Hypoxia-inducible factor 1 (HIF-1), and Arnt-containing, heterodimeric basic helix-loop-helix transcription factor that regulates hypoxia-inducible genes

In response to hypoxia, the hypoxia-inducible factor-1 (HIF-1) mediates transcriptional activation of anetwork of genes encoding erythropoietin, vascular endothelial growth factor, and several glycolyticenzymes. HIF-1 consists of a heterodimer of two basic helix-loop-helix PAS (Per/Arnt/Sim) proteins,HIF-1alpha (Drosophila homolog: Similar) and Arnt. HIF-1alpha and Arnt mRNAs are constitutively expressed and are not alteredupon exposure of HeLa or HepG2 cells to hypoxia, suggesting that the activity of the HIF-1alpha-Arntcomplex may be regulated by some as yet unknown posttranscriptional mechanism. In support of thismodel, it has been demonstrated that Arnt protein levels are not increased under conditions that inducean hypoxic response in HeLa and HepG2 cells. However, under identical conditions, HIF-1alphaprotein levels are rapidly and dramatically up-regulated, as assessed by immunoblot analysis. Inaddition, HIF-1alpha acquires a new conformational state upon dimerization with Arnt, renderingHIF-1alpha more resistant to proteolytic digestion in vitro. Dimerization as such is not sufficient toelicit the conformational change in HIF-1alpha, since truncated forms of Arnt that are capable ofdimerizing with HIF-1alpha do not induce this effect. The high affinity DNA binding form ofthe HIF-1alpha-Arnt complex is only generated by forms of Arnt capable of eliciting the allostericchange in conformation. In conclusion, the combination of enhanced protein levels and allostericchange by dimerization defines a novel mechanism for modulation of transcription factor activity (Kallio, 1997).

Hypoxia-inducible factor-1 (HIF-1), a DNA-binding complex implicated in the regulation of geneexpression by oxygen, has been shown to consist of a heterodimer of two basic helix-loop-helix PAS proteins, HIF-1alpha, and HIF-1beta. One partner, HIF-1beta, is the aryl hydrocarbon receptor nuclear translocator (Arnt), an essential component of the xenobiotic response. In the present work, Arnt-deficient mutant cells, originally derived from the mouse hepatoma line Hepa1c1c7, have been used to analyze the role ofArnt/HIF-1beta in oxygen-regulated gene expression. Two stimuli were examined: hypoxia itself anddesferrioxamine, an iron-chelating agent that also activates HIF-1. Induction of the DNA binding andtranscriptional activity of HIF-1 is absent in the mutant cells, indicating an essential role forArnt/HIF-1beta. Analysis of deleted Arnt/HIF-1beta genes indicates that the basic,helix-loop-helix, and PAS domains, but not the amino or carboxyl termini, are necessary for functionin the response to hypoxia. Comparison of gene expression in wild type and mutant cells demonstratesthe critical importance of Arnt/HIF-1beta in the hypoxic induction of a wide variety of genes.Nevertheless, for some genes a reduced response to hypoxia and desferrioxamine persists in thesemutant cells, clearly distinguishing Arnt/HIF-1beta-dependent and Arnt/HIF-1beta-independentmechanisms of gene activation by both these stimuli (Wood, 1996).

Hypoxia-inducible factor 1 (HIF-1) is a DNA-binding heterodimeric protein complex originallydescribed in the transcriptional activation of the erythropoietin gene by hypoxia. This protein complex iscomposed of two subunits, HIF-1alpha and HIF-1beta (synonymous with aryl hydrocarbon receptor nuclear translocator, Arnt). In this study, Arnt-deficient cells, derived from the mouse hepatoma cell lineHepa1c1c7, were used to further characterize HIF-1 complex formation and its relationship with gene activation by hypoxia and desferrioxamine (Df). Gel shift assays reveal that Arnt is absolutely required forthe formation of the HIF-1 DNA-binding complex. Results from RNase protection assays andNorthern blots show that the lack of functional HIF-1 complex completely abrogates the response tohypoxia of three genes, vascular endothelial growth factor (VEGF), the glycolytic enzymes aldolase A (ALDA),and phosphoglycerate kinase 1 (PGK-1), each of which is known to be upregulated by low oxygen tension.Desferrioxamine induction of VEGF and PGK-1 genes is reduced in the Arnt-deficient cells, butunlike the response to hypoxia, the induction is not completely suppressed. These results suggest that Df is able toactivate gene transcription through HIF-1-independent mechanisms. Exposure to either hypoxia or Df does notinduce any changes in HIF-1alpha and -1beta mRNA levels, suggesting that posttranscriptionalmechanisms are involved in HIF-1 complex activation (Salceda, 1996).

Hypoxia-inducible factor 1 (HIF-1) is a heterodimeric basic helix-loop-helix transcription factor thatregulates hypoxia-inducible genes, including the human erythropoietin (EPO) gene. Expression of vascular endothelial growth factor (VEGF) is induced in cells exposed to hypoxia orischemia. Neovascularization stimulated by VEGF occurs in several important clinical contexts,including myocardial ischemia, retinal disease, and tumor growth. Hypoxia-inducible factor 1 (HIF-1) isa heterodimeric basic helix-loop-helix protein that activates transcription of the human erythropoietingene in hypoxic cells. HIF-1 is involved in the activation of VEGFtranscription. VEGF 5'-flanking sequences mediate transcriptional activation of reporter geneexpression in hypoxic Hep3B cells. A 47-bp sequence located 985 to 939 bp 5' to the VEGFtranscription initiation site mediates hypoxia-inducible reporter gene expression directed by a simianvirus 40 promoter element, which is otherwise minimally responsive to hypoxia. When reporterscontaining VEGF sequences, in the context of the native VEGF or heterologous simian virus 40promoter, are cotransfected with expression vectors encoding HIF-1alpha and Arnt, reporter gene transcription is much greater in bothhypoxic and nonhypoxic cells than in cells transfected with the reporter alone. A HIF-1 binding siteis present in the 47-bp hypoxia response element; a 3-bp substitution eliminates the abilityof the element to bind HIF-1 and to activate transcription in response to hypoxia and/or recombinantHIF-1. Cotransfection of cells with an expression vector encoding a dominant negative form ofHIF-1alpha inhibits the activation of reporter transcription in hypoxic cells in a dose-dependentmanner. VEGF mRNA is not induced by hypoxia in mutant cells that do not express the Arnt subunit. These findings implicate HIF-1 in the activation of VEGF transcription in hypoxiccells (Forsythe, 1996).

Hypoxia-inducible factor 1 alpha (HIF-1 alpha) and the intracellular dioxin receptor mediate hypoxiaand dioxin signaling, respectively. Both proteins are conditionally regulated basic helix-loop-helix(bHLH) transcription factors that, in addition to the bHLH motif, share a Per-Arnt-Sim (PAS) regionof homology and form heterodimeric complexes with the common bHLH/PAS partner factor Arnt.HIF-1 alpha requires Arnt for DNA binding in vitro and functional activityin vivo. Both the bHLH and PAS motifs of Arnt are critical for dimerization with HIF-1 alpha.Strikingly, HIF-1 alpha exhibits very high affinity for Arnt in coimmunoprecipitation assays in vitro,resulting in competition with the ligand-activated dioxin receptor for recruitment of Arnt. Consistentwith these observations, activation of HIF-1 alpha function in vivo or overexpression of HIF-1 alphainhibits ligand-dependent induction of DNA binding activity by the dioxin receptor and dioxin receptorfunction on minimal reporter gene constructs. However, HIF-1 alpha- and dioxin receptor-mediatedsignaling pathways are not mutually exclusive, since activation of dioxin receptor function does notimpair HIF-1 alpha-dependent induction of target gene expression. Both HIF-1 alpha and Arnt mRNAsare expressed constitutively in a large number of human tissues and cell lines, and these steady-stateexpression levels are not affected by exposure to hypoxia. Thus, HIF-1 alpha may be conditionallyregulated by a mechanism that is distinct from induced expression levels, the prevalent model ofactivation of HIF-1 alpha function. HIF-1 alpha is associated withthe molecular chaperone hsp90. Given the critical role of hsp90 for ligand binding activity and activationof the dioxin receptor, it is therefore possible that HIF-1 alpha is regulated by a similar mechanism,possibly by binding to an as yet unknown class of ligands (Gradin, 1996).

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).

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

Hypoxia-inducible factor (HIF) is a heterodimeric transcription factorcomposed of HIFalpha and the arylhydrocarbon receptor nuclear translocator(ARNT/HIF1ß). ARNT function is requiredfor murine placental development. Cultured trophoblast stem (TS)cells were used to investigate the molecular basis of this requirement. Invitro, wild-type TS cell differentiation is largely restricted tospongiotrophoblasts and giant cells. Interestingly, Arnt-null TScells differentiate into chorionic trophoblasts and syncytiotrophoblasts, asdemonstrated by their expression of Tfeb, glial cells missing 1 (Gcm1) and theHIV receptor CXCR4. During this process, a region of the differentiatingArnt-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 celldifferentiation in 20% O2; additionally, pVHL levels are modulatedduring the same time period. These results suggest that oxygen-independent HIFfunctions 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 subcellularlocalization. In wild-type TS cells, inhibition of HDAC activity recapitulatesthe Arnt-null phenotype, suggesting that crosstalk between the HIFsand the HDACs is required for normal trophoblast differentiation. Thus, theHIFs play important roles in modulating the developmental plasticity of stemcells by integrating physiological, transcriptional and epigenetic inputs (Maltepe, 2005).

Characterization of murine ARNT interacting protein (AINT)

Basic helix-loop-helix-PER-ARNT-SIM (bHLH-PAS) proteins form dimeric transcription factors to mediate diverse biological functions including xenobiotic metabolism, hypoxic response, circadian rhythm and central nervous system midline development. The Ah receptor nuclear translocator protein (ARNT) plays a central role as a common heterodimerization partner. A novel, embryonically expressed, ARNT interacting protein (AINT) is described that may be a member of a larger coiled-coil PAS interacting protein family. The AINT C-terminus mediates interaction with the PAS domain of ARNT in yeast and interacts in vitro with ARNT and ARNT2 specifically. AINTlocalizes to the cytoplasm and overexpression leads to non-nuclear localization of ARNT. A dynamic pattern of AINT mRNA expression during embryogenesis and cerebellum ontogeny supports a role for AINT in development (Sadek, 2000).

The AINT C-terminus is homologous to two human proteins, TACC1 andTACC2. TACC1 (accession no. AF049910) is embryonically expressed and constitutive expression results in transformation and anchorage independent growth of mousefibroblasts. The TACC2 (accession no.AF095791) gene is located on chromosome 10 and has yetto be described further. Three additional TACC-related proteins have been described. The coiled-coil region of Xenopus maskin is most closely related to AINT andTACC3 and interacts with CPEB and eIF-4E to restrictpolyadenylation-induced translation during oocyte matura-tion. Drosophila TACC (D-TACC) has been shown to interact with and stabilize centrosomal microtubules in embryo extracts via the C-terminalregion (Gergely, 2000). Finally, AZU-1, a variant ofTACC2, is implicated in tumor suppression and tissuemorphogenesis of epithelial cells. A C. elegans sequence cloned from chromosomeIII (accession no. Q9XWJ0) also appears to encode theconserved coiled-coil domain of TACC proteins althoughfunctionally this has not been characterized. To comparethis growing protein family and determine importantconserved sequences in the coiled-coil domains, the C-terminal ends of each member were aligned anda phylogenetic tree was constucted based on this conservedregion. The existing TACC proteins can be aligned into two distinct clades, one consisting of TACC1 and TACC2 proteins and a second containing TACC3proteins, to which AINT belongs. With the sudden growthin TACC family members it will be interesting to see iffuture members will resolve the first clade into two separategroups for TACC1 and TACC2 proteins (Sadek, 2000).

AINT is not a bHLH-PAS protein. Many recent studies inthe field of bHLH-PAS proteins have served to identify novelfamily members and most mechanisms described thus far areexamples of heterodimerization of two bHLH-PAS proteinsvia the PAS domain. However, it has become increasinglyapparent that PAS proteins also interact heterotypically withnon-PAS proteins such as AINT. Examples of this type ofinteraction may be seen at different stages of the signal transduction pathway. Early in the transduction pathway the AhR interacts with HSP90 and AIP/ARA9/XAP in its non-liganded state, perhaps to maintain a conformation that recognizes ligand. The Drosophila PAS protein Period (Per) forms heterodimers with thecircadian clock protein Timeless (Tim), which does notcontain a PAS domain. This heterodimerization is importantfor the regulation of circadian rhythms and is mediated by thePAS domain of Per. In the later stages of signal transduction,HIF-1alpha actively recruits CBP to the transcriptional complex. In addition, SRC-1, SRC-2, and SRC-3 have not been widely studied in the context of PAS interactions and, instead, have been shown to bridge interactionsbetween nuclear receptors and basal transcription machineryvia conserved LXXLL motifs termed nuclear receptor boxes. Fromthese many examples, it might not be unusual to haveisolated a non-bHLH-PAS protein in the screening forARNT interacting proteins. Of the heterotypic interactions mentioned above, thePer-Tim interaction may be most relevant in the contextof AINT. In the same way that Tim interacts with the PASdomain of Per, evidence is presented that AINT requires an intact PAS domain of ARNT for interaction. Because the PAS domain provides the dimerization surface between many proteins, this could be a clue to the function of AINT in either enhancing or disrupting homotypic interactions between ARNT and other PAS proteins (Sadek, 2000).

Hypoxia requires notch signaling to maintain the undifferentiated cell state

In addition to controlling a switch to glycolytic metabolism and induction of erythropoiesis and angiogenesis, hypoxia promotes the undifferentiated cell state in various stem and precursor cell populations. The latter process requires Notch signaling. Hypoxia blocks neuronal and myogenic differentiation in a Notch-dependent manner. Hypoxia activates Notch-responsive promoters and increases expression of Notch direct downstream genes. The Notch intracellular domain (ICD) interacts with HIF-1alpha, a global regulator of oxygen homeostasis, and HIF-1alpha is recruited to Notch-responsive promoters upon Notch activation under hypoxic conditions. Taken together, these data provide molecular insights into how reduced oxygen levels control the cellular differentiation status and demonstrate a role for Notch in this process (Gustafsson, 2005).

The data presented here indicate that Notch ICD and HIF-1α are important at the convergence point between the two signaling mechanisms. The importance of Notch ICD is underlined by the ability of γ-secretase inhibitors, which block the S3 cleavage of the Notch receptor and thus liberation of Notch ICD, to strongly reduce the hypoxic response on Notch downstream genes and promoters. Furthermore, the signaling output from an exogenously introduced Notch 1 ICD was modified by hypoxia, leading to increased activation of 12XCSL-luc and Hes-luc in a Notch 1 ICD-dependent manner. The importance of HIF-1α in this process receives support from the observed direct physical interaction between HIF-1α and Notch 1 ICD, the lack of an hypoxia-induced effect on Notch signaling in fibroblasts devoid of HIF-1α, and that both the amount and activity status of HIF-1α correlate with the level of Notch activation. The latter notion is based on the observations that: (1) transfected HIF-1α elevates the Notch downstream response; (2) a transactivation-inactive form of HIF-1α leaves the Notch response unchanged, and (3) the response is augmented in cells lacking VHL. Finally, HIF-1α is recruited to the Hey-2 promoter in C2C12 cells in a Notch- and hypoxia-dependent manner. This suggests a mechanism involving an effect of direct transcriptional activation of a Notch-responsive promoter by HIF-1α, probably as part of a Notch ICD/CSL transcriptional complex. This model is consistent with the observation that a transcriptionally inactive form of HIF-1α, which was capable of interacting with Notch 1 ICD, does not augment the Notch downstream response. It is therefore reasoned that hypoxia-dependent stabilization of Notch 1CD is not sufficient for activation of the Notch response but may require the recruitment of a form of HIF-1α containing the C-terminal transactivation domain to the Notch ICD/CSL regulatory complex, possibly potentiating the interaction with transcriptional coactivators. This hypothesis is based on the finding that HIF-1α is recruited to promoters of Notch downstream genes and the observation that a mutated form of Notch ICD, unable to interact with CBP/p300, is transcriptionally active at hypoxia. In this context, it will be interesting to learn whether HIF-1β also participates in such a regulatory complex (Gustafsson, 2005).

The link between hypoxia and Notch described here may have ramifications for other aspects of hypoxia, such as tumor development, in which deregulation of both HIF-1α- and Notch-mediated signaling events have been implicated. Since many tumors show elevated expression of HIF-1α, caused by hypoxia inherent to growing tumors and/or genetic loss of VHL, it will be interesting to investigate whether the elevated levels of HIF-1α are paralleled by increased Notch signaling, and whether the ensuing Notch induction contributes to tumor development (Gustafsson, 2005).

In conclusion, the data presented here demonstrate a link between hypoxia and Notch signaling and provide insights into how hypoxia maintains the undifferentiated cell state, by using the Notch signaling mechanism. The data also point to an important role for HIF-1α in this process and to the fact that it can interact with the Notch intracellular domain to link hypoxic information to a Notch response. These data advance the understanding of how Notch crosstalks with other signaling mechanisms and may open up possibilities to control various aspects of the hypoxic response by experimentally manipulating Notch signaling (Gustafsson, 2005).

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tango: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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