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Mol Cell Biol. Sep 1996; 16(9): 4604–4613.
PMCID: PMC231459

Activation of vascular endothelial growth factor gene transcription by hypoxia-inducible factor 1.

Abstract

Expression of vascular endothelial growth factor (VEGF) is induced in cells exposed to hypoxia or ischemia. Neovascularization stimulated by VEGF occurs in several important clinical contexts, including myocardial ischemia, retinal disease, and tumor growth. Hypoxia-inducible factor 1 (HIF-1) is a heterodimeric basic helix-loop-helix protein that activates transcription of the human erythropoietin gene in hypoxic cells. Here we demonstrate the involvement of HIF-1 in the activation of VEGF transcription. VEGF 5'-flanking sequences mediated transcriptional activation of reporter gene expression in hypoxic Hep3B cells. A 47-bp sequence located 985 to 939 bp 5' to the VEGF transcription initiation site mediated hypoxia-inducible reporter gene expression directed by a simian virus 40 promoter element that was otherwise minimally responsive to hypoxia. When reporters containing VEGF sequences, in the context of the native VEGF or heterologous simian virus 40 promoter, were cotransfected with expression vectors encoding HIF-1alpha and HIF-1beta (ARNT [aryl hydrocarbon receptor nuclear translocator]), reporter gene transcription was much greater in both hypoxic and nonhypoxic cells than in cells transfected with the reporter alone. A HIF-1 binding site was demonstrated in the 47-bp hypoxia response element, and a 3-bp substitution eliminated the ability of the element to bind HIF-1 and to activate transcription in response to hypoxia and/or recombinant HIF-1. Cotransfection of cells with an expression vector encoding a dominant negative form of HIF-1alpha inhibited the activation of reporter transcription in hypoxic cells in a dose-dependent manner. VEGF mRNA was not induced by hypoxia in mutant cells that do not express the HIF-1beta (ARNT) subunit. These findings implicate HIF-1 in the activation of VEGF transcription in hypoxic cells.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Banai S, Shweiki D, Pinson A, Chandra M, Lazarovici G, Keshet E. Upregulation of vascular endothelial growth factor expression induced by myocardial ischaemia: implications for coronary angiogenesis. Cardiovasc Res. 1994 Aug;28(8):1176–1179. [PubMed]
  • Beck I, Ramirez S, Weinmann R, Caro J. Enhancer element at the 3'-flanking region controls transcriptional response to hypoxia in the human erythropoietin gene. J Biol Chem. 1991 Aug 25;266(24):15563–15566. [PubMed]
  • Beck I, Weinmann R, Caro J. Characterization of hypoxia-responsive enhancer in the human erythropoietin gene shows presence of hypoxia-inducible 120-Kd nuclear DNA-binding protein in erythropoietin-producing and nonproducing cells. Blood. 1993 Aug 1;82(3):704–711. [PubMed]
  • Burbach KM, Poland A, Bradfield CA. Cloning of the Ah-receptor cDNA reveals a distinctive ligand-activated transcription factor. Proc Natl Acad Sci U S A. 1992 Sep 1;89(17):8185–8189. [PMC free article] [PubMed]
  • Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. [PubMed]
  • Dolwick KM, Swanson HI, Bradfield CA. In vitro analysis of Ah receptor domains involved in ligand-activated DNA recognition. Proc Natl Acad Sci U S A. 1993 Sep 15;90(18):8566–8570. [PMC free article] [PubMed]
  • Ebert BL, Firth JD, Ratcliffe PJ. Hypoxia and mitochondrial inhibitors regulate expression of glucose transporter-1 via distinct Cis-acting sequences. J Biol Chem. 1995 Dec 8;270(49):29083–29089. [PubMed]
  • Fandrey J, Bunn HF. In vivo and in vitro regulation of erythropoietin mRNA: measurement by competitive polymerase chain reaction. Blood. 1993 Feb 1;81(3):617–623. [PubMed]
  • Ferrara N, Henzel WJ. Pituitary follicular cells secrete a novel heparin-binding growth factor specific for vascular endothelial cells. Biochem Biophys Res Commun. 1989 Jun 15;161(2):851–858. [PubMed]
  • Ferrara N, Houck K, Jakeman L, Leung DW. Molecular and biological properties of the vascular endothelial growth factor family of proteins. Endocr Rev. 1992 Feb;13(1):18–32. [PubMed]
  • Finkenzeller G, Technau A, Marmé D. Hypoxia-induced transcription of the vascular endothelial growth factor gene is independent of functional AP-1 transcription factor. Biochem Biophys Res Commun. 1995 Mar 8;208(1):432–439. [PubMed]
  • Firth JD, Ebert BL, Pugh CW, Ratcliffe PJ. Oxygen-regulated control elements in the phosphoglycerate kinase 1 and lactate dehydrogenase A genes: similarities with the erythropoietin 3' enhancer. Proc Natl Acad Sci U S A. 1994 Jul 5;91(14):6496–6500. [PMC free article] [PubMed]
  • Firth JD, Ebert BL, Ratcliffe PJ. Hypoxic regulation of lactate dehydrogenase A. Interaction between hypoxia-inducible factor 1 and cAMP response elements. J Biol Chem. 1995 Sep 8;270(36):21021–21027. [PubMed]
  • Folkman J, Shing Y. Angiogenesis. J Biol Chem. 1992 Jun 5;267(16):10931–10934. [PubMed]
  • Gleadle JM, Ebert BL, Firth JD, Ratcliffe PJ. Regulation of angiogenic growth factor expression by hypoxia, transition metals, and chelating agents. Am J Physiol. 1995 Jun;268(6 Pt 1):C1362–C1368. [PubMed]
  • Goldberg MA, Gaut CC, Bunn HF. Erythropoietin mRNA levels are governed by both the rate of gene transcription and posttranscriptional events. Blood. 1991 Jan 15;77(2):271–277. [PubMed]
  • Goldberg MA, Schneider TJ. Similarities between the oxygen-sensing mechanisms regulating the expression of vascular endothelial growth factor and erythropoietin. J Biol Chem. 1994 Feb 11;269(6):4355–4359. [PubMed]
  • Gospodarowicz D, Abraham JA, Schilling J. Isolation and characterization of a vascular endothelial cell mitogen produced by pituitary-derived folliculo stellate cells. Proc Natl Acad Sci U S A. 1989 Oct;86(19):7311–7315. [PMC free article] [PubMed]
  • Hoffman EC, Reyes H, Chu FF, Sander F, Conley LH, Brooks BA, Hankinson O. Cloning of a factor required for activity of the Ah (dioxin) receptor. Science. 1991 May 17;252(5008):954–958. [PubMed]
  • Ikeda E, Achen MG, Breier G, Risau W. Hypoxia-induced transcriptional activation and increased mRNA stability of vascular endothelial growth factor in C6 glioma cells. J Biol Chem. 1995 Aug 25;270(34):19761–19766. [PubMed]
  • Jelkmann W. Erythropoietin: structure, control of production, and function. Physiol Rev. 1992 Apr;72(2):449–489. [PubMed]
  • Klagsbrun M, Soker S. VEGF/VPF: the angiogenesis factor found? Curr Biol. 1993 Oct 1;3(10):699–702. [PubMed]
  • Krantz SB. Erythropoietin. Blood. 1991 Feb 1;77(3):419–434. [PubMed]
  • Ladoux A, Frelin C. Hypoxia is a strong inducer of vascular endothelial growth factor mRNA expression in the heart. Biochem Biophys Res Commun. 1993 Sep 15;195(2):1005–1010. [PubMed]
  • Leung DW, Cachianes G, Kuang WJ, Goeddel DV, Ferrara N. Vascular endothelial growth factor is a secreted angiogenic mitogen. Science. 1989 Dec 8;246(4935):1306–1309. [PubMed]
  • Levy AP, Levy NS, Goldberg MA. Post-transcriptional regulation of vascular endothelial growth factor by hypoxia. J Biol Chem. 1996 Feb 2;271(5):2746–2753. [PubMed]
  • Levy AP, Levy NS, Loscalzo J, Calderone A, Takahashi N, Yeo KT, Koren G, Colucci WS, Goldberg MA. Regulation of vascular endothelial growth factor in cardiac myocytes. Circ Res. 1995 May;76(5):758–766. [PubMed]
  • Levy AP, Levy NS, Wegner S, Goldberg MA. Transcriptional regulation of the rat vascular endothelial growth factor gene by hypoxia. J Biol Chem. 1995 Jun 2;270(22):13333–13340. [PubMed]
  • Li H, Dong L, Whitlock JP., Jr Transcriptional activation function of the mouse Ah receptor nuclear translocator. J Biol Chem. 1994 Nov 11;269(45):28098–28105. [PubMed]
  • Liu Y, Cox SR, Morita T, Kourembanas S. Hypoxia regulates vascular endothelial growth factor gene expression in endothelial cells. Identification of a 5' enhancer. Circ Res. 1995 Sep;77(3):638–643. [PubMed]
  • Ma Q, Dong L, Whitlock JP., Jr Transcriptional activation by the mouse Ah receptor. Interplay between multiple stimulatory and inhibitory functions. J Biol Chem. 1995 May 26;270(21):12697–12703. [PubMed]
  • Madan A, Curtin PT. A 24-base-pair sequence 3' to the human erythropoietin gene contains a hypoxia-responsive transcriptional enhancer. Proc Natl Acad Sci U S A. 1993 May 1;90(9):3928–3932. [PMC free article] [PubMed]
  • Melillo G, Musso T, Sica A, Taylor LS, Cox GW, Varesio L. A hypoxia-responsive element mediates a novel pathway of activation of the inducible nitric oxide synthase promoter. J Exp Med. 1995 Dec 1;182(6):1683–1693. [PMC free article] [PubMed]
  • Miller JW, Adamis AP, Shima DT, D'Amore PA, Moulton RS, O'Reilly MS, Folkman J, Dvorak HF, Brown LF, Berse B, et al. Vascular endothelial growth factor/vascular permeability factor is temporally and spatially correlated with ocular angiogenesis in a primate model. Am J Pathol. 1994 Sep;145(3):574–584. [PMC free article] [PubMed]
  • Minchenko A, Salceda S, Bauer T, Caro J. Hypoxia regulatory elements of the human vascular endothelial growth factor gene. Cell Mol Biol Res. 1994;40(1):35–39. [PubMed]
  • Nomura M, Yamagishi S, Harada S, Hayashi Y, Yamashima T, Yamashita J, Yamamoto H. Possible participation of autocrine and paracrine vascular endothelial growth factors in hypoxia-induced proliferation of endothelial cells and pericytes. J Biol Chem. 1995 Nov 24;270(47):28316–28324. [PubMed]
  • Pierce EA, Avery RL, Foley ED, Aiello LP, Smith LE. Vascular endothelial growth factor/vascular permeability factor expression in a mouse model of retinal neovascularization. Proc Natl Acad Sci U S A. 1995 Jan 31;92(3):905–909. [PMC free article] [PubMed]
  • Plate KH, Breier G, Millauer B, Ullrich A, Risau W. Up-regulation of vascular endothelial growth factor and its cognate receptors in a rat glioma model of tumor angiogenesis. Cancer Res. 1993 Dec 1;53(23):5822–5827. [PubMed]
  • Plate KH, Breier G, Weich HA, Risau W. Vascular endothelial growth factor is a potential tumour angiogenesis factor in human gliomas in vivo. Nature. 1992 Oct 29;359(6398):845–848. [PubMed]
  • Pugh CW, Tan CC, Jones RW, Ratcliffe PJ. Functional analysis of an oxygen-regulated transcriptional enhancer lying 3' to the mouse erythropoietin gene. Proc Natl Acad Sci U S A. 1991 Dec 1;88(23):10553–10557. [PMC free article] [PubMed]
  • Ratcliffe PJ. Molecular biology of erythropoietin. Kidney Int. 1993 Oct;44(4):887–904. [PubMed]
  • Reisz-Porszasz S, Probst MR, Fukunaga BN, Hankinson O. Identification of functional domains of the aryl hydrocarbon receptor nuclear translocator protein (ARNT). Mol Cell Biol. 1994 Sep;14(9):6075–6086. [PMC free article] [PubMed]
  • Reyes H, Reisz-Porszasz S, Hankinson O. Identification of the Ah receptor nuclear translocator protein (Arnt) as a component of the DNA binding form of the Ah receptor. Science. 1992 May 22;256(5060):1193–1195. [PubMed]
  • Schuster SJ, Badiavas EV, Costa-Giomi P, Weinmann R, Erslev AJ, Caro J. Stimulation of erythropoietin gene transcription during hypoxia and cobalt exposure. Blood. 1989 Jan;73(1):13–16. [PubMed]
  • Semenza GL, Nejfelt MK, Chi SM, Antonarakis SE. Hypoxia-inducible nuclear factors bind to an enhancer element located 3' to the human erythropoietin gene. Proc Natl Acad Sci U S A. 1991 Jul 1;88(13):5680–5684. [PMC free article] [PubMed]
  • Semenza GL, Roth PH, Fang HM, Wang GL. Transcriptional regulation of genes encoding glycolytic enzymes by hypoxia-inducible factor 1. J Biol Chem. 1994 Sep 23;269(38):23757–23763. [PubMed]
  • Semenza GL, Wang GL. A nuclear factor induced by hypoxia via de novo protein synthesis binds to the human erythropoietin gene enhancer at a site required for transcriptional activation. Mol Cell Biol. 1992 Dec;12(12):5447–5454. [PMC free article] [PubMed]
  • Shima DT, Kuroki M, Deutsch U, Ng YS, Adamis AP, D'Amore PA. The mouse gene for vascular endothelial growth factor. Genomic structure, definition of the transcriptional unit, and characterization of transcriptional and post-transcriptional regulatory sequences. J Biol Chem. 1996 Feb 16;271(7):3877–3883. [PubMed]
  • Shweiki D, Itin A, Soffer D, Keshet E. Vascular endothelial growth factor induced by hypoxia may mediate hypoxia-initiated angiogenesis. Nature. 1992 Oct 29;359(6398):843–845. [PubMed]
  • Sogawa K, Nakano R, Kobayashi A, Kikuchi Y, Ohe N, Matsushita N, Fujii-Kuriyama Y. Possible function of Ah receptor nuclear translocator (Arnt) homodimer in transcriptional regulation. Proc Natl Acad Sci U S A. 1995 Mar 14;92(6):1936–1940. [PMC free article] [PubMed]
  • Stein I, Neeman M, Shweiki D, Itin A, Keshet E. Stabilization of vascular endothelial growth factor mRNA by hypoxia and hypoglycemia and coregulation with other ischemia-induced genes. Mol Cell Biol. 1995 Oct;15(10):5363–5368. [PMC free article] [PubMed]
  • Swanson HI, Chan WK, Bradfield CA. DNA binding specificities and pairing rules of the Ah receptor, ARNT, and SIM proteins. J Biol Chem. 1995 Nov 3;270(44):26292–26302. [PubMed]
  • Tan CC, Ratcliffe PJ. Effect of inhibitors of oxidative phosphorylation on erythropoietin mRNA in isolated perfused rat kidneys. Am J Physiol. 1991 Dec;261(6 Pt 2):F982–F987. [PubMed]
  • Tischer E, Mitchell R, Hartman T, Silva M, Gospodarowicz D, Fiddes JC, Abraham JA. The human gene for vascular endothelial growth factor. Multiple protein forms are encoded through alternative exon splicing. J Biol Chem. 1991 Jun 25;266(18):11947–11954. [PubMed]
  • Wang GL, Jiang BH, Rue EA, Semenza GL. Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O2 tension. Proc Natl Acad Sci U S A. 1995 Jun 6;92(12):5510–5514. [PMC free article] [PubMed]
  • Wang GL, Semenza GL. General involvement of hypoxia-inducible factor 1 in transcriptional response to hypoxia. Proc Natl Acad Sci U S A. 1993 May 1;90(9):4304–4308. [PMC free article] [PubMed]
  • Wang GL, Semenza GL. Characterization of hypoxia-inducible factor 1 and regulation of DNA binding activity by hypoxia. J Biol Chem. 1993 Oct 15;268(29):21513–21518. [PubMed]
  • Wang GL, Semenza GL. Desferrioxamine induces erythropoietin gene expression and hypoxia-inducible factor 1 DNA-binding activity: implications for models of hypoxia signal transduction. Blood. 1993 Dec 15;82(12):3610–3615. [PubMed]
  • Wang GL, Semenza GL. Purification and characterization of hypoxia-inducible factor 1. J Biol Chem. 1995 Jan 20;270(3):1230–1237. [PubMed]
  • Wenger RH, Rolfs A, Marti HH, Bauer C, Gassmann M. Hypoxia, a novel inducer of acute phase gene expression in a human hepatoma cell line. J Biol Chem. 1995 Nov 17;270(46):27865–27870. [PubMed]
  • Whitelaw ML, Gustafsson JA, Poellinger L. Identification of transactivation and repression functions of the dioxin receptor and its basic helix-loop-helix/PAS partner factor Arnt: inducible versus constitutive modes of regulation. Mol Cell Biol. 1994 Dec;14(12):8343–8355. [PMC free article] [PubMed]

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