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 basic helixloop-helix transcription factor which is expressed when mammalian cells are subjected to hypoxia and which activates transcription of genes encoding erythropoietin, vascular endothelial growth factor, and other proteins that are important for maintaining oxygen homeostasis. Previous studies have provided indirect evidence that HIF-1 also regulates transcription of genes encoding glycolytic enzymes. In this paper we characterize hypoxia response elements in the promoters of the ALDA, ENO1, and Ldha genes. We demonstrate that HIF-1 plays an essential role in activating transcription via these elements and show that although absolutely necessary, the presence of a HIF-1 binding site alone is not sufficient to mediate transcriptional responses to hypoxia. Analysis of hypoxia response elements in the ENO1 and Ldha gene promoters revealed that each contains two functionally-essential HIF-1 sites arranged as direct and inverted repeats, respectively. Our data establish that functional hypoxia-response elements consist of a pair of contiguous transcription factor binding sites at least one of which contains the core sequence 5-RCGTG-3 and is recognized by HIF-1. These results provide further evidence that the coordinate transcriptional activation of genes encoding glycolytic enzymes which occurs in hypoxic cells is mediated by HIF-1.Multiple homeostatic mechanisms are employed by mammals to respond to chronic hypoxia. In the case of systemic hypoxia due to decreased environmental O 2 (hypobaric hypoxia) or decreased blood O 2 -carrying capacity (anemia), erythropoiesis is stimulated by the production of erythropoietin (EPO).