Regulation of the hypoxia-inducible transcription factor 1alpha by the ubiquitin-proteasome pathway. (1/3793)

HIF-1alpha (hypoxia-inducible factor 1alpha) is a basic-helix-loop-helix PAS (Per/Arnt/Sim) transcription factor that, under hypoxic conditions, dimerizes with a partner factor, the basic-helix-loop-helix/PAS protein Arnt, to recognize hypoxia-responsive elements of target genes. It has recently been demonstrated that HIF-1alpha protein but not mRNA levels are dramatically up-regulated in response to hypoxia. Here we show that inhibitors of 26 S proteasome activity produced a dramatic accumulation of endogenous as well as transfected HIF-1alpha protein under normoxic conditions, whereas the levels of Arnt protein were not affected. HIF-1alpha was polyubiquitinated in vivo under normoxic conditions, indicating rapid degradation via the ubiquitin-proteasome pathway. This degradation process appeared to target a region within the C terminus of HIF-1alpha. Importantly, HIF-1alpha ubiquitination was drastically decreased under hypoxic conditions. Up-regulation of HIF-1alpha protein by proteasome inhibitors did not result in transcriptional activation of reporter genes, indicating either the requirement of additional regulatory steps to induce functional activity of HIF-1alpha or the inability of polyubiquitinated forms of HIF-1alpha to mediate hypoxic signal transduction. In support of both these notions, we demonstrate that HIF-1alpha showed hypoxia-dependent translocation from the cytoplasm to the nucleus and that this regulatory mechanism was severely impaired in the presence of proteasome inhibitors. Taken together, these data demonstrate that the mechanism of hypoxia-dependent activation of HIF-1alpha is a complex multistep process and that stabilization of HIF-1alpha protein levels is not sufficient to generate a functional form.  (+info)

Impaired physiological responses to chronic hypoxia in mice partially deficient for hypoxia-inducible factor 1alpha. (2/3793)

Chronic hypoxia induces polycythemia, pulmonary hypertension, right ventricular hypertrophy, and weight loss. Hypoxia-inducible factor 1 (HIF-1) activates transcription of genes encoding proteins that mediate adaptive responses to hypoxia, including erythropoietin, vascular endothelial growth factor, and glycolytic enzymes. Expression of the HIF-1alpha subunit increases exponentially as O2 concentration is decreased. Hif1a-/- mouse embryos with complete deficiency of HIF-1alpha due to homozygosity for a null allele at the Hif1a locus die at midgestation, with multiple cardiovascular malformations and mesenchymal cell death. Hif1a+/- heterozygotes develop normally and are indistinguishable from Hif1a+/+ wild-type littermates when maintained under normoxic conditions. In this study, the physiological responses of Hif1a+/- and Hif1a+/+ mice exposed to 10% O2 for one to six weeks were analyzed. Hif1a+/- mice demonstrated significantly delayed development of polycythemia, right ventricular hypertrophy, pulmonary hypertension, and pulmonary vascular remodeling and significantly greater weight loss compared with wild-type littermates. These results indicate that partial HIF-1alpha deficiency has significant effects on multiple systemic responses to chronic hypoxia.  (+info)

Inhibition of hypoxia-inducible factor 1 activation by carbon monoxide and nitric oxide. Implications for oxygen sensing and signaling. (3/3793)

It has been proposed that cells sense hypoxia by a heme protein, which transmits a signal that activates the heterodimeric transcription factor hypoxia-inducible factor 1 (HIF-1), thereby inducing a number of physiologically relevant genes such as erythropoietin (Epo). We have investigated the mechanism by which two heme-binding ligands, carbon monoxide and nitric oxide, affect oxygen sensing and signaling. Two concentrations of CO (10 and 80%) suppressed the activation of HIF-1 and induction of Epo mRNA by hypoxia in a dose-dependent manner. In contrast, CO had no effect on the induction of HIF-1 activity and Epo expression by either cobalt chloride or the iron chelator desferrioxamine. The affinity of CO for the putative sensor was much lower than that of oxygen (Haldane coefficient, approximately 0.5). Parallel experiments were done with 100 microM sodium nitroprusside, a nitric oxide donor. Both NO and CO inhibited HIF-1 DNA binding by abrogating hypoxia-induced accumulation of HIF-1alpha protein. Moreover, both NO and CO specifically targeted the internal oxygen-dependent degradation domain of HIF-1alpha, and also repressed the C-terminal transactivation domain of HIF-1alpha. Thus, NO and CO act proximally, presumably as heme ligands binding to the oxygen sensor, whereas desferrioxamine and perhaps cobalt appear to act at a site downstream.  (+info)

Induction and nuclear translocation of hypoxia-inducible factor-1 (HIF-1): heterodimerization with ARNT is not necessary for nuclear accumulation of HIF-1alpha. (4/3793)

Hypoxia-inducible factor-1 (HIF-1) is a master regulator of mammalian oxygen homeostasis. HIF-1 consists of two subunits, HIF-1alpha and the aryl hydrocarbon receptor nuclear translocator (ARNT). Whereas hypoxia prevents proteasomal degradation of HIF-1alpha, ARNT expression is thought to be oxygen-independent. We and others previously showed that ARNT is indispensable for HIF-1 DNA-binding and transactivation function. Here, we have used ARNT-mutant mouse hepatoma and embryonic stem cells to examine the requirement of ARNT for accumulation and nuclear translocation of HIF-1alpha in hypoxia. As shown by immunofluorescence, HIF-1alpha accumulation in the nucleus of hypoxic cells was independent of the presence of ARNT, suggesting that nuclear translocation is intrinsic to HIF-1alpha. Co-immunoprecipitation of HIF-1alpha together with ARNT could be performed in nuclear extracts but not in cytosolic fractions, implying that formation of the HIF-1 complex occurs in the nucleus. A proteasome inhibitor and a thiol-reducing agent could mimic hypoxia by inducing HIF-1alpha in the nucleus, indicating that escape from proteolytic degradation is sufficient for accumulation and nuclear translocation of HIF-1alpha. During biochemical separation, both HIF-1alpha and ARNT tend to leak from the nuclei in the absence of either subunit, suggesting that heterodimerization is required for stable association within the nuclear compartment. Nuclear stabilization of the heterodimer might also explain the hypoxically increased total cellular ARNT levels observed in some of the cell lines examined.  (+info)

Molecular mechanisms of transcription activation by HLF and HIF1alpha in response to hypoxia: their stabilization and redox signal-induced interaction with CBP/p300. (5/3793)

Hypoxia-inducible factor 1 alpha (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 delineated 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 revealed that NADs, which were rarely detectable at normoxia, became stabilized and accumulated at hypoxia, whereas CADs were constitutively expressed. In the mammalian two-hybrid system, CAD and NAD baits enhanced the luciferase expression from a reporter gene by co-transfection with CREB-binding protein (CBP) prey, whereas CAD, but not NAD, enhanced 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 revealed that expression of Ref-1 and thioredoxin further enhanced the luciferase activity expressed by CAD, but not by NAD. Amino acid replacement in the sequences of CADs revealed a specific cysteine to be essential for their hypoxia-inducible interaction with CBP. Nuclear translocation of thioredoxin from cytoplasm was observed upon reducing O2 concentrations.  (+info)

Repression of dioxin signal transduction in fibroblasts. Identification Of a putative repressor associated with Arnt. (6/3793)

Heterodimeric complexes of basic helix-loop-helix/PAS transcription factors are involved in regulation of diverse physiological phenomena such as circadian rhythms, reaction to low oxygen tension, and detoxification. In fibroblasts, the basic helix-loop-helix/PAS heterodimer consisting of the ligand-inducible dioxin receptor and Arnt shows DNA-binding activity, and the receptor and Arnt are able to activate transcription when fused to a heterologous DNA-binding domain. However, fibroblasts are nonresponsive to dioxin with regard to induction mediated by the DNA response element recognized by the receptor and Arnt. Here we demonstrate that Arnt is associated with a fibroblast-specific factor, forming a complex that is capable of binding the dioxin response element. This factor may function as a repressor since negative regulation of target gene induction appears to be abolished by inhibition of histone deacetylase activity by trichostatin A. Finally, the negative regulatory function of this factor appears to be restricted for dioxin signaling since Arnt was able to mediate, together with hypoxia-inducible factor-1alpha, transcriptional activation in hypoxic cells. Taken together, these data suggest that fibroblast-specific inhibition of dioxin responsiveness involves recruitment by Arnt of a cell type- and signaling pathway-specific corepressor associated with a histone deacetylase.  (+info)

Molecular cloning of cDNAs encoding hypoxia-inducible factor (HIF)-1alpha and -2alpha of bovine arterial endothelial cells. (7/3793)

Hypoxia-inducible factor (HIF)-1alpha and -2alpha are two basic helix-loop-helix/PAS domain transcriptional factors that mediate hypoxia-induced gene expression. We found that bovine arterial endothelial cells (BAEC) expressed both HIF-1alpha and -2alpha by RT-PCR and then isolated cDNAs encoding these two transcriptional factors of BAEC. The deduced amino acid sequences of both HIF-1alpha and -2alpha showed high homologies among mammalian species. Northern blot analysis indicated that the mRNAs for HIF-1alpha and -2alpha from BAEC showed a size of approx. 5.5 and 6.2 kilobases, respectively and that both mRNAs were constitutively expressed and not induced by hypoxia in BAEC.  (+info)

Defective vascularization of HIF-1alpha-null embryos is not associated with VEGF deficiency but with mesenchymal cell death. (8/3793)

Hypoxia-inducible factor 1 (HIF-1) is a dimeric transcription factor composed of HIF-1alpha and HIF-1beta subunits that plays an essential role in mammalian O2 homeostasis. In Hif1a-/- knockout mice, complete deficiency of HIF-1alpha resulted in cardiac and vascular malformations and embryonic lethality at E10.5. Between E8. 75 and E9.25 striking vascular regression and abnormal remodeling occurred in the cephalic region concomitant with marked mesenchymal cell death. Similar vascular defects were observed in HIF-1alpha- and VEGF-deficient embryos and VEGF mRNA expression was not induced by hypoxia in Hif1a-/- embryonic stem cells. Surprisingly, Hif1a-/- embryos demonstrated increased VEGF mRNA expression compared to wild-type embryos. In tissue culture cells, VEGF mRNA expression was induced by glucose deprivation independent of HIF-1alpha, providing a mechanism for increased VEGF mRNA expression in Hif1a-/- embryos, in which absence of adequate tissue perfusion resulted in both O2 and glucose deprivation. Rather than being associated with VEGF deficiency, the vascular defects in Hif1a-/- embryos were spatially correlated with cell death, the onset of which preceded vascular regression.  (+info)