Expression of vascular endothelial growth factor (VEGF) is induced in cells exposedUnder normal physiologic conditions, each of the approximately 10 14 cells in the adult human body is provided with an adequate supply of O 2 to meet its metabolic demands through the concerted function of the pulmonary, hematopoietic, and cardiovascular systems. O 2 is transported through the circulation by erythrocytes, the production of which is controlled by the glycoprotein hormone/growth factor erythropoietin (EPO) (reviewed in references 20, 23, and 43). Cells in the liver and kidney that produce EPO are able to sense O 2 concentration and respond to systemic hypoxia with increased EPO gene transcription (8,15,46). A hypoxia-inducible enhancer element was identified in the 3Ј-flanking region of the human and mouse EPO genes (2,3,33,42,48,50). Hypoxia-inducible factor 1 (HIF-1) was detected in nuclear extracts of hypoxic Hep3B cells (exposed to 1% O 2 for 4 h) and was undetectable in extracts from nonhypoxic cells (maintained at 20% O 2 ). HIF-1 bound to the EPO enhancer, and mutations that eliminated HIF-1 binding also eliminated enhancer function (50). Exposure of hypoxic cells to inhibitors of protein synthesis (cycloheximide) or phosphorylation (2-aminopurine) inhibited the induction of both EPO mRNA and HIF-1 DNA-binding activity, and other inducers of EPO expression (CoCl 2 and desferrioxamine) also induced HIF-1 activity (50,58,60). Methylation interference analysis revealed that HIF-1 bound to the EPO enhancer sequence 5Ј-TACGTGCT-3Ј by making major groove contacts with both guanine residues on each strand (59).Protein purification indicated that HIF-1 was a heterodimeric protein (61). Peptide and nucleic acid sequence analysis demonstrated that both subunits were basic helixloop-helix (bHLH) proteins (57). HIF-1␣ was a novel 826-amino-acid polypeptide, whereas HIF-1␤ was identical to the 774-and 789-amino-acid products of the ARNT (aryl hydrocarbon receptor nuclear translocator) gene previously shown to heterodimerize with the aryl hydrocarbon receptor (AHR) (57). HIF-1␣, HIF-1␤ (ARNT), and AHR are all members of a subfamily of bHLH proteins that contain a conserved PAS domain following the bHLH motif (4,18,57). In all three polypeptides, the basic domain is required for DNA binding following heterodimerization mediated by the HLH and PAS domains, and the C terminus contains one or more transactivation domains (6,21,29,32,44,63). Forced expression of HIF-1␣ and HIF-1␤ (ARNT) in cultured cells transfected with a reporter plasmid containing the EPO enhancer resulted in significantly higher levels of transcription, both at 1% and at 20% O 2 , than in cells transfected with the reporter plasmid alone, demonstrating that transcriptional activation via the EPO enhancer is mediated by HIF-1 (21).In contrast to systemic hypoxia, which elicits increased EPO synthesis, hypoxia can also be restricted to cells within a localized region of a specific organ, usually as a result of insufficient perfusion, as in the case of myocard...

Hypoxia is an essential developmental and physiological stimulus that plays a key role in the pathophysiology of cancer, heart attack, stroke, and other major causes of mortality. Hypoxia-inducible factor 1 (HIF-1) is the only known mammalian transcription factor expressed uniquely in response to physiologically relevant levels of hypoxia. We now report that in Hif1a −/− embryonic stem cells that did not express the O 2 -regulated HIF-1␣ subunit, levels of mRNAs encoding glucose transporters and glycolytic enzymes were reduced, and cellular proliferation was impaired. Vascular endothelial growth factor mRNA expression was also markedly decreased in hypoxic Hif1a −/− embryonic stem cells and cystic embryoid bodies. Complete deficiency of HIF-1␣ resulted in developmental arrest and lethality by E11 of Hif1a −/− embryos that manifested neural tube defects, cardiovascular malformations, and marked cell death within the cephalic mesenchyme. In Hif1a +/+ embryos, HIF-1␣ expression increased between E8.5 and E9.5, coincident with the onset of developmental defects and cell death in Hif1a −/− embryos. These results demonstrate that HIF-1␣ is a master regulator of cellular and developmental O 2 homeostasis.

Hypoxia-inducible factor-1 (HIF-1) is a transcription factor that regulates adaptive responses to the lack of oxygen in mammalian cells. HIF-1 consists of two proteins, HIF-1alpha and HIF-1beta. HIF-1alpha accumulates under hypoxic conditions, whereas HIF-1beta is constitutively expressed. HIF-1alpha and HIF-1beta expression were measured during adaptation to hypobaric hypoxia (0.5 atm) in rat cerebral cortex. Western blot analyses indicated that HIF-1alpha rapidly accumulated during the onset of hypoxia and did not fall for 14 days but fell to normal by 21 days despite the continuous low arterial oxygen tension. Immunostaining showed that neurons, astrocytes, ependymal cells, and possibly endothelial cells were the cell types expressing HIF-1alpha. Genes with hypoxia-responsive elements were activated under these conditions, as evidenced by elevated vascular endothelial growth factor and glucose transporter-1 mRNA levels. When 21-day-adapted rats were exposed to a more severe hypoxic challenge (8% oxygen), HIF-1alpha accumulated again. On the basis of these results, we speculate that the vascular remodeling and metabolic changes triggered during prolonged hypoxia are capable of restoring normal tissue oxygen levels.

Hypoxia-inducible factor 1 (HIF-1) is a basic helix-loop-helix protein that activates transcription of hypoxia-inducible genes, including those encoding: erythropoietin, vascular endothelial growth factor, heme oxygenase-1, inducible nitric oxide synthase, and the glycolytic enzymes aldolase A, enolase 1, lactate dehydrogenase A, phosphofructokinase I, and phosphoglycerate kinase 1. Hypoxia response elements from these genes consist of a HIF-1 binding site (that contains the core sequence 5'-CGTG-3') as well as additional DNA sequences that are required for function, which in some elements include a second HIF-1 binding site. HIF-1 is a heterodimer. The HIF-1 alpha subunit is unique to HIF-1, whereas HIF-1 beta (ARNT) can dimerize with other bHLH-PAS proteins. Structural analysis of HIF-1 alpha revealed that dimerization with HIF-1 beta (ARNT) requires the HLH and PAS domains, DNA binding is mediated by the basic domain, and that HIF-1 alpha contains a carboxyl-terminal transactivation domain. Co-transfection of HIF-1 alpha and HIF-1 beta (ARNT) expression vectors and a reporter gene containing a wild-type hypoxia response element resulted in increased transcription in non-hypoxic cells and a superinduction of transcription in hypoxic cells, whereas HIF-1 expression vectors had no effect on the transcription of reporter genes containing a mutation in the HIF-1 binding site. HIF-1 alpha and HIF-1 beta (ARNT) protein levels were induced by hypoxia in all primary and transformed cell lines examined. In HeLa cells, the levels of HIF-1 alpha and HIF-1 beta protein and HIF-1 DNA-binding activity increased exponentially as cellular oxygen tension decreased, with maximum values at 0.5% oxygen and half-maximal values at 1.5 to 2% oxygen. HIF-1 alpha and HIF-1 beta (ARNT) mRNAs were detected in all human, mouse, and rat organs assayed and mRNA expression was modestly induced in rodents subjected to hypoxia. HIF-1 alpha protein levels were induced in vivo when animals were subjected to anemia or hypoxia. The HIF1A gene was mapped to human chromosome 14q21-q24 and mouse chromosome 12.