Despite the pivotal role the hypoxia-inducible factor-1α (HIF-1α) plays in physiological and pathological processes, little is known regarding the timeframe and mechanisms involved in its regulation. We determined the onset, accumulation, and degradation of HIF-1α and a number of redox-sensitive nuclear factors over a range of pathophysiological oxygen concentrations. Experiments were carried out on nonadherent human HeLaS3 cells placed in tonometers to achieve rapid equilibration between the cell suspension and the various hypoxic/reoxygenation conditions. Exposure to hypoxia for less than 2 min already revealed nuclear HIF-1α protein induction on Western blots and HIF-1 DNA binding in EMSAs. One hour after anoxic/hypoxic exposure, nuclear HIF-1α proteins reached maximal levels, which were maintained for 4 h. Reoxygenation reduced HIF-1 DNA binding within 2 min, and nuclear HIF-1α protein levels within 4 to 8 min, down to a level below the detection limit within 32 min. Western blot analysis of the redox sensitive nuclear factors NF-κB, c-Fos, c-Jun, Ref-1, and thioredoxin showed no alteration in their nuclear levels in response to anoxia/hypoxia, but reoxygenation rapidly caused a transient increase in nuclear NF-κB and thioredoxin protein levels. The instant initiation of HIF-1α accumulation shown here limits the hypoxic signaling pathway to below 2 min.Key words: anoxia • reoxygenation • redox factor • ROS issue hypoxia occurs during physiological and pathological processes such as embryogenesis, tumorigenesis, transplantation, and wounding and has recently attracted attention as a target for gene therapy since its correlation with tumor malignancy (1). The hypoxia-inducible factor-1 (HIF-1) is the molecular key player in the hypoxic response: It induces specific expression of several hypoxically regulated genes, such as vascular endothelial growth factor, transferrin, and glycolytic enzymes, and thereby restores blood supply and energy availability to the hypoxic tissue. HIF-1 is a heterodimeric transcription factor consisting of HIF-1α and HIF-1β/ARNT (aryl hydrocarbon receptor nuclear translocator). Both ubiquitously expressed subunits are efficiently translated under normoxic and hypoxic conditions (2), they belong to the Per-ARNT/AhR-Sim subfamily of basic helix-loop-helix transcription factors (3), and they are capable of nuclear translocation (4). Inside the nucleus, the two subunits dimerize to form the HIF-1 complex, which binds to the conserved consensus sequence (A)CGTG within the hypoxia response element present in oxygen-regulated target genes (5) (personal T communications, Camenisch G. et al.). The DNA-binding domain of HIF-1α lies within the Nterminal region of the protein, whereas the C-terminal region holds the two transactivation domains. The central region of HIF-1α contains an oxygen-dependent degradation (ODD) domain located between amino acids 401 and 603 (6), which confers oxygen-sensitivity to the HIF-1α subunit. As a result, HIF-1α is rapidly degraded in normoxic conditions. Degr...

Hypoxia-inducible factor 1 alpha (HIF-1 alpha) and the intracellular dioxin receptor mediate hypoxia and dioxin signalling, 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) region of homology and form heterodimeric complexes with the common bHLH/PAS partner factor Arnt. Here we demonstrate that HIF-1 alpha required Arnt for DNA binding in vitro and functional activity in vivo. Both the bHLH and PAS motifs of Arnt were critical for dimerization with HIF-1 alpha. Strikingly, HIF-1 alpha exhibited very high affinity for Arnt in coimmunoprecipitation assays in vitro, resulting in competition with the ligand-activated dioxin receptor for recruitment of Arnt. Consistent with these observations, activation of HIF-1 alpha function in vivo or overexpression of HIF-1 alpha inhibited ligand-dependent induction of DNA binding activity by the dioxin receptor and dioxin receptor function on minimal reporter gene constructs. However, HIF-1 alpha- and dioxin receptor-mediated signalling pathways were not mutually exclusive, since activation of dioxin receptor function did not impair HIF-1 alpha-dependent induction of target gene expression. Both HIF-1 alpha and Arnt mRNAs were expressed constitutively in a large number of human tissues and cell lines, and these steady-state expression levels were not affected by exposure to hypoxia. Thus, HIF-1 alpha may be conditionally regulated by a mechanism that is distinct from induced expression levels, the prevalent model of activation of HIF-1 alpha function. Interestingly, we observed that HIF-1 alpha was associated with the molecular chaperone hsp90. Given the critical role of hsp90 for ligand binding activity and activation of the dioxin receptor, it is therefore possible that HIF-1 alpha is regulated by a similar mechanism, possibly by binding an as yet unknown class of ligands.

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The hypoxia-inducible factor-I (HIF-1) was first described as a DNA binding activity that specifically recognizes an 8 bp motif known to be essential for hypoxia-inducible erythropoietin gene transcription.Subsequently HIF-1 activity has also been found in cell lines which do not express erythropoietin, suggesting that HIF-1 is part of a widespread oxygen sensing mechanism. In electrophoretic mobility shift assays HIF-1 DNA binding activity is only detectable in nuclear extracts of cells cultivated in a low oxygen atmosphere. In addition to HIF-1, a constitutive DNA binding activity also specifically binds the HIFI probe. Here we report that CRE and AP1 oligonucleotides efficiently competed for binding of the HIF1 probe to this constitutive factor, whereas HIF-1 activity itself remained unaffected. Monoclonal antibodies raised against the CRE binding factors ATF-1 and CREB-1 supershifted the constitutive factor, while Jun and Fos family members, which constitute the AP-1 factor, were immunologically undetectable. Recombinant ATF-1 and CREB-1 proteins bound HIF1 probes either as homodimers or as heterodimers, indicating a new binding specificity for ATF-1/CREB-1. Finally, reporter gene assays in HeLa cells treated with either a cAMP analogue or a phorbol ester suggest that the PKA, but not the PKC signalling pathway is involved in oxygen sensing.

The hypoxia-inducible factor-1 (HIF-1) is a transcriptional activator involved in the expression of oxygen-regulated genes such as that for erythropoietin. Following exposure to low oxygen partial pressure (hypoxia), HIF-1 binds to an hypoxia-response element located 3′ to the erythropoietin gene and confers activation of erythropoietin expression. The conserved core HIF-1 binding site (HBS) of the erythropoietin 3′ enhancer (CGTG) contains a CpG dinucleotide known to be a potential target of cytosine methylation. We found that methylation of the HBS abolishes HIF-1 DNA binding as well as hypoxic reporter gene activation, suggesting that a methylation-free HBS is mandatory for HIF-1 function. The in vivo methylation pattern of the erythropoietin 3′ HBS in various human cell lines and mouse organs was assessed by genomic Southern blotting using a methylation-sensitive restriction enzyme. Whereas this site was essentially methylation-free in the erythropoietin-producing cell line Hep3B, a direct correlation between erythropoietin protein expression and the degree of erythropoietin 3′ HBS methylation was found in different HepG2 sublines. However, the finding that this site is partially methylation-free in human cell lines and mouse tissues that do not express erythropoietin suggests that there might be a general selective pressure to keep this site methylation-free, independent of erythropoietin expression.