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).
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).
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).
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).
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. 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).
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).
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 is 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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.
The Interactive Fly resides on the
Society for Developmental Biology's Web server.