal cells is a critical step of the angiogenic-like response of the tracheal system in hypoxic larvae (Dekanty, 2008).

In summary, it is proposed that tracheal cells respond to hypoxia in an autonomous manner, by promoting the accumulation of Sima, which induces expression of the receptor Btl, thereby increasing sensitivity of these cells to the ligand Bnl. Concomitantly, Bnl is induced in hypoxic target tissues through a mechanism that also involves the participation of Sima, and serves to cue the outgrowth of terminal branches toward O2-starved areas (Dekanty, 2008).

During angiogenesis, vertebrate VEGF receptors are upregulated in endothelial cells of blood vessels that invade hypoxic tissues, and, particularly, Flt-1 induction is HIF dependent. Endothelial-specific overexpression of VEGF receptors might reveal to what extent this induction is a cardinal step in the angiogenic response to hypoxia (Dekanty, 2008).

Dose-dependent modulation of HIF-1alpha/sima controls the rate of cell migration and invasion in Drosophila ovary border cells

The role of the hypoxic response during metastasis was analysed in migrating border cells of the Drosophila ovary. Acute exposure to 1% O2 delayed or blocked border cell migration (BCM), whereas prolonged exposure resulted in the first documented accelerated BCM phenotype. Similarly, manipulating the expression levels of sima, the Drosophila hypoxia-inducible factor (HIF)-1alpha ortholog, revealed that Sima can either block or restore BCM in a dose-dependent manner. In contrast, over-expression of Vhl (Drosophila von Hippel-Lindau) generated a range of phenotypes, including blocked, delayed and accelerated BCM, whereas over-expression of hph (Drosophila HIF prolyl hydroxylase) only accelerated BCM. Mosaic clone analysis of sima or tango (HIF-1beta ortholog) mutants revealed that cells lacking Hif-1 transcriptional activity were preferentially detected in the leading cell position of the cluster, resulting in either a delay or acceleration of BCM. Moreover, in sima mutant cell clones, there was reduced expression of nuclear slow border cells (Slbo) and basolateral DE-cadherin, proteins essential for proper BCM. These results show that Sima levels define the rate of BCM in part through regulation of Slbo and DE-cadherin, and suggest that dynamic regulation of Hif-1 activity is necessary to maintain invasive potential of migrating epithelial cells (Doronkin, 2010).

HIF- and non-HIF-regulated hypoxic responses require the estrogen-related receptor in Drosophila melanogaster

Low-oxygen tolerance is supported by an adaptive response that includes a coordinate shift in metabolism and the activation of a transcriptional program that is driven by the hypoxia-inducible factor (HIF) pathway. The precise contribution of HIF-1a in the adaptive response, however, has not been determined. This study investigate how HIF influences hypoxic adaptation throughout Drosophila melanogaster development. Hypoxic-induced transcriptional changes were found to be comprised of HIF-dependent and HIF-independent pathways that are distinct and separable. Normoxic set-points of carbohydrate metabolites are significantly altered in sima mutants, and these animals are unable to mobilize glycogen in hypoxia. Furthermore, it was found that the estrogen-related receptor (dERR), which is a global regulator of aerobic glycolysis in larvae, is required for a competent hypoxic response. dERR binds to dHIFa and participates in the HIF-dependent transcriptional program in hypoxia. In addition, dERR acts in the absence of dHIFa in hypoxia and a significant portion of HIF-independent transcriptional responses can be attributed to dERR actions, including upregulation of glycolytic transcripts. These results indicate that competent hypoxic responses arise from complex interactions between HIF-dependent and -independent mechanisms, and that dERR plays a central role in both of these programs (Li, 2013).

These results underscore the complexities of adaptive responses in hypoxia, which are life-stage specific and controlled by multiple hypoxic-sensitive (H-sensitive) pathways. Although the data confirm that HIF is a major transcriptional driver of hypoxic responses, distinct HIF-independent responses were also defined. These data raise new questions about dHIFa collaboration and challenge the notion that the HIF complex has little or no normoxic role. In addition, it was shown that a significant fraction of HIF-independent pathways can be attributed to the ERR nuclear receptor (Li, 2013).

Among the HIF-independent genes were numerous glycolytic transcripts that are well-known responders to hypoxia. The fact that these genes are as effectively upregulated in sima mutants as they are in a control response was surprising, particularly considering the known role of HIF-1a in this process. This study found that dERR is the overriding factor that mediates hypoxic upregulation of glycolytic genes (Pgi, Pfk, GAPDH2, enolase) just prior to metamorphic onset (Li, 2013).

The findings, however, do not exclude dHIFa contribution in hypoxic expression of HI genes at other developmental times. The super-induction of LDH during metamorphosis in w1118 animals versus sima mutants is consistent with this scenario. These temporal- and context-specific differences may explain the wide variability in hypoxic responses that have been seen between cell-types, despite the ubiquitous presence of the HIF pathway. Furthermore, they may account for discrepancies between the current data collected on Drosophila and reports on mammalian systems. For example, LDH is a HIF-independent hypoxia-regulated gene in late-L3 animals. However, loss of dERR has a greater effect on the diminution of hypoxic induction at this developmental time than does loss of dHIFa. But, this effect is short-lived, because just hours later, when the larva transitions into a pupa, dHIFa appears to work in combination with a non-HIF pathway to elicit hypoxic responsiveness. This combinatorial response during Drosophila metamorphosis is consistent with vertebrate studies that show LDH expression is the product of HIF-1 action that also requires the presence a cAMP response element for full hypoxic induction. Thus, in addition to different pools of potential coregulatory molecules that may significantly alter HIF-dependent transcription, entirely different transcriptional pathways, with their own triggers of hypoxic induction, refine the H response. Given the right spatiotemporal setting, HIF-independent pathways may displace (or substitute for) the HIF pathway altogether, a result that is consistent with the data. Further support of this idea is evident in the expression of Alas2, the rate-limiting enzyme for heme production. Alas2 has been identified as a HIF-dependent and a HIF-independent hypoxia-regulated gene in mammals. In this study, ALAS is H-responsive, and displays HIF-independent and ERR-dependent upregulation, which may be subject to dHIFa negative regulation in dERR's absence (Li, 2013).

The dynamic patterns of temporal expression of HIF-independent (HI) and HIF-dependent (HD) genes raise the fundamental question of how hypoxic responses are regulated through development and into the adult. Low-oxygen responses are not one-size-fits-all programs that mitigate oxidative damage and metabolic imbalance; they must be coordinated with developmental progression and metabolic state. In particular, late-L3 wandering larvae exhibit a hypersensitive transcriptional response to hypoxia for HIF-independent/ERR-dependent glycolytic genes. This includes a robust LDH induction. Paradoxically, however, late-L3 larvae do not produce lactate in the 6-hr hypoxic challenge. In contrast, at other developmental times (L1, adult), animals correspondingly produce lactate in hypoxia, even though they remain transcriptionally incompetent to induce LDH transcript. It is speculated that the atypical transcriptional and metabolic hypoxic profiles of the late-L3 larva are a product of its developmentally programmed energetic state, which at this time is transitioning from low to high efficiency. Just prior to the wandering L3 time, larvae are prolifically growing, and in a state of metabolism that is fueled by aerobic glycolysis. This metabolic program is ERR-dependent. Just after this developmental time, larvae initiate metamorphosis, which will impose 5 days of developmentally forced starvation. During this lipid-driven phase, metabolism is characterized by high efficiency OXPHOS (Li, 2013).

In contrast to the switch-like hypoxic expression of HIF-independent glycolytic transcripts, the HIF prolyl hydroxylase fatiga displays relatively uniform expression throughout development, suggesting that regulation of the HIF pathway, by HIF itself, is equally important at all times for the animal. Such disparities in induction are only understood in context. While these studies provide a framework with which to view H responses, they indicate that further developmental analysis is needed to more fully appreciate hypoxic response pathways and the mechanisms that specifically support their activities (Li, 2013).

Although this study has emphasized the transcriptional and metabolic impacts of hypoxia on carbohydrate catabolism, the breadth of the data sets indicate that many important hypoxia-induced changes are thus far unappreciated and await further investigation. What is the significance, for instance, of the greater than 10-fold increase of HIF-dependent expression of dDPH-1 (CG11652) in hypoxia? DPH-1 is a tumor suppressor that is responsible for the first step of the unique protein modification that occurs on elongation factor 2 (eEF2), which converts a histidine residue to diphthamide. This residue is the target of diphtheria toxin that can shut down protein synthesis through ADP-ribosylation. Although diphthamide formation is conserved from archaea to human, its significance on cellular function is not clear, as it is dispensable for protein elongation However, it has been implicated in translational fidelity and is likely an asset under stress. GO analysis performed on HD H-regulated genes indicate that dHIFa is important in replenishing select protein translation/RNA processing transcripts. From this perspective, DPH-1 induction by dHIFa may be indicative of a regulatory role of hypoxic translation for HIFs. Such a role would be consistent with a recent report from mammals that demonstrates a HIF-2a-dependent association with ribosomal/translational control proteins and the selective hypoxic translation of transcripts containing an RNA hypoxic response element in the 3'UTR via a mechanism involving eIF4E2 (Li, 2013).

This analysis of carbohydrate catabolism identifies amylase-mediated breakdown of glycogen as the fuel of first resort in hypoxia). This catabolic pathway feeds into glycolysis and supplies needed glucose for increased glycolytic flux, obviating the need to draw on circulating sugar in the form of trehalose, which did not change in the 6-hr challenge. The strategy of glycogen mobilization allows animals to maintain a remarkably stable profile for a wide variety of carbohydrate catabolites (Li, 2013).

Trehalose levels are substantially elevated in sima mutants, regardless of oxygen status. These data may indicate a role for dHIFa in the insulin receptor pathway. Numerous studies demonstrate that trehalose levels are altered by genetic disruptions of the insulin-signaling components. Alternatively, elevated trehalose levels may be the result of constitutively high expression of amylase. Although the increased amylase expression does not translate into a depleted level of glycogen in the sima mutant, it is conceivable that increased glycogen deposition compensates for increased glycogenolysis (Li, 2013).

It is important to note that post-transcriptional control mechanisms are well known to impact glycolytic enzymes. Although these were not documented in this study, such influences on hypoxic glycolytic flux are likely to have genotype-specific effects (Li, 2013).

sima mutants do not mobilize glycogen in hypoxia, but they are able to initiate H-induced changes for other carbohydrates. This is the case for the glycolytic intermediate DHAP, which more than doubles in a control hypoxic response and significantly accumulates in mutants. These findings are consistent with appropriate transcriptional responses that were noted for glycolytic transcripts in sima animals, which are upregulated in hypoxia by dERR, not dHIFa. The results for glycogen notwithstanding, it is the widespread derangement of normoxic set points for metabolites that characterizes the metabolic incompetency of the sima mutant. The data indicate that dHIFa has it greatest impact on metabolism in the unchallenged normoxic state, rather than in hypoxia (Li, 2013).

The mechanism whereby dERR participates in hypoxic responses needs to be explored further. This study identified dERR as a potential player in hypoxic responses through its association with dHIFa, suggesting that it acts in a collaborative role with the HIF complex through direct recruitment to HREs. This model was favored by the Ao report for ERR participation in hypoxic responses in vertebrates (Ao, 2008). Additionally, dERR may recruit dHIFa to ERR-specific response elements to facilitate H responses. Another possibility is that dERR actively regulates hypoxic transcription without dHIFa at all; or, in parallel to the actions of dHIFa, which may occur independently, yet simultaneously. Each of these scenarios is consistent with hypoxic expression analysis that were performed to generate HD, HI, ED, and DM gene sets. Moreover, in the presence of dERR, dHIFa may act as a negative regulator of hypoxic responses at select hypoxia-regulated sites. Of further interest also, will be the identification of the triggers for ERR participation in hypoxic-induced responses (Li, 2013).

Apart from dERR and dHIFa, these data indicate that at least one more hypoxic-sensitive pathway is active and important for mediating hypoxic adaptation, as many H-sensitive transcripts were found that fall outside the regulation of either factor. The nature of the alternate pathway(s) is unknown. The results shown in this study suggest that identifying the sensors and effectors that regulate these HIF- and ERR-independent hypoxic response pathways will have profound impacts on understanding of hypoxic signaling, and will undoubtedly provide new avenues with which to approach the complex problem of metabolic transition (Li, 2013).


EVOLUTIONARY HOMOLOGS

Interaction of HIF-1 with ARNT

Hypoxia-inducible factor 1 (HIF-1) is found in mammalian cells cultured under reduced O2 tension and is necessary for transcriptional activation mediated by the erythropoietin gene enhancer in hypoxic cells. Both HIF-1 subunits are basic-helix-loop-helix proteins containing a PAS domain, defined by its presence in the Drosophila Per and Sim proteins and in the mammalian ARNT and AHR proteins. HIF-1 alpha is most closely related to Sim. HIF-1 beta is a series of ARNT gene products, which can thus heterodimerize with either HIF-1 alpha or AHR. HIF-1 alpha and HIF-1 beta (ARNT) RNA and protein levels are induced in cells exposed to 1% O2 and decay rapidly upon return of the cells to 20% O2, consistent with the role of HIF-1 as a mediator of transcriptional responses to hypoxia (Wang, 1995a).

Hypoxia-inducible factor 1 (HIF-1) is a DNA-binding protein that activates erythropoietin (Epo) gene transcription in Hep3B cells subjected to hypoxia or cobalt chloride treatment. HIF-1 DNA binding activity is also induced by hypoxia or cobalt in non-Epo-producing cells, suggesting a general role for HIF-1 in hypoxia signal transduction and transcriptional regulation. This study reports the biochemical purification of HIF-1 from Epo-producing Hep3B cells and non-Epo-producing HeLa S3 cells. HIF-1 protein was purified 11,250-fold by DEAE ion-exchange and DNA affinity chromatography. Analysis of HIF-1 isolated from a preparative gel shift assay revealed four polypeptides. Peptide mapping of these HIF-1 components demonstrates that 91-, 93-, and 94-kDa polypeptides had similar tryptic maps, whereas the 120-kDa polypeptide had a distinct profile. Glycerol gradient sedimentation analysis suggested that HIF-1 exists predominantly in a heterodimeric form and to a lesser extent as a heterotetramer. Partially purified HIF-1 binds specifically to the wild-type HIF-1 binding site from the EPO enhancer but not to a mutant sequence that lacks hypoxia-inducible enhancer activity. UV cross-linking analysis with purified HIF-1 indicates that both subunits of HIF-1 contact DNA directly. It is concluded that in both cobalt chloride-treated HeLa cells and hypoxic Hep3B cells, HIF-1 is composed of two different subunits: 120-kDa HIF-1 alpha and 91-94-kDa HIF-1 beta (Wang, 1995b).

Hypoxia-inducible factor-1 (HIF-1) is a potent cellular survival factor contributing to tumorigenesis in a broad range of cancers. The functional transcription factor exists as a heterodimeric complex consisting of HIF-1alpha and the aryl hydrocarbon receptor nuclear translocator (ARNT). Association of HIF-1 with ARNT is required for its activity; however, no other role has been ascribed to this interaction. Pharmacologic inhibition of Hsp90 by geldanamycin (GA) impairs HIF transcription and promotes VHL (Von Hippel-Lindau)-independent degradation of the protein, thus implicating Hsp90 as an essential interacting partner for HIF. This study further explores the physiological role for Hsp90 in HIF function. The PAS (Per-ARNT-Sim) domain of HIF is required both to promote association with Hsp90 and confer sensitivity to GA. Coincidentally, this domain also associates with ARNT. Overexpression of ARNT in a VHL-deficient background results in substantially increased HIF-1 protein concomitant with increased protein stability. Conversely, down-regulation of endogenous ARNT protein by RNA interference decreases the steady-state HIF protein. ARNT-mediated stabilization of HIF is specific for the Hsp90-dependent pathway; ARNT was unable to protect HIF from VHL-mediated degradation. The ability of ARNT to up-regulate HIF and diminish HIF sensitivity to GA is due to its ability to compete for the Hsp90 binding site on HIF. These data elucidate novel functions for ARNT and Hsp90 in regulating HIF function and further illustrate that cofactor association may significantly impact upon the sensitivity of Hsp90 clients to chaperone inhibitors (Isaacs, 2004).

Function of the PAS dimerization domain

bHLH PAS transcriptional regulators control critical developmental and metabolic processes, including transcriptional responses to stimuli such as hypoxia and environmental pollutants, mediated respectively by hypoxia inducible factors (HIF-alpha) and the dioxin (aryl hydrocarbon) receptor (DR). The bHLH proteins contain a basic DNA binding sequence adjacent to a helix-loop-helix dimerization domain. Dimerization among bHLH.PAS proteins is additionally regulated by the PAS region, which controls the specificity of partner choice such that HIF-alpha and DR must dimerize with the aryl hydrocarbon nuclear translocator (Arnt) to form functional DNA binding complexes. Purified bacterially expressed proteins encompassing the N-terminal bHLH and bHLH.PAS regions of Arnt, DR, and HIF-1alpha have been analyzed and the contribution of the PAS domains to DNA binding in vitro was examined. Recovery of functional DNA binding proteins from bacteria was dramatically enhanced by coexpression of the bHLH.PAS regions of DR or HIF-1alpha with the corresponding region of Arnt. Formation of stable protein-DNA complexes by DR/Arnt and HIF-1alpha/Arnt heterodimers with their cognate DNA sequences requires the PAS A domains and exhibits KD values of 0.4 nM and approximately 50 nM, respectively. In contrast, the presence of the PAS domains of Arnt has little effect on DNA binding by Arnt homodimers, and these bind DNA with a KD of 45 nM. In the case of the DR, both high affinity DNA binding and dimer stability are specific to the Arnt native PAS domain, since a chimera in which the PAS A domain was substituted with the equivalent domain of Arnt generates a destabilized protein that binds DNA poorly (Chapman-Smith, 2004).

The structure and interactions of the C-terminal PAS domain of human HIF-2alpha has been studied by NMR spectroscopy. HIF-2alpha PAS-B binds the analogous ARNT domain in vitro, showing that residues involved in this interaction are located on the solvent-exposed side of the HIF-2alpha central beta-sheet. Mutating residues at this surface not only disrupts the interaction between isolated PAS domains in vitro but also interferes with the ability of full-length HIF to respond to hypoxia in living cells. Extending these findings to other PAS domains, this beta-sheet interface is found to be widely used for both intra- and inter-molecular interactions, suggesting a basis of specificity and regulation of many types of PAS-containing signaling proteins (Erbel, 2003).

The inhibitory PAS (Per/Arnt/Sim) domain protein, IPAS, functions as a dominant negative regulator of hypoxia-inducible transcription factors (HIFs) by forming complexes with those proteins that fail to bind to hypoxia response elements of target genes. IPAS is predominantly expressed in mice in Purkinje cells of the cerebellum and in corneal epithelium of the eye where it appears to play a role in negative regulation of angiogenesis and maintenance of an avascular phenotype. Sequencing of the mouse IPAS genomic structure has revealed that IPAS is a splicing variant of the HIF-3alpha locus. Thus, in addition to three unique exons (1a, 4a, and 16) IPAS shares three exons (2, 4, and 5) with HIF-3alpha as well as alternatively spliced variants of exons 3 and 6. In experiments using normal mice and mice exposed to hypoxia (6% O2) for 6 h, alternative splicing of the HIF-3alpha transcript is observed in the heart and lung. The alternatively spliced transcript is only observed under hypoxic conditions, thus defining a novel mechanism of hypoxia-dependent regulation of gene expression. Importantly, this mechanism may establish negative feedback loop regulation of adaptive responses to hypoxia/ischemia in these tissues (Makino, 2002).

HIFalpha subcellular localization and activation

In response to decreased cellular oxygen concentrations the basic helix-loop-helix (bHLH)/PAS (Per, Arnt, Sim) hypoxia-inducible transcription factor, HIF-1alpha, mediates activation of networks of target genes involved in angiogenesis, erythropoiesis and glycolysis. The mechanism of activation of HIF-1alpha is a multi-step process that includes hypoxia-dependent nuclear import and activation (derepression) of the transactivation domain, resulting in recruitment of the CREB-binding protein (CBP)/p300 coactivator. Inducible nuclear accumulation is dependent on a nuclear localization signal (NLS) within the C-terminal end of HIF-1alpha which also harbors the hypoxia-inducible transactivation domain. Nuclear import of HIF-1alpha is inhibited by either deletion or a single amino acid substitution within the NLS sequence motif and, within the context of the full-length protein, these mutations also resulted in inhibition of the transactivation activity of HIF-1alpha and recruitment of CBP. However, nuclear localization per se is not sufficient for transcriptional activation, since fusion of HIF-1alpha to the heterologous GAL4 DNA-binding domain generated a protein that showed constitutive nuclear localization but required hypoxic stimuli for function as a CBP-dependent transcription factor. Thus, hypoxia-inducible nuclear import and transactivation by recruitment of CBP can be functionally separated from one another and play critical roles in signal transduction by HIF-1alpha (Kallio, 1998).

Eukaryotic cells sense oxygen and adapt to hypoxia by regulating a number of genes. HIF-1 is the 'master' in this pleiotypic response. HIF-1 comprises two members of the basic helix-loop-helix transcription factor family, HIF-1 alpha and HIF-1 beta. The HIF-1 alpha protein is subject to drastic O2-dependent proteasomal control. However, the signalling components regulating the 'switch' for 'escaping' proteasomal degradation under hypoxia are still largely unknown. The rapid nuclear translocation of HIF-1 alpha could represent an efficient way to escape from this degradation. It was therefore asked where in the cell is HIF-1 alpha degraded? To address this question, HIF-1 alpha was trapped either in the cytoplasm, by fusing HIF-1 alpha to the cytoplasmic domain of the Na(+)-H(+) exchanger (NHE-1), or in the nucleus, by treatment with leptomycin B. Surprisingly, HIF-1 alpha was found to be stabilized by hypoxia and undergo O2-dependent proteasomal degradation with an identical half-life (5-8 min) in both cellular compartments. Therefore, HIF-1 alpha entry into the nucleus is not, as proposed, a key event that controls its stability. This result markedly contrasts with the mechanism that controls p53 degradation via MDM2 (Berra, 2001).

Hypoxia inducible factors (HIF1, 2 and 3), consisting of alpha and beta subunits, play an essential role in various responses to hypoxia. Nuclear entry of alpha subunits is a necessary step for the formation of DNA-binding complex with beta subunit, which is constitutively localized in the nucleus. The nuclear accumulation of HIF2alpha induced by hypoxia is mediated through a novel variant of bipartite-type nuclear localization signal (NLS) in the C-terminus of the protein, which has an unusual length of spacer sequence between two adjacent basic domains. When the ubiquitin-proteasome system is deficient or inhibited, HIF2alpha accumulates in the nucleus even under normoxia, also mediated through the bipartite NLS. These findings indicate that the protein stability is critical for the nuclear localization of HIF2alpha and hypoxia is not a necessary factor for the process. Importantly, the NLS of HIF2alpha is also conserved in the other HIF family members, HIF1alpha and HIF3alpha. Mutational analyses prove that the NLS mediating the nuclear localization of HIF1alpha is indeed bipartite-type, but not monopartite-type as thought before. These results suggest that the newly identified NLS is crucial for the functional regulation of HIF family (Luo, 2001).

Regulation of mTOR function in response to hypoxia by REDD1 and the TSC1/TSC2 tumor suppressor complex

Mammalian target of rapamycin (mTOR) is a central regulator of protein synthesis whose activity is modulated by a variety of signals. Energy depletion and hypoxia result in mTOR inhibition. While energy depletion inhibits mTOR through a process involving the activation of AMP-activated protein kinase (AMPK) by LKB1 and subsequent phosphorylation of TSC2, the mechanism of mTOR inhibition by hypoxia is not known. This study shows that mTOR inhibition by hypoxia requires the TSC1/TSC2 tumor suppressor complex and the hypoxia-inducible gene REDD1/RTP801 (Drosophila homologs: scylla and charybde). Disruption of the TSC1/TSC2 complex through loss of TSC1 or TSC2 blocks the effects of hypoxia on mTOR, as measured by changes in the mTOR targets S6K and 4E-BP1, and results in abnormal accumulation of Hypoxia-inducible factor (HIF). In contrast to energy depletion, mTOR inhibition by hypoxia does not require AMPK or LKB1. Down-regulation of mTOR activity by hypoxia requires de novo mRNA synthesis and correlates with increased expression of the hypoxia-inducible REDD1 gene. Disruption of REDD1 abrogates the hypoxia-induced inhibition of mTOR, and REDD1 overexpression is sufficient to down-regulate S6K phosphorylation in a TSC1/TSC2-dependent manner. Inhibition of mTOR function by hypoxia is likely to be important for tumor suppression as TSC2-deficient cells maintain abnormally high levels of cell proliferation under hypoxia (Brugarolas, 2004).

HIF degradation by the ubiquitin-proteasome pathway

The hypoxia-inducible factor 1 transcriptional activator complex (HIF-1) is involved in the activation of the erythropoietin and several other hypoxia-responsive genes. The HIF-1 complex is composed of two protein subunits: HIF-1beta/ARNT (aryl hydrocarbon receptor nuclear translocator), which is constitutively expressed, and HIF-1alpha, which is not present in normal cells but induced under hypoxic conditions. The HIF-1alpha subunit is continuously synthesized and degraded under normoxic conditions, while it accumulates rapidly following exposure to low oxygen tensions. The involvement of the ubiquitin-proteasome system in the proteolytic destruction of HIF-1 in normoxia was studied by the use of specific inhibitors of the proteasome system. Lactacystin and MG-132 were found to protect the degradation of the HIF-1 complex in cells transferred from hypoxia to normoxia. The same inhibitors were able to induce HIF-1 complex formation when added to normoxic cells. Final confirmation of the involvement of the ubiquitin-proteasome system in the regulated degradation of HIF-1alpha was obtained by the use of ts20TGR cells, which contain a temperature-sensitive mutant of E1, the ubiquitin-activating enzyme. Exposure of ts20 cells, under normoxic conditions, to the non-permissive temperature induced a rapid and progressive accumulation of HIF-1. The effect of proteasome inhibitors on the normoxic induction of HIF-1 binding activity was mimicked by the thiol reducing agent N-(2-mercaptopropionyl)-glycine and by the oxygen radical scavenger 2-acetamidoacrylic acid. Furthermore, N-(2-mercaptopropionyl)-glycine induced gene expression as measured by the stimulation of a HIF-1-luciferase expression vector and by the induction of erythropoietin mRNA in normoxic Hep 3B cells. These last findings strongly suggest that the hypoxia induced changes in HIF-1alpha stability and subsequent gene activation are mediated by redox-induced changes (Salceda, 1997).

An oxygen-dependent degradation (ODD) domain within HIF-1alpha that controls its degradation by the ubiquitin-proteasome pathway has been identifed. The ODD domain consists of approximately 200 amino acid residues, located in the central region of HIF-1alpha. Because portions of the domain independently confer degradation of HIF-1alpha, deletion of this entire region is required to give rise to a stable HIF-1alpha, capable of heterodimerization, DNA-binding, and transactivation in the absence of hypoxic signaling. Conversely, the ODD domain alone confers oxygen-dependent instability when fused to a stable protein, Gal4. Hence, the ODD domain plays a pivotal role for regulating HIF-1 activity and thereby may provide a means of controlling gene expression by changes in oxygen tension (Huang, 1998).

Oxygen-dependent proteolytic destruction of hypoxia-inducible factor-alpha (HIF-alpha) subunits plays a central role in regulating transcriptional responses to hypoxia. The von Hippel-Lindau tumor suppressor E3 ubiquitin ligase (VHLE3) plays a key role in this process; an interaction with HIF-1 alpha has been defined that is regulated by prolyl hydroxylation. Two independent regions within the HIF-alpha oxygen-dependent degradation domain (ODDD) are targeted for ubiquitylation by VHLE3 in a manner dependent upon prolyl hydroxylation. In a series of in vitro and in vivo assays, independent and non-redundant operation of each site in regulation of the HIF system has been demonstrated. Both sites contain a common core motif, but differ both in overall sequence and in the conditions under which they bind to the VHLE3 ligase complex. The definition of two independent destruction domains implicates a more complex system of pVHL-HIF-alpha interactions, but reinforces the role of prolyl hydroxylation as an oxygen-dependent destruction signal (Masson, 2001).

Hypoxia-inducible factor-1alpha (HIF1alpha) is a central regulator of the cellular response to hypoxia. Prolyl-hydroxylation of HIF1alpha by PHD enzymes is prerequisite for HIF1alpha degradation. The abundance of PHD1 and PHD3 are regulated via their targeting for proteasome-dependent degradation by the E3 ubiquitin ligases Siah1a/2 (Drosophila homolog: Seven in absentia), under hypoxia conditions. Siah2 null fibroblasts exhibit prolonged PHD3 half-life, resulting in lower levels of HIF1alpha expression during hypoxia. Significantly, hypoxia-induced HIF1alpha expression is completely inhibited in Siah1a/2 null cells, yet can be rescued upon inhibition of PHD3 by RNAi. Siah2 targeting of PHD3 for degradation increases upon exposure to even mild hypoxic conditions, which coincides with increased Siah2 transcription. Siah2 null mice subjected to hypoxia display an impaired hyperpneic respiratory response and reduced levels of hemoglobin. Thus, the control of PHD1/3 by Siah1a/2 constitutes another level of complexity in the regulation of HIF1alpha during hypoxia (Nakayama, 2004).

SUMO conjugation to proteins is involved in the regulation of diverse cellular functions (see Drosophila SUMO). A protein, RWD-containing sumoylation enhancer (RSUME), has been identified that enhances overall SUMO-1, -2, and -3 conjugation by interacting with the SUMO conjugase Ubc9. RSUME increases noncovalent binding of SUMO-1 to Ubc9 and enhances Ubc9 thioester formation and SUMO polymerization. RSUME enhances the sumoylation of IkappaB in vitro and in cultured cells, leading to an inhibition of NF-kappaB transcriptional activity. RSUME is induced by hypoxia and enhances the sumoylation of HIF-1α, promoting its stabilization and transcriptional activity during hypoxia. Disruption of the RWD domain structure of RSUME demonstrates that this domain is critical for RSUME action. Together, these findings point to a central role of RSUME in the regulation of sumoylation and, hence, several critical regulatory pathways in mammalian cells (Carbia-Nagashima, 2007).

HIF prolyl-hydroxylases are oxygen sensors that are required for the degradation of HIF

Posttranslational modification by prolyl hydroxylation is a key regulatory event that targets HIF-alpha subunits for proteasomal destruction via the von Hippel-Lindau ubiquitylation complex. A conserved HIF-VHL-prolyl hydroxylase pathway has been defined in C. elegans, and a genetic approach has been used to identify EGL-9 as a dioxygenase that regulates HIF by prolyl hydroxylation. In mammalian cells, HIF-prolyl hydroxylases are represented by a series of isoforms bearing a conserved 2-histidine-1-carboxylate iron coordination motif at the catalytic site. Direct modulation of recombinant enzyme activity by graded hypoxia, iron chelation, and cobaltous ions mirrors the characteristics of HIF induction in vivo, fulfilling requirements for these enzymes being oxygen sensors that regulate HIF (Epstein, 2001).

Rat Sm-20 is a homolog of the Caenorhabditis elegans gene egl-9 and has been implicated in the regulation of growth, differentiation and apoptosis in muscle and nerve cells. Null mutants in egl-9 result in a complete tolerance to an otherwise lethal toxin produced by Pseudomonas aeruginosa. This study describes the conserved Egl-Nine (EGLN) gene family of which rat SM-20 and C. elegans Egl-9 are members and characterizes the mouse and human homologs. Each of the human genes (EGLN1, EGLN2 and EGLN3) are of a conserved genomic structure consisting of five coding exons. Phylogenetic analysis and domain organization show that EGLN1 represents the ancestral form of the gene family and that EGLN3 is the human ortholog of rat Sm-20. The previously observed mitochondrial targeting of rat SM-20 is unlikely to be a general feature of the protein family and may be a feature specific to rats. An EGLN gene is unexpectedly found in the genome of P. aeruginosa, a bacterium known to produce a toxin that acts through the Egl-9 protein. The pathogenic bacterium Vibrio cholerae is also shown to have an EGLN gene suggesting that it is an important pathogenicity factor. These results provide new insights into host-pathogen interactions and a basis for further functional characterization of the gene family and resolve discrepancies in annotation between gene family members (Taylor, 2001).

Hypoxia-inducible factor (HIF) is a heterodimeric transcription factor induced by hypoxia. Under normoxic conditions, site-specific proline hydroxylation of the alpha subunits of HIF allows recognition by the von Hippel-Lindau tumor suppressor protein (VHL), a component of an E3 ubiquitin ligase complex that targets these subunits for degradation by the ubiquitin-proteasome pathway. Under hypoxic conditions, this hydroxylation is inhibited, allowing the alpha subunits of HIF to escape VHL-mediated degradation. Three enzymes, prolyl hydroxylase domain-containing proteins 1, 2, and 3 (PHD1, -2, and -3; also known as HIF prolyl hydroxylase 3, 2, and 1, respectively), have been identified that catalyze proline hydroxylation of HIF alpha subunits. These enzymes hydroxylate specific prolines in HIF alpha subunits in the context of a strongly conserved LXXLAP sequence motif (where X indicates any amino acid and P indicates the hydroxylacceptor proline). PHD2 has the highest specific activity toward the primary hydroxylation site of HIF-1alpha. Furthermore, and unexpectedly, mutations can be tolerated at the -5, -2, and -1 positions (relative to proline) of the LXXLAP motif. Thus, these results provide evidence that the only obligatory residue for proline hydroxylation in HIF-1alpha is the hydroxylacceptor proline itself (Huang, 2002).

HIF plays a pivotal role in cellular adaptation to low oxygen availability. In normoxia, the HIF-alpha subunits are targeted for destruction by prolyl hydroxylation, a specific modification that provides recognition for the E3 ubiquitin ligase complex containing the von Hippel-Lindau tumor suppressor protein (pVHL). Three HIF prolyl-hydroxylases (PHD1, 2 and 3) were identified recently in mammals and shown to hydroxylate HIF-alpha subunits. Specific 'silencing' of PHD2 with short interfering RNAs is sufficient to stabilize and activate HIF-1alpha in normoxia in all the human cells investigated. 'Silencing' of PHD1 and PHD3 has no effect on the stability of HIF-1alpha either in normoxia or upon re-oxygenation of cells briefly exposed to hypoxia. It is therefore conclude that, in vivo, PHDs have distinct assigned functions, PHD2 being the critical oxygen sensor setting the low steady-state levels of HIF-1alpha in normoxia. Interestingly, PHD2 is upregulated by hypoxia, providing an HIF-1-dependent auto-regulatory mechanism driven by the oxygen tension (Berra, 2003).

The hypoxia-inducible factors (HIFs) play a central role in oxygen homeostasis. Hydroxylation of one or two critical prolines by specific hydroxylases (P4Hs) targets their HIF-alpha subunits for proteasomal degradation. By studying the three human HIF-P4Hs, it was found that the longest and shortest isoenzymes have major transcripts encoding inactive polypeptides; this suggests novel regulation by alternative splicing. Recombinant HIF-P4Hs expressed in insect cells require peptides of more than 8 residues, distinct differences being found between isoenzymes. All the HIF-P4Hs work enzymatically to hydroxylate peptides corresponding to Pro564 in HIF-1alpha, whereas a Pro402 peptide had 20-50-fold Km values for two isoenzymes but was not hydroxylated by the shortest isoenzyme at all: this difference was not explained by the two prolines being in a -Pro402-Ala- and -Pro564-Tyr-sequence. All the HIF-P4Hs-hydroxylated peptides that corresponded to two of three potential sites in HIF-2alpha and one in HIF-3alpha. The Km values for O2 were slightly above its atmospheric concentration, indicating that the HIF-P4Hs are effective oxygen sensors. Small molecule inhibitors of collagen P4Hs also inhibited the HIF-P4Hs, but with distinctly different Ki values, indicating that it should be possible to develop specific inhibitors for each class of P4Hs and possibly even for the individual HIF-P4Hs (Hirsala, 2003).

Hypoxia-inducible factor1 is an essential transcription factor for cellular adaptation to decreased oxygen availability. In normoxia the oxygen-sensitive alpha-subunit of HIF-1 is hydroxylated on Pro564 and Pro402 and thus targeted for proteasomal degradation. Three human oxygen-dependent HIF-1 alpha prolyl hydroxylases (PHD1, PHD2, and PHD3) function as oxygen sensors in vivo. Furthermore, the asparagine hydroxylase FIH-1 (factor inhibiting HIF) has been found to hydroxylate Asp803 of the HIF-1 C-terminal transactivation domain, which results in the decreased ability of HIF-1 to bind to the transcriptional coactivator p300/CBP. These enzymes have been fused to the N-terminus of fluorescent proteins and transiently transfected the fusion proteins into human osteosarcoma cells (U2OS). Three-dimensional 2-photon confocal fluorescence microscopy shows that PHD1 is exclusively present in the nucleus; PHD2 and FIH-1 are mainly located in the cytoplasm and PHD3 is homogeneously distributed in cytoplasm and nucleus. Hypoxia did not influence the localization of any enzyme under investigation. In contrast to FIH-1, each PHD inhibited nuclear HIF-1 alpha accumulation in hypoxia. All hydroxylases suppressed activation of a cotransfected hypoxia-responsive luciferase reporter gene. Endogenous PHD2mRNA and PHD3mRNA are hypoxia-inducible, whereas expression of PHD1mRNA and FIH-1mRNA is oxygen independent. It is proposed that PHDs and FIH-1 form an oxygen sensor cascade of distinct subcellular localization (Metzen, 2003).

von Hippel-Lindau tumor suppressor protein, a component of an E3 ubiquitin ligase complex, is required for degradation of HIF-1

Hypoxia-inducible factor-1 (HIF-1) has a key role in cellular responses to hypoxia, including the regulation of genes involved in energy metabolism, angiogenesis and apoptosis. The alpha subunits of HIF are rapidly degraded by the proteasome under normal conditions, but are stabilized by hypoxia. Cobaltous ions or iron chelators mimic hypoxia, indicating that the stimuli may interact through effects on a ferroprotein oxygen sensor. A critical role is demonstrated for the von Hippel-Lindau (VHL) tumor suppressor gene product pVHL in HIF-1 regulation. In VHL-defective cells, HIF alpha-subunits are constitutively stabilized and HIF-1 is activated. Re-expression of pVHL restores oxygen-dependent instability. pVHL and HIF alpha-subunits co-immunoprecipitate, and pVHL is present in the hypoxic HIF-1 DNA-binding complex. In cells exposed to iron chelation or cobaltous ions, HIF-1 is dissociated from pVHL. These findings indicate that the interaction between HIF-1 and pVHL is iron dependent, and that it is necessary for the oxygen-dependent degradation of HIF alpha-subunits. Thus, constitutive HIF-1 activation may underlie the angiogenic phenotype of VHL-associated tumors. The pVHL/HIF-1 interaction provides a new focus for understanding cellular oxygen sensing (Maxwell, 1999).

The von Hippel-Lindau tumor suppressor protein (pVHL) has emerged as a key factor in cellular responses to oxygen availability, being required for the oxygen-dependent proteolysis of alpha subunits of hypoxia inducible factor-1. Mutations in VHL cause a hereditary cancer syndrome associated with dysregulated angiogenesis, and up-regulation of hypoxia inducible genes. The mechanisms underlying these processes have been investigated; extracts from VHL-deficient renal carcinoma cells have a defect in HIF-alpha ubiquitylation activity that is complemented by exogenous pVHL. This defect was specific for HIF-alpha among a range of substrates tested. Furthermore, HIF-alpha subunits are the only pVHL-associated proteasomal substrates identified by comparison of metabolically labeled anti-pVHL immunoprecipitates from proteosomally inhibited cells and normal cells. Analysis of pVHL/HIF-alpha interactions has defined short sequences of conserved residues within the internal transactivation domains of HIF-alpha molecules sufficient for recognition by pVHL. In contrast, while full-length pVHL and the p19 variant interact with HIF-alpha, the association was abrogated by further N-terminal and C-terminal truncations. The interaction was also disrupted by tumor-associated mutations in the beta-domain of pVHL and loss of interaction was associated with defective HIF-alpha ubiquitylation and regulation, defining a mechanism by which these mutations generate a constitutively hypoxic pattern of gene expression promoting angiogenesis. The findings indicate that pVHL regulates HIF-alpha proteolysis by acting as the recognition component of a ubiquitin ligase complex, and support a model in which its beta domain interacts with short recognition sequences in HIF-alpha subunits (Cockman, 2000).

In normoxic cells the hypoxia-inducible factor-1 alpha (HIF-1 alpha) is rapidly degraded by the ubiquitin-proteasome pathway, and activation of HIF-1 alpha to a functional form requires protein stabilization. The product of the von Hippel-Lindau (VHL) tumor suppressor gene mediates ubiquitylation and proteasomal degradation of HIF-1 alpha under normoxic conditions via interaction with the core of the oxygen-dependent degradation domain of HIF-1 alpha. The region of VHL mediating interaction with HIF-1 alpha overlaps with a putative macromolecular binding site observed within the crystal structure of VHL. This motif of VHL also represents a mutational hotspot in tumors, and one of these mutations impairs interaction with HIF-1 alpha and subsequent degradation. Interestingly, the VHL binding site within HIF-1 alpha overlaps with one of the minimal transactivation domains. Protection of HIF-1 alpha against degradation by VHL is a multistep mechanism, including hypoxia-induced nuclear translocation of HIF-1 alpha and an intranuclear hypoxia-dependent signal. VHL was not released from HIF-1 alpha during this process. Finally, stabilization of HIF-1 alpha protein levels per se does not totally bypass the need of the hypoxic signal for generating the transactivation response (Tanimoto, 2000).

Hypoxia-inducible factor (HIF) is a transcriptional complex that plays a central role in the regulation of gene expression by oxygen. In oxygenated and iron replete cells, HIF-alpha subunits are rapidly destroyed by a mechanism that involves ubiquitylation by the von Hippel-Lindau tumor suppressor (pVHL) E3 ligase complex. This process is suppressed by hypoxia and iron chelation, allowing transcriptional activation. The interaction between human pVHL and a specific domain of the HIF-1alpha subunit is regulated through hydroxylation of a proline residue (HIF-1alpha P564) by an enzyme termed HIF-alpha prolyl-hydroxylase (HIF-PH). An absolute requirement for dioxygen as a cosubstrate and iron as cofactor suggests that HIF-PH functions directly as a cellular oxygen sensor (Jaakkola, 2001).

HIF (hypoxia-inducible factor) is a transcription factor that plays a pivotal role in cellular adaptation to changes in oxygen availability. In the presence of oxygen, HIF is targeted for destruction by an E3 ubiquitin ligase containing the von Hippel-Lindau tumor suppressor protein (pVHL). Human pVHL binds to a short HIF-derived peptide when a conserved proline residue at the core of this peptide is hydroxylated. Because proline hydroxylation requires molecular oxygen and Fe(2+), this protein modification may play a key role in mammalian oxygen sensing (Ivan, 2001).

The hypoxia-inducible factor (HIF) activates the expression of genes that contain a hypoxia response element. The alpha-subunits of the HIF transcription factors are degraded by proteasomal pathways during normoxia but are stabilized under hypoxic conditions. The von Hippel-Lindau protein (pVHL) mediates the ubiquitination and rapid degradation of HIF-alpha (including HIF-1alpha and HIF-2alpha). Post-translational hydroxylation of a proline residue in the oxygen-dependent degradation (ODD) domain of HIF-alpha is required for the interaction between HIF and VHL. Cobalt mimics hypoxia and causes accumulation of HIF-1alpha and HIF-2alpha. However, little is known about the mechanism by which this occurs. Cobalt binds directly to the ODD domain of HIF-2alpha. Cobalt inhibits pVHL binding to HIF-alpha even when HIF-alpha is hydroxylated. Deletion of 17 amino acids within the ODD domain of HIF-2alpha that are required for pVHL binding prevents the binding of cobalt and stabilizes HIF-2alpha during normoxia. These findings show that cobalt mimics hypoxia, at least in part, by occupying the VHL-binding domain of HIF-alpha and thereby preventing the degradation of HIF-alpha (Yuan, 2003).

Functional inactivation of the von Hippel-Lindau (VHL) tumor suppressor protein is the cause of familial VHL disease and sporadic kidney cancer. The VHL gene product (pVHL) is a component of an E3 ubiquitin ligase complex that targets the hypoxia-inducible factor (HIF) 1 and 2 alpha subunits for polyubiquitylation. This process is dependent on the hydroxylation of conserved proline residues on the alpha subunits of HIF-1/2 in the presence of oxygen. In an effort to identify orphan HIF-like proteins in the data base that are potential targets of the pVHL complex, multiple splice variants of the human HIF-3 alpha locus are reported as follows: hHIF-3 alpha 1, hHIF-3 alpha 2 (also referred to as hIPAS; human inhibitory PAS domain protein), hHIF-3 alpha 3, hHIF-3 alpha 4, hHIF-3 alpha 5, and hHIF-3 alpha 6. The common oxygen-dependent degradation domain of hHIF-3 alpha 1-3 splice variants is targeted for ubiquitylation by the pVHL complex in vitro and in vivo. This activity is enhanced in the presence of prolyl hydroxylase and is dependent on a proline residue at position 490. Furthermore, the ubiquitin conjugation occurs on lysine residues at position 465 and 568 within the oxygen-dependent degradation domain. These results demonstrate additional targets of the pVHL complex and suggest a growing complexity in the regulation of hypoxia-inducible genes by the HIF family of transcription factors (Maynard, 2003).

Mutation HIF isoforms

In Hif1a-/- embryonic stem cells that do not express the O2-regulated HIF-1alpha subunit, levels of mRNAs encoding glucose transporters and glycolytic enzymes are reduced, and cellular proliferation is impaired. Vascular endothelial growth factor mRNA expression is also markedly decreased in hypoxic Hif1a-/- embryonic stem cells and cystic embryoid bodies. Complete deficiency of HIF-1alpha results in developmental arrest and lethality by E11 of Hif1a-/- embryos that manifest neural tube defects, cardiovascular malformations, and marked cell death within the cephalic mesenchyme. In Hif1a+/+ embryos, HIF-1alpha expression increases between E8.5 and E9.5, coincident with the onset of developmental defects and cell death in Hif1a-/- embryos. These results demonstrate that HIF-1alpha is a master regulator of cellular and developmental O2 homeostasis (Iyer, 1998).

Hypoxia-inducible factor (HIF) transcription factors respond to multiple environmental stressors, including hypoxia and hypoglycemia. Mice lacking the HIF family member HIF-2alpha (encoded by Epas1) have a syndrome of multiple-organ pathology, biochemical abnormalities and altered gene expression patterns. Histological and ultrastructural analyses show retinopathy, hepatic steatosis, cardiac hypertrophy, skeletal myopathy, hypocellular bone marrow, azoospermia and mitochondrial abnormalities in these mice. Serum and urine metabolite studies show hypoglycemia, lactic acidosis, altered Krebs cycle function and dysregulated fatty acid oxidation. Biochemical assays show enhanced generation of reactive oxygen species (ROS), whereas molecular analyses indicate reduced expression of genes encoding the primary antioxidant enzymes (AOEs). Transfection analyses show that HIF-2alpha could efficiently transactivate the promoters of the primary AOEs. Prenatal or postnatal treatment of Epas1-/- mice with a superoxide dismutase (SOD) mimetic reversed several aspects of the null phenotype. A rheostat role is proposed for HIF-2alpha that allows for the maintenance of ROS as well as mitochondrial homeostasis (Scortegagna, 2003).

The neurohormone orexin stimulates hypoxia-inducible factor-1 activity

Orexin A and Orexin B (also known as hypocretins) are neuropeptides that bind two related G-coupled protein receptors (OXR1 and OXR2) and thus induce wakefulness, food consumption, and locomotion. Conversely, deletion of the orexin gene in mice produces a condition similar to canine and human narcolepsy. Despite the central importance of the orexin system in regulating wakefulness and feeding behavior, little is known about the downstream signaling mechanisms that achieve these effects. In this study, genomics techniques were used to probe this question and reveal that orexin activates the hypoxia-inducible factor 1 (HIF-1), a heterodimeric transcription factor whose pathogenic role in stimulating angiogenesis in hypoxic tumors has been the focus of intense investigation. Orexin-stimulated HIF-1 activity is due to both increased HIF-1α gene transcription and a down-regulation of von Hippel-Lindau (VHL), the E3 ubiquitin ligase that mediates the turnover of HIF-1 via the ubiquitin-proteasome pathway. Orexin-mediated activation of HIF-1 results in increased glucose uptake and higher glycolytic activity, as expected from studies of hypoxic cells. However, orexin receptor-expressing cells somehow override the HIF-1-mediated preference for funneling pyruvate into anaerobic glycolysis and instead favor ATP production through the tricarboxylic acid cycle and oxidative phosphorylation. These findings implicate HIF-1 as an important transcription factor in the hormone-mediated regulation of hunger and wakefulness (Sikder, 2007).

Multiple functions and differential regulation of HIF isoforms

Deprivation of oxygen and/or glucose (hypoglycemia) represents a serious stress that affects cellular survival. The hypoxia-inducible transcription factor-1alpha (HIF-1alpha), which has been implicated in the cellular response to hypoxia, mediates apoptosis during hypoxia, but the function of its homolog HIF-2alpha remains unknown. Therefore, the role of HIF-2alpha in cellular survival was studied by targeted inactivation of the HIF-2alpha gene (HIF-2alpha-/-) in murine embryonic stem (ES) cells. In contrast to HIF-1alpha deficiency, loss of HIF-2alpha does not protect ES cells against apoptosis during hypoxia. Both HIF-1alpha-/- and HIF-2alpha-/- ES cells are, however, resistant to apoptosis in response to hypoglycemia. When co-cultured with wild type ES cells, HIF-2alpha-/- ES cells become rapidly and progressively enriched in hypoglycemia but not in hypoxia. Thus, HIF-1alpha and HIF-2alpha may have distinct roles in responses to environmental stress, and despite its name, HIF-2alpha may be more important in the survival response to environmental variables other than the level of oxygen (Brusselmans, 2001).

Transcriptional responses to hypoxia are primarily mediated by hypoxia-inducible factor (HIF), a heterodimer of HIF-alpha and the aryl hydrocarbon receptor nuclear translocator subunits. The HIF-1alpha and HIF-2alpha subunits are structurally similar in their DNA binding and dimerization domains but differ in their transactivation domains, implying they may have unique target genes. Previous studies using Hif-1alpha-/- embryonic stem and mouse embryonic fibroblast cells show that loss of HIF-1alpha eliminates all oxygen-regulated transcriptional responses analyzed, suggesting that HIF-2alpha is dispensable for hypoxic gene regulation. In contrast, HIF-2alpha has been shown to regulate some hypoxia-inducible genes in transient transfection assays and during embryonic development in the lung and other tissues. To address this discrepancy, and to identify specific HIF-2alpha target genes, DNA microarray analysis was used to evaluate hypoxic gene induction in cells expressing HIF-2alpha but not HIF-1alpha. In addition, HEK293 cells were engineered to express stabilized forms of HIF-1alpha or HIF-2alpha via a tetracycline-regulated promoter. In this first comparative study of HIF-1alpha and HIF-2alpha target genes, it has been demonstrated that HIF-2alpha does regulate a variety of broadly expressed hypoxia-inducible genes, suggesting that its function is not restricted, as initially thought, to endothelial cell-specific gene expression. Importantly, HIF-1alpha (and not HIF-2alpha) stimulates glycolytic gene expression in both types of cells, clearly showing for the first time that HIF-1alpha and HIF-2alpha have unique targets (Hu, 2003).

The hypoxia-inducible factors 1alpha (HIF-1alpha) and 2alpha (HIF-2alpha) have extensive structural homology and have been identified as key transcription factors responsible for gene expression in response to hypoxia. They play critical roles not only in normal development, but also in tumor progression. This study reports on the differential regulation of protein expression and transcriptional activity of HIF-1alpha and -2alpha by hypoxia in immortalized mouse embryo fibroblasts (MEFs). Oxygen-dependent protein degradation is restricted to HIF-1alpha; HIF-2alpha protein is detected in MEFs regardless of oxygenation and is localized primarily to the cytoplasm. Endogenous HIF-2alpha remains transcriptionally inactive under hypoxic conditions; however, ectopically overexpressed HIF-2alpha translocates into the nucleus and can stimulate expression of hypoxia-inducible genes. The factor inhibiting HIF-1 can selectively inhibit the transcriptional activity of HIF-1alpha but has no effect on HIF-2alpha-mediated transcription in MEFs. It is proposed that HIF-2alpha is not a redundant transcription factor of HIF-1alpha for hypoxia-induced gene expression and show evidence that there is a cell type-specific modulator(s) that enables selective activation of HIF-1alpha but not HIF-2alpha in response to low-oxygen stress (Park, 2003).

Transcriptional adaptations to hypoxia are mediated by hypoxia-inducible factor (HIF)-1, a heterodimer of HIF-alpha and aryl hydrocarbon receptor nuclear translocator subunits. The HIF-1alpha and HIF-2alpha subunits both undergo rapid hypoxia-induced protein stabilization and bind identical target DNA sequences. When coexpressed in similar cell types, discriminating control mechanisms may exist for their regulation, explaining why HIF-1alpha and HIF-2alpha do not substitute during embryogenesis. In a human lung epithelial cell line (A549), HIF-1alpha and HIF-2alpha proteins were similarly induced by acute hypoxia (4 h, 0.5% O2) at the translational or posttranslational level. However, HIF-1alpha and HIF-2alpha were differentially regulated by prolonged hypoxia (12 h, 0.5% O2) since HIF-1alpha protein stimulation disappears because of a reduction in its mRNA stability, whereas HIF-2alpha protein stimulation remains high and stable. Prolonged hypoxia also induces an increase in the quantity of natural antisense HIF-1alpha (aHIF), whose gene promoter contains several putative hypoxia response elements to which (as is confirmed here) the HIF-1alpha or HIF-2alpha protein can bind. Finally, transient transfection of A549 cells by dominant-negative HIF-2alpha, also acting as a dominant-negative for HIF-1alpha, prevents both the decrease in the HIF-1alpha protein and the increase in the aHIF transcript. Taken together, these data indicate that, during prolonged hypoxia, HIF-alpha proteins negatively regulate HIF-1alpha expression through an increase in aHIF and destabilization of HIF-1alpha mRNA. This trans-regulation between HIF-1alpha and HIF-2alpha during hypoxia likely conveys target gene specificity (Uchida, 2004).

Differential activation and antagonistic function of HIF-α isoforms in macrophages are essential for NO homeostasis

Hypoxic response and inflammation both involve the action of the hypoxia-inducible transcription factors HIF-1α and HIF-2α. Previous studies have revealed that both HIF-α proteins are in a number of aspects similarly regulated post-translationally. However, the functional interrelationship of these two isoforms remains largely unclear. The polarization of macrophages controls functionally divergent processes; one of these is nitric oxide (NO) production, which in turn is controlled in part by HIF factors. This study shows that the HIF-α isoforms can be differentially activated: HIF-1α is induced by Th1 cytokines in M1 macrophage polarization, whereas HIF-2α is induced by Th2 cytokines during an M2 response. This differential response was most evident in polarized macrophages through HIF-α isoform-specific regulation of the inducible NO synthase gene by HIF-1α, and the arginase1 gene by HIF-2α. In silico modeling predicted that regulation of overall NO availability is due to differential regulation of HIF-1α versus HIF-2α, acting to, respectively, either increase or suppress NO synthesis. An in vivo model of endotoxin challenge confirmed this; thus, these studies reveal that the two homologous transcription factors, HIF-1α and HIF-2α, can have physiologically antagonistic functions, but that their antiphase regulation allows them to coordinately regulate NO production in a cytokine-induced and transcription-dependent fashion (Takeda, 2010).

Functional characterization of myeloid response has allowed macrophage activation to be classified as responsive to Lipopolysaccharide (LPS) or Th1 cytokines such as IFNγ, or Th2 cytokines, including IL-4 and IL-13. Macrophages polarized with Th1 cytokines are called M1 macrophages, and are considered to be classically activated. M1 macrophage polarization is important for the clearance of phagocytosed or intracellular pathogens; this is mediated by production of proinflammatory cytokines, reactive oxygen species, and nitric oxide (NO). Macrophages activated by Th2 cytokines are considered M2-polarized, or alternatively activated, and are important in humoral immunity and repair processes (Takeda, 2010).

Macrophages are often present in hypoxic tissues, and hypoxia strongly affects macrophage functions. Much of the overall transcriptional response to hypoxia is mediated by a group of transcription factors known as hypoxia-inducible factors (HIF). One of these, HIF-1α, is expressed ubiquitously, and is tightly linked to inflammatory response and microbicidal activities of myeloid cells. Another oxygen-responsive component of the HIF family, HIF-2α, is expressed in a more limited fashion, although it is also found in myeloid cells. Among the transcriptional targets of HIF-1α, the inducible NO synthase gene (iNOS) is regulated by both hypoxia and a number of other factors. It is also expressed primarily in macrophages that are M1-polarized. iNOS produces NO by metabolizing its substrate, the amino acid L-arginine. Macrophages also have another arginine-metabolizing enzyme, arginase1, which generates ornithine and urea. Arginase1 is highly expressed in M2 macrophages, and competes with iNOS for their common substrate, L-arginine. Arginase1 activity can thus regulate NO production via the limitation of arginine availability in the extracellular environment. Arginase1 gene expression is also induced by hypoxia; this raises the question of how these two differing metabolic fates of arginine, NO synthesis and arginase1 activity, are regulated under hypoxia, and how the two transcription factors, HIF-1α and HIF-2α, participate in that regulation (Takeda, 2010).

In this study, HIF-1α and HIF-2α mRNA is expressed differentially in M1- and M2-polarized macrophages, due to differential induction of the two isoforms by Th1 and Th2 cytokines. Through computational analysis of transcription rates, mRNA, and protein half-lives, it was determined that these dynamic changes of mRNA levels could strongly influence overall protein levels in the absence of classical effects on post-translational stability. It was then found that HIF-1α and HIF-2α act in this way to cooperatively maintain NO homeostasis, and that they act through a functional antagonism to accomplish this by differential action on their two target genes: iNOS and arginase1 (Takeda, 2010).

Role of HIF-1alpha in hypoxia-mediated apoptosis, cell proliferation, and tumor angiogenesis

As a result of hypoxia and nutrients, the growth and viability of cells is reduced. HIF-1alpha helps to restore oxygen homeostasis by inducing glycolysis, erythropoiesis and angiogenesis. Hypoxia and hypoglycaemia reduce proliferation and increase apoptosis in wild-type (HIF-1alpha+/+) embryonic stem (ES) cells, but not in ES cells with inactivated HIF-1alpha genes (HIF-1alpha-/-); however, a deficiency of HIF-1alpha does not affect apoptosis induced by cytokines. Hypoxia/hypoglycaemia-regulated genes involved in controlling the cell cycle are either HIF-1alpha-dependent (those encoding the proteins p53, p21, Bcl-2) or HIF-1alpha-independent (p27, GADD153), suggesting that there are at least two different adaptive responses to being deprived of oxygen and nutrients. Loss of HIF-1alpha reduces hypoxia-induced expression of vascular endothelial growth factor, prevents formation of large vessels in ES-derived tumors, and impairs vascular function, resulting in hypoxic microenvironments within the tumor mass. However, growth of HIF-1alpha tumors was not retarded but was accelerated, owing to decreased hypoxia-induced apoptosis and increased stress-induced proliferation. Because hypoxic stress contributes to many (patho)biological disorders, this new role for HIF-1alpha in hypoxic control of cell growth and death may be of general pathophysiological importance (Carmeliet, 1998).

HIF-1alpha interaction with transcriptional coactivators and covalent modification of the HIF C-terminal transactivation domain

HIF1alpha and its related factor, HLF, activate expression of a group of genes such as erythropoietin in response to low oxygen. Transfection analysis using fusion genes of GAL4DBD with various fragments of the two factors delineates two transcription activation domains which are inducible in response to hypoxia and are localized in the C-terminal half. Their sequences are conserved between HLF and HIF1alpha. One is designated NAD (N-terminal activation domain), while the other is CAD (C-terminal activation domain). Immunoblot analysis has revealed that NADs, which are rarely detectable at normoxia, became stabilized and accumulated at hypoxia, whereas CADs are constitutively expressed. In the mammalian two-hybrid system, CAD and NAD baits enhance the luciferase expression from a reporter gene by co-transfection with CREB-binding protein (CBP) prey, whereas CAD, but not NAD, enhances beta-galactosidase expression in yeast by CBP co-expression, suggesting that NAD and CAD interact with CBP/p300 by a different mechanism. Co-transfection experiments reveal that expression of Ref-1 and thioredoxin further enhance the luciferase activity expressed by CAD, but not by NAD. Amino acid replacement in the sequences of CADs reveal a specific cysteine to be essential for their hypoxia-inducible interaction with CBP. Nuclear translocation of thioredoxin from cytoplasm is observed upon reducing O2 concentrations (Ema, 1999).

Two members of the SRC-1/p160 family of transcriptional coactivators harboring histone acetyltransferase activity, SRC-1 and transcription intermediary factor 2 (TIF2), are able to interact with HIF-1alpha and enhance its transactivation potential in a hypoxia-dependent manner. HIF-1alpha contains within its C terminus two transactivation domains. The hypoxia-inducible activity of both these domains is enhanced by either SRC-1 or the CREB-binding protein (CBP)/p300 coactivator. Moreover, at limiting concentrations, SRC-1 produces this effect in synergy with CBP. Interestingly, this effect is strongly potentiated by the redox regulatory protein Ref-1, a dual-function protein harboring DNA repair endonuclease and cysteine reducing activities. These data indicate that all three proteins, CBP, SRC-1, and Ref-1, are important components of the hypoxia signaling pathway and have a common function in regulation of HIF-1alpha function in hypoxic cells. Given the absence of cysteine residues in one of the Ref-1-regulated transactivation domains of HIF-1alpha, it is thus possible that Ref-1 functions in hypoxic cells by targeting critical steps in the recruitment of the CBP-SRC-1 coactivator complex (Carrero, 2000).

The hypoxia-inducible factors (HIFs) 1alpha and 2alpha are key mammalian transcription factors that exhibit dramatic increases in both protein stability and intrinsic transcriptional potency during low-oxygen stress. This increased stability is due to the absence of proline hydroxylation, which in normoxia promotes binding of HIF to the von Hippel-Lindau (VHL tumor suppressor) ubiquitin ligase. Hypoxic induction of the COOH-terminal transactivation domain (CAD) of HIF occurs through abrogation of hydroxylation of a conserved asparagine in the CAD. Inhibitors of Fe(II)- and 2-oxoglutarate-dependent dioxygenases prevents hydroxylation of the Asn, thus allowing the CAD to interact with the p300 transcription coactivator. Replacement of the conserved Asn by Ala results in constitutive p300 interaction and strong transcriptional activity. Full induction of HIF-1alpha and -2alpha, therefore, relies on the abrogation of both Pro and Asn hydroxylation, which during normoxia occur at the degradation and COOH-terminal transactivation domains, respectively (Lando, 2002a).

Mammalian cells adapt to hypoxic conditions through a transcriptional response pathway mediated by the hypoxia-inducible factor, HIF. HIF transcriptional activity is suppressed under normoxic conditions by hydroxylation of an asparagine residue within its C-terminal transactivation domain, blocking association with coactivators. The protein FIH-1, previously shown to interact with HIF, is an asparaginyl hydroxylase. Like known hydroxylase enzymes, FIH-1 is an Fe(II)-dependent enzyme that uses molecular O2 to modify its substrate. Together with the recently discovered prolyl hydroxylases that regulate HIF stability, this class of oxygen-dependent enzymes comprises critical regulatory components of the hypoxic response pathway (Lando, 2002b).

Hypoxia-inducible factors (HIF) are a family of heterodimeric transcriptional regulators that play pivotal roles in the regulation of cellular utilization of oxygen and glucose and are essential transcriptional regulators of angiogenesis in solid tumor and ischemic disorders. The transactivation activity of HIF complexes requires the recruitment of p300/CREB-binding protein (CBP) by HIF-1 alpha and HIF-2 alpha that undergo oxygen-dependent degradation. HIF activation in tumors is caused by several factors including mitogen-activated protein kinase (MAPK) signaling. This study investigates the molecular basis for HIF activation by MAPK. MAPK is required for the transactivation activity of HIF-1 alpha. Furthermore, inhibition of MAPK disrupts the HIF-p300 interaction and suppresses the transactivation activity of p300. Overexpression of MEK1, an upstream MAPK activator, stimulates the transactivation of both p300 and HIF-1 alpha. Interestingly, the C-terminal transactivation domain of HIF-1 alpha is not a direct substrate of MAPK, and HIF-1 alpha phosphorylation is not required for HIF-CAD/p300 interaction. Taken together, these data suggest that MAPK signaling facilitates HIF activation through p300/CBP (Sang, 2003).

Targets of activity of HIF

The oxygen-regulated control system responsible for the induction of erythropoietin (Epo) by hypoxia is present in most (if not all) cells and operates on other genes, including those involved in energy metabolism. To understand the organization of cis-acting sequences that are responsible for oxygen-regulated gene expression, the 5' flanking region of the mouse gene encoding the hypoxically inducible enzyme lactate dehydrogenase A (LDH) was studied. Deletional and mutational analysis of the function of mouse LDH-reporter fusion gene constructs in transient transfection assays defined three domains, between -41 and -84 base pairs upstream of the transcription initiation site, that were crucial for oxygen-regulated expression. The most important of these, although not capable of driving hypoxic induction in isolation, has the consensus of a hypoxia-inducible factor 1 (HIF-1) site, and cross-competes for the binding of HIF-1 with functionally active Epo and phosphoglycerate kinase-1 sequences. The second domain is positioned close to the HIF-1 site, in an analogous position to one of the critical regions in the Epo 3' hypoxic enhancer. The third domain has the motif of a cAMP response element (CRE). Activation of cAMP by forskolin has no effect on the level of LDH mRNA in normoxia, but produces a magnified response to hypoxia that is dependent upon the integrity of the CRE, indicating an interaction between inducible factors binding the HIF-1 and CRE sites (Firth, 1995).

The vasopressin gene is expressed in the suprachiasmatic nucleus where the basic helix-loop-helix (bHLH)-PAS factors CLOCK and MOP3 regulate circadian expression through interactions with E-box sequences. Vasopressin gene regulation by HIF-1alpha, a bHLH-PAS factor involved in responses to hypoxia, has been studied. By transfecting Neuro-2A cells with 5' flanking regions of the vasopressin gene driving a luciferase reporter, it has been shown that CLOCK and HIF-1alpha cooperate in the induction of expression from 1000 bp and 350 bp of the vasopressin promoter but do not activate a 120-bp promoter fragment. The region between -191 and -128 contains an E-box A that appears to be essential for HIF-1alpha/CLOCK-mediated transcriptional activity. However, gel-shift analysis shows that the cooperative effect of HIF-1alpha and CLOCK results in MOP3 binding, but does not involve heterodimerization of HIF-1alpha/CLOCK, at E-box A. These data indicate that cross-talk between mediators of hypoxic and circadian pathways can regulate target genes (Ghorbel, 2003).

Bi-allelic-inactivating mutations of the VHL tumor suppressor gene are found in the majority of clear cell renal cell carcinomas (VHL-/- RCC). VHL-/- RCC cells overproduce hypoxia-inducible genes as a consequence of constitutive, oxygen-independent activation of hypoxia inducible factor (HIF). While HIF activation explains the highly vascularized nature of VHL loss lesions, the relative role of HIF in oncogenesis and loss of growth control remains unknown. HIF plays a central role in promoting unregulated growth of VHL-/- RCC cells by activating the transforming growth factor-alpha (TGF-alpha)/epidermal growth factor receptor (EGF-R) pathway. Dominant-negative HIF and enzymatic inhibition of EGF-R were equally efficient at abolishing EGF-R activation and serum-independent growth of VHL-/- RCC cells. TGF-alpha is the only known EGF-R ligand that has a VHL-dependent expression profile and its overexpression by VHL-/- RCC cells is a direct consequence of HIF activation. In contrast to TGF-alpha, other HIF targets, including vascular endothelial growth factor (VEGF), were unable to stimulate serum-independent growth of VHL-/- RCC cells. VHL-/- RCC cells expressing reintroduced type 2C mutants of VHL, and which retain the ability to degrade HIF, fail to overproduce TGF-alpha and proliferate in serum-free media. These data link HIF with the overproduction of a bona fide renal cell mitogen leading to activation of a pathway involved in growth of renal cancer cells. Moreover, these results suggest that HIF might be involved in oncogenesis to a much higher extent than previously appreciated (Gunaratnam, 2003).

The human endothelial nitric-oxide synthase gene (heNOS) is constitutively expressed in endothelial cells, and its expression is induced under hypoxia. The goal of this study was to search for regulatory elements of the endothelial nitric-oxide synthase (eNOS) gene responsive to hypoxia. Levels of eNOS mRNA, measured by real time reverse transcriptase-PCR analysis, are increased, and heNOS promoter activity is enhanced by hypoxia as compared with normoxia control experiments. Promoter truncation followed by footprint analysis allows the mapping and identification of the hypoxia-responsive elements at position -5375 to -5366, closely related to hypoxia-inducible factor (HIF)-responsive element (HRE). To test whether known HIF-1 and HIF-2 are involved in hypoxia-induced heNOS promoter activation, HMEC-1 and HUVEC were transiently transfected with HIF-1alpha and HIF-1beta or HIF-2alpha and HIF-1beta expression vectors. Exogenous HIF-2 markedly increases luciferase reporter activity driven by the heNOS promoter in its native location. The induction of luciferase was conserved with the antisense construct and was increased in cotransfection experiments when this fragment was cloned 5' to the proximal 785-bp fragment of the eNOS promoter. Deletion analysis and site-directed mutagenesis has demonstrated that the two contiguous HIF consensus binding sites spanning bp -5375 to -5366 relative to the transcription start site are both functional for heNOS promoter activity induction by hypoxia and by HIF-2 overexpression. In conclusion, heNOS is a hypoxia-inducible gene, whose transcription is stimulated through HIF-2 interaction with two contiguous HRE sites located at -5375 to -5366 of the heNOS promoter (Coulet, 2003).

Interactions between Ets family members and a variety of other transcription factors serve important functions during development and differentiation processes, e.g. in the hematopoietic system. The endothelial basic helix-loop-helix PAS domain transcription factor, hypoxia-inducible factor-2alpha (HIF-2alpha) (but not its close relative HIF-1alpha), cooperates with Ets-1 in activating transcription of the vascular endothelial growth factor receptor-2 (VEGF-2) gene (Flk-1). The receptor tyrosine kinase Flk-1 is indispensable for angiogenesis, and its expression is closely regulated during development. Consistent with the hypothesis that HIF-2alpha controls the expression of Flk-1 in vivo, HIF-2alpha and Flk-1 are co-regulated in postnatal mouse brain capillaries. A tandem HIF-2alpha/Ets binding site was identified within the Flk-1 promoter that acted as a strong enhancer element. Based on the analysis of transgenic mouse embryos, these motifs are essential for endothelial cell-specific reporter gene expression. A single HIF-2alpha/Ets element confers strong cooperative induction by HIF-2alpha and Ets-1 when fused to a heterologous promoter and is most active in endothelial cells. The physical interaction of HIF-2alpha with Ets-1 was demonstrated and localized to the HIF-2alpha carboxyl terminus and the autoinhibitory exon VII domain of Ets-1, respectively. The deletion of the DNA binding and carboxyl-terminal transactivation domains of HIF-2alpha, respectively, created dominant negative mutants that suppressed transactivation by the wild type protein and failed to synergize with Ets-1. These results suggest that the interaction between HIF-2alpha and endothelial Ets factors is required for the full transcriptional activation of Flk-1 in endothelial cells and may therefore represent a future target for the manipulation of angiogenesis (Elvert, 2003).

Hypoxia plays a key role in the pathophysiology of many disease states, and expression of the retinoic acid receptor-related orphan receptor alpha (RORalpha) gene increases under hypoxia. The mechanism for this transient hypoxia-induced increase in RORalpha expression was investigated. Reverse transcription-coupled PCR analysis revealed that the steady-state level of mRNA for the RORalpha4 isoform, but not the RORalpha1 isoform, increases in HepG2 cells after 3 h of hypoxia. Transient transfection studies showed that the hypoxia-induced increase in RORalpha4 mRNA occurs at the transcriptional level and is dependent on a hypoxia-responsive element (HRE) located downstream of the promoter. A dominant-negative mutant of hypoxia-inducible factor-1alpha (HIF-1alpha) abrogates the transcription activated by hypoxia as well as the transcription activated by exogenously expressed HIF-1alpha, demonstrating the direct involvement of HIF-1alpha in the transcriptional activation. However, HIF-1 alone is not sufficient to activate transcription in hypoxic conditions but, rather, required Sp1/Sp3, which binds to a cluster of GC-rich sequences adjacent to the HRE. Deletion of one or more of these GC boxes reduced or eliminated the HIF-1-dependent transcription. Together, these results suggest that the hypoxia-responsive region of the RORalpha4 promoter is composed of the HRE and GC-rich sequences and that the transcriptional activation under hypoxia is conferred through the cooperation of HIF-1 with Sp1/Sp3 (Miki, 2004).

HIF regulation of cell cycle

Hypoxia induces angiogenesis and glycolysis for cell growth and survival, and also leads to growth arrest and apoptosis. HIF-1alpha, a basic helix-loop-helix PAS transcription factor, acts as a master regulator of oxygen homeostasis by upregulating various genes under low oxygen tension. Although genetic studies have indicated the requirement of HIF-1alpha for hypoxia-induced growth arrest and activation of p21(cip1), a key cyclin-dependent kinase inhibitor controlling cell cycle checkpoint, the mechanism underlying p21(cip1) activation has been elusive. HIF-1alpha, even in the absence of hypoxic signal, induces cell cycle arrest by functionally counteracting Myc, thereby derepressing p21(cip1). The HIF-1alpha antagonism is mediated by displacing Myc binding from p21(cip1) promoter. Neither HIF-1alpha transcriptional activity nor its DNA binding is essential for cell cycle arrest, indicating a divergent role for HIF-1alpha. In keeping with its antagonism of Myc, HIF-1alpha also downregulates Myc-activated genes such as hTERT and BRCA1. Hence, it is propose that Myc is an integral part of a novel HIF-1alpha pathway, which regulates a distinct group of Myc target genes in response to hypoxia (Koshiji, 2004a).

In hypoxic cells, HIF-1alpha escapes from oxygen-dependent proteolysis and binds to the hypoxia-responsive element (HRE) for transcriptional activation of target genes involved in angiogenesis and glycolysis. The G1 checkpoint gene p21(cip1)is activated by HIF-1alpha with a novel mechanism that involves the HIF-1alpha PAS domains to displace Myc binding from p21(cip1) promoter. This HIF-1alpha-Myc pathway may account for up- and down-regulation of other hypoxia-responsive genes that lack the HRE. Moreover, the role of HIF-1alpha in cell cycle control indicates a dual, yet seemingly conflicting, nature of HIF-1alpha: promoting cell growth and arrest in concomitance. It is speculated that a dynamic balance between the two processes is achieved by a 'stop-and-go' strategy to maintain cell growth and survival. Tumor cells may adopt such a scheme to evade the killing by chemotherapeutic agents (Koshiji, 2004b).

Phenotypic effects of HIF overexpression

HIF-1alpha transactivates genes required for energy metabolism and tissue perfusion and is necessary for embryonic development and tumor explant growth. HIF-1alpha is overexpressed during carcinogenesis, myocardial infarction, and wound healing; however, the biological consequences of HIF-1alpha overexpression are unknown. Transgenic mice expressing constitutively active HIF-1alpha in epidermis display a 66% increase in dermal capillaries, a 13-fold elevation of total vascular endothelial growth factor (VEGF) expression, and a six- to ninefold induction of each VEGF isoform. Despite marked induction of hypervascularity, HIF-1alpha did not induce edema, inflammation, or vascular leakage, phenotypes developing in transgenic mice overexpressing VEGF cDNA in skin. Remarkably, blood vessel leakage resistance induced by HIF-1alpha overexpression was not caused by up-regulation of angiopoietin-1 or angiopoietin-2. Hypervascularity induced by HIF-1alpha could improve therapy of tissue ischemia (Datar, 2000).

A conserved family of prolyl-4-hydroxylases that modify HIF

Mammalian cells respond to changes in oxygen availability through a conserved pathway that is regulated by the hypoxia-inducible factor (HIF). The alpha subunit of HIF is targeted for degradation under normoxic conditions by a ubiquitin-ligase complex that recognizes a hydroxylated proline residue in HIF. A conserved family of HIF prolyl hydoxylase (HPH) enzymes has been identified that appears to be responsible for this posttranslational modification. In cultured mammalian cells, inappropriate accumulation of HIF caused by forced expression of the HIF-1alpha subunit under normoxic conditions was attenuated by coexpression of HPH. Suppression of HPH in cultured Drosophila melanogaster cells by RNA interference resulted in elevated expression of a hypoxia-inducible gene (LDH, encoding lactate dehydrogenase) under normoxic conditions. These findings indicate that HPH is an essential component of the pathway through which cells sense oxygen (Bruick, 2001).

This study examined the role of HPH in Drosophila. A single HPH gene was identified in the Drosophila genome (gene CG1114), hereby designated dmHPH. Drosophila homologs of HIF-1alpha (Sima), ARNT (dARNT), and pVHL (dVHL; a constituent of a protein-ubiquitin ligase complex containing the product of the von Hippel Lindau tumor suppressor protein) have been previously identified and the activity and stability of Sima have been shown to be regulated by oxygen. Together, these observations suggest that Drosophila has a hypoxic response pathway analogous to that in mammalian cells. Double-stranded RNAs corresponding to Sima or dmHPH with the KC167 cell line derived from Drosophila embryos to eliminate expression of these genes by RNA interference (RNAi). After incubation under normoxic conditions, total RNA was prepared from the cells and examined by Northern blotting. These results confirmed that RNAi substantially reduces the levels of mRNAs encoding Sima or dmHPH (Bruick, 2001).

To determine the effects of the partial loss of function of these gene products, the expression of the gene encoding lactate dehydrogenase (dmLDH), which is a HIF-dependent hypoxia-inducible gene in mammalian cells, was examined. Untreated KC167 cells incubated in 1% O2 for 15 hours accumulated 7.5 times more dmLDH mRNA than cells maintained under normoxic conditions. KC167 cells treated with double-stranded Sima RNA showed a twofold reduction in dmLDH mRNA levels. By contrast, RNAi-mediated reduction of dmHPH mRNA resulted in a 2.5-fold increase in dmLDH mRNA levels under normoxic conditions. These data suggest that the HPH enzymes are bona fide HIF prolyl hydroxylases and act as integral regulators of the HIF-dependent hypoxia response pathway (Bruick, 2001).

Hypoxia-inducible factor-dependent histone deacetylase activity determines stem cell fate in the placenta

Hypoxia-inducible factor (HIF) is a heterodimeric transcription factor composed of HIFalpha and the arylhydrocarbon receptor nuclear translocator (ARNT/HIF1ß). ARNT function is required for murine placental development. Cultured trophoblast stem (TS) cells were used to investigate the molecular basis of this requirement. In vitro, wild-type TS cell differentiation is largely restricted to spongiotrophoblasts and giant cells. Interestingly, Arnt-null TS cells differentiate into chorionic trophoblasts and syncytiotrophoblasts, as demonstrated by their expression of Tfeb, glial cells missing 1 (Gcm1) and the HIV receptor CXCR4. During this process, a region of the differentiating Arnt-null TS cells undergo granzyme B-mediated apoptosis, suggesting a role for this pathway in murine syncytiotrophoblast turnover. Surprisingly, HIF1alpha and HIF2alpha are induced during TS cell differentiation in 20% O2; additionally, pVHL levels are modulated during the same time period. These results suggest that oxygen-independent HIF functions are crucial to this differentiation process. Since histone deacetylase (HDAC) activity has been linked to HIF-dependent gene expression, whether ARNT deficiency affects this epigenetic regulator was investigated. Interestingly, Arnt-null TS cells have reduced HDAC activity, increased global histone acetylation, and altered class II HDAC subcellular localization. In wild-type TS cells, inhibition of HDAC activity recapitulates the Arnt-null phenotype, suggesting that crosstalk between the HIFs and the HDACs is required for normal trophoblast differentiation. Thus, the HIFs play important roles in modulating the developmental plasticity of stem cells by integrating physiological, transcriptional and epigenetic inputs (Maltepe, 2005).

The transcription factor HIF-1 plays a critical role in the growth factor-dependent regulation of both aerobic and anaerobic glycolysis

Mammalian cells are believed to have a cell-intrinsic ability to increase glucose metabolism in response to hypoxia. The ability of hematopoietic cells to up-regulate anaerobic glycolysis in response to hypoxia is dependent on receptor-mediated signal transduction. In the absence of growth factor signaling, hematopoietic cells fail to express hypoxia-inducible transcription factor (Hif-1α) mRNA. Growth factor-deprived hematopoietic cells do not engage in glucose-dependent anabolic synthesis and neither express Hif-1α mRNA nor require HIF-1α protein to regulate cell survival in response to hypoxia. However, HIF-1α is adaptive for the survival of growth factor-stimulated cells; suppression of HIF-1α results in death when growing cells are exposed to hypoxia. Growth factor-dependent HIF-1α expression reprograms the intracellular fate of glucose, resulting in decreased glucose-dependent anabolic synthesis and increased lactate production, an effect that is enhanced when HIF-1α protein is stabilized by hypoxia. Together, these data suggest that HIF-1α contributes to the regulation of growth factor-stimulated glucose metabolism even in the absence of hypoxia (Lum, 2007).

Increased adipocyte O2 consumption triggers HIF-1alpha, causing inflammation and insulin resistance in obesity

Adipose tissue hypoxia and inflammation have been causally implicated in obesity-induced insulin resistance. This study reports that, early in the course of high-fat diet (HFD) feeding and obesity, adipocyte respiration becomes uncoupled, leading to increased oxygen consumption and a state of relative adipocyte hypoxia. These events are sufficient to trigger HIF-1alpha induction, setting off the chronic adipose tissue inflammatory response characteristic of obesity. At the molecular level, these events involve saturated fatty acid stimulation of the adenine nucleotide translocase 2 (ANT2), an inner mitochondrial membrane protein, which leads to the uncoupled respiratory state. Genetic or pharmacologic inhibition of either ANT2 or HIF-1alpha can prevent or reverse these pathophysiologic events, restoring a state of insulin sensitivity and glucose tolerance. These results reveal the sequential series of events in obesity-induced inflammation and insulin resistance (Lee, 2014).

HIF1alpha regulation of Sox9 is necessary to maintain differentiation of hypoxic prechondrogenic cells during early skeletogenesis

During early stages of limb development, the vasculature is subjected to extensive remodeling that leaves the prechondrogenic condensation avascular and hypoxic. Numerous studies on a variety of cell types have reported that hypoxia has an inhibitory effect on cell differentiation. In order to investigate the mechanism that supports chondrocyte differentiation under hypoxic conditions, the transcription factor hypoxia-inducible factor 1α) was inactivated in mouse limb bud mesenchyme. Developmental analysis of Hif1α-depleted limbs revealed abnormal cartilage and joint formation in the autopod, suggesting that HIF1α is part of a mechanism that regulates the differentiation of hypoxic prechondrogenic cells. Dramatically reduced cartilage formation in Hif1α-depleted micromass culture cells under hypoxia provided further support for the regulatory role of HIF1α in chondrogenesis. Reduced expression of Sox9, a key regulator of chondrocyte differentiation, followed by reduction of Sox6, collagen type II and aggrecan in Hif1α-depleted limbs raised the possibility that HIF1α regulation of Sox9 is necessary under hypoxic conditions for differentiation of prechondrogenic cells to chondrocytes. To study this possibility, Hif1α micromass cultures were targeted. Under hypoxic conditions, Sox9 expression was increased twofold relative to its expression in normoxic condition; this increment was lost in the Hif1α-depleted cells. Chromatin immunoprecipitation demonstrated direct binding of HIF1α to the Sox9 promoter, thus supporting direct regulation of HIF1α on Sox9 expression. This work establishes for the first time HIF1α as a key component in the genetic program that regulates chondrogenesis by regulating Sox9 expression in hypoxic prechondrogenic cells (Amarilio, 2007).

PTEN deficiency causes dyschondroplasia in mice by enhanced hypoxia-inducible factor 1alpha signaling and endoplasmic reticulum stress

Chondrocytes within the growth plates acclimatize themselves to a variety of stresses that might otherwise disturb cell fate. The tumor suppressor PTEN has been implicated in the maintenance of cell homeostasis. However, the functions of PTEN in regulating chondrocytic adaptation to stresses remain largely unknown. This study created chondrocyte-specific Pten knockout mice (Ptenco/co;Col2a1-Cre) using the Cre-loxP system. Following AKT activation, Pten mutant mice exhibit dyschondroplasia resembling human enchondroma. Cartilaginous nodules originate from Pten mutant resting chondrocytes that suffer from impaired proliferation and differentiation, and this is coupled with enhanced endoplasmic reticulum (ER) stress. It was further found that ER stress in Pten mutant chondrocytes only occurs under hypoxic stress, characterized by an upregulation of unfolded protein response-related genes as well as an engorged and fragmented ER in which collagens are trapped. An upregulation of hypoxia-inducible factor 1alpha (HIF1alpha) and downstream targets followed by ER stress induction was also observed in Pten mutant growth plates and in cultured chondrocytes, suggesting that PI3K/AKT signaling modulates chondrocytic adaptation to hypoxic stress via regulation of the HIF1alpha pathway. These data demonstrate that PTEN function in chondrocytes is essential for their adaptation to stresses and for the inhibition of dyschondroplasia (Yang, 2008).

HIF and neural stem cells

Insufficient oxygen and nutrient supply often restrain solid tumor growth, and the hypoxia-inducible factors (HIF) 1 alpha and HIF-2 alpha are key transcription regulators of phenotypic adaptation to low oxygen levels. Moreover, mouse gene disruption studies have implicated HIF-2 alpha in embryonic regulation of tyrosine hydroxylase, a hallmark gene of the sympathetic nervous system. Neuroblastoma tumors originate from immature sympathetic cells, and therefore the effect of hypoxia on the differentiation status of human neuroblastoma cells was investigated. Hypoxia stabilizes HIF-1 alpha and HIF-2 alpha proteins and activates the expression of known hypoxia-induced genes, such as vascular endothelial growth factor and tyrosine hydroxylase. These changes in gene expression also occur in hypoxic regions of experimental neuroblastoma xenografts grown in mice. In contrast, hypoxia decreases the expression of several neuronal/neuroendocrine marker genes but induces genes expressed in neural crest sympathetic progenitors, for instance c-kit and Notch-1. Thus, hypoxia apparently causes dedifferentiation both in vitro and in vivo. These findings suggest a novel mechanism for selection of highly malignant tumor cells with stem-cell characteristics (Jogi, 2002).

HIF-alpha and tumor angiogenesis

Reactive oxygen species (ROS) are implicated in the pathophysiology of various diseases, including cancer. In this study, JunD, a member of the AP-1 family of transcription factors, is shown to reduce tumor angiogenesis by limiting Ras-mediated production of ROS. Using junD-deficient cells, JunD is demonstrated to regulate genes involved in antioxidant defense, H2O2 production, and angiogenesis. The accumulation of H2O2 in junD-/- cells decreases the availability of FeII and reduces the activity of HIF prolyl hydroxylases (PHDs) that target hypoxia-inducible factors-alpha (HIFalpha) for degradation. Subsequently, HIF-alpha proteins accumulate and enhance the transcription of VEGF-A, a potent proangiogenic factor. This study uncovers the mechanism by which JunD protects cells from oxidative stress and exerts an antiangiogenic effect. Furthermore, new insights are provided into the regulation of PHD activity, allowing immediate reactive adaptation to changes in O2 or iron levels in the cell (Gerald, 2004).

Production of ROS and hypoxic response are key players in the occurrence and progression of cancers. The junD-/- adult mice do not develop tumors spontaneously, suggesting that the protective effect of JunD may only be uncovered under stress conditions. A protective effect of JunD has been demonstrated in cells transformed by the Ras oncogene, one of the most frequently mutated oncogenes in human cancers. Ras-mediated transformation enhances ROS production, and treatment with antioxidant molecules decreases the proliferation rate of Ras-transformed cell lines. JunD has been shown to antagonize Ras-mediated transformation by modulating cell proliferation. The present study shows that overexpression of JunD decreases ROS production in Ras-transformed cells. Thus, it is proposed that the inhibitory effect of JunD on the proliferation of Ras-transformed cell lines is mediated in part through the decreased level of ROS. Moreover, Ras oncogene contributes to the growth of solid tumors by a direct effect on cell proliferation and by facilitating tumor angiogenesis. Indeed, transformation by Ras stabilizes HIF-1α and upregulates VEGF-A expression as well as other HIF target genes. Furthermore, Ras-induced stabilization of HIF-1α is mediated through inhibition of HIF hydroxylation. The data argue that ROS accumulation in Ras-transformed cells triggers PHD inhibition. JunD has a major effect on this process. Indeed, JunD decreases ROS production, restores PHD activity, and subsequently reduces significantly Ras-dependent tumor angiogenesis in vivo. Thus, JunD displays a protective role against Ras-mediated transformation by buffering cells to maintain the redox balance (Gerald, 2004).

The hypoxia-responsive transcription factor HIF-1alpha was deleted in endothelial cells (EC) to determine its role during neovascularization. It was found that loss of HIF-1alpha inhibits a number of important parameters of EC behavior during angiogenesis: these include proliferation, chemotaxis, extracellular matrix penetration, and wound healing. Most strikingly, loss of HIF-1alpha in EC results in a profound inhibition of blood vessel growth in solid tumors. These phenomena are all linked to a decreased level of VEGF expression and loss of autocrine response of VEGFR-2 in HIF-1alpha null EC. It is thus shown that a HIF-1alpha-driven, VEGF-mediated autocrine loop in EC is an essential component of solid tumor angiogenesis (Tang, 2004).


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