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).
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 (gene CG1114) (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).
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).
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 organismís 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: 5í B(A/G)CGTGVBBB 3í (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), Drosophilaís 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 Drosophilaís 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 Simaís partner protein Tango (Gorr, 2004).
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).
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).
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