Molecular Regulation of the Endothelin-1 Gene by Hypoxia

Endothelin-1 (ET-1) is a peptide hormone with potent vasoconstrictor properties which is synthesized and secreted predominantly by vascular endothelial cells. Its production is regulated by numerous stimuli including ischemia and hypoxia, and the enhanced levels that occur during myocardial ischemia may contribute to the progression of heart failure. We reported previously a preliminary characterization of a hypoxia-inducible factor-1 (HIF-1) binding site in the human ET-1 promoter which contributed to the activation of ET-1 expression in endothelial cells. We report here that the HIF-1 binding site alone is not sufficient for the response to hypoxia but requires an additional 50 base pairs of flanking sequence that includes binding sites for the factors activator protein-1 (AP-1), GATA-2, and CAAT-binding factor (NF-1). Mutation of any one of these sites or the HIF-1 site eliminated induction by hypoxia. Mutations of the AP-1 and GATA-2 sites, but not the HIF-1 site, were complemented by overexpressing AP-1, GATA-2, HIF-1α, or the activator protein p300/CBP, restoring the response to hypoxia. Binding studies in vitro confirmed physical associations among GATA-2, AP-1, and HIF-1 factors. Overexpression or depletion of p300/CBP modulated the level of ET-1 promoter expression as well as the endogenous ET-1 transcript but did not change the fold induction by hypoxia in either case. Regulation of the ET-1 promoter by hypoxia in non-endothelial cells required overexpression of GATA-2 and HIF-1α. The results support essential roles for AP-1, GATA-2, and NF-1 in stabilizing the binding of HIF-1 and promoting recruitment of p300/CBP to the ET-1 hypoxia response complex. Endothelin-1 (ET-1) is a peptide hormone with potent vasoconstrictor properties which is synthesized and secreted predominantly by vascular endothelial cells. Its production is regulated by numerous stimuli including ischemia and hypoxia, and the enhanced levels that occur during myocardial ischemia may contribute to the progression of heart failure. We reported previously a preliminary characterization of a hypoxia-inducible factor-1 (HIF-1) binding site in the human ET-1 promoter which contributed to the activation of ET-1 expression in endothelial cells. We report here that the HIF-1 binding site alone is not sufficient for the response to hypoxia but requires an additional 50 base pairs of flanking sequence that includes binding sites for the factors activator protein-1 (AP-1), GATA-2, and CAAT-binding factor (NF-1). Mutation of any one of these sites or the HIF-1 site eliminated induction by hypoxia. Mutations of the AP-1 and GATA-2 sites, but not the HIF-1 site, were complemented by overexpressing AP-1, GATA-2, HIF-1α, or the activator protein p300/CBP, restoring the response to hypoxia. Binding studies in vitro confirmed physical associations among GATA-2, AP-1, and HIF-1 factors. Overexpression or depletion of p300/CBP modulated the level of ET-1 promoter expression as well as the endogenous ET-1 transcript but did not change the fold induction by hypoxia in either case. Regulation of the ET-1 promoter by hypoxia in non-endothelial cells required overexpression of GATA-2 and HIF-1α. The results support essential roles for AP-1, GATA-2, and NF-1 in stabilizing the binding of HIF-1 and promoting recruitment of p300/CBP to the ET-1 hypoxia response complex. Endothelin-1 (ET-1) 1The abbreviations used are:ET-1endothelin-1AP-1activator protein-1NF-1nuclear factor-1HIF-1hypoxia-inducible factor-1EC(s)endothelial cell(s)HUVEC(s)human umbilical venous endothelial cell(s)HEKhuman embryonic kidneyLucluciferaseEpoerythropoietinWTwild typeMmutatedLDHlactate dehydrogenaseHREhypoxia response element is a 21-residue peptide synthesized and secreted by vascular endothelial cells. It is a member of a family of structurally related peptide hormones and is the most potent endogenously produced vasoconstrictor known (for review, see Ref. 1Rubanyi G.M. Polokoff M.A. Pharmacol. Rev. 1994; 46: 325-415PubMed Google Scholar). The enhanced secretion of ET-1 during myocardial ischemia has been linked with enhanced contractility in the failing heart (2Sakai S. Miyauchi T. Sakurai T. Kasuya Y. Ihara M. Yamaguchi I. Goto K. Sugishita Y. Circulation. 1996; 93: 1214-1222Crossref PubMed Scopus (287) Google Scholar) as well as with the progression of heart failure (3Sakai S. Miyauchi T. Kobayashi M. Yamaguchi I. Goto K. Sugishita Y. Nature. 1996; 384: 353-355Crossref PubMed Scopus (575) Google Scholar). The combined actions of ET-1 and the endothelial cell relaxing factor nitric oxide may be important in regulating vascular tone and blood pressure (1Rubanyi G.M. Polokoff M.A. Pharmacol. Rev. 1994; 46: 325-415PubMed Google Scholar). In addition to its vasomodulating activity, ET-1 has been shown to modulate multiple cell functions including proliferation, proto-oncogene and protein kinase activity (1Rubanyi G.M. Polokoff M.A. Pharmacol. Rev. 1994; 46: 325-415PubMed Google Scholar), induction of inotropy and hypertrophy in cardiac muscle, and activation of cardiac specific genes (4Hu J.R. Von Harsdorf R. Lang R.E. Eur. J. Pharmacol. 1988; 158: 275-278Crossref PubMed Scopus (109) Google Scholar, 5Ito H. Hirata Y. Hiroe M. Tsujino M. Adachi S. Takamoto T. Nitta M. Taniguchi K. Marumo F. Circ. Res. 1991; 69: 209-215Crossref PubMed Scopus (464) Google Scholar). At least two cell surface receptors for the endothelins are present on multiple cell types, and most of the actions of the peptides are probably relayed through these receptors (for review, see Ref. 6Sokolovsky M. J. 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The promoter region contains activator protein-1 (AP-1), GATA-2, nuclear factor-1 (NF-1), and acute phase regulatory element sites upstream from classical CAAT and TATAA boxes. The AP-1 sites have been shown to mediate transcriptional induction by Fos and Jun M.-E. Yoshizumi M. Quertermous T. J. Biol. Chem. 1991; 266: Full Text PDF PubMed Google Scholar) as well as the response to (13Oliver F.J. Rubia G. Feener E.P. Lee M.-E. Loeken M.R. Shiba T. Quertermous T. King G.L. J. Biol. Chem. 1991; 266: 23251-23256Abstract Full Text PDF PubMed Google Scholar) and thrombin (7Kitazumi K. Tasaka K. Biochem. Pharmacol. 1993; 46: 455-464Crossref PubMed Scopus (26) Google Scholar). The GATA-2 and AP-1 are essential for level expression of ET-1 in endothelial cells M.-E. Quertermous T. J. Biol. Chem. 1990; Full Text PDF PubMed Google Scholar). In addition to the the ET-1 gene contains two sequences in the region which probably the of the ET-1 and its induction by protein (21Inoue A. Yanagisawa M. Takuwa Y. Mitsui Y. Kobayashi M. Masaki T. J. Biol. Chem. 1989; 264: 14954-14959Abstract Full Text PDF PubMed Google Scholar). is one of the most potent of ET-1 gene expression in endothelial cells and may be the of the production of ET-1 during myocardial ischemia T. M. A. Nature. 1990; Scopus Google Scholar, Biochem. Biophys. Res. Commun. 1993; PubMed Scopus Google Scholar, S. A. H. K. J. Life Sci. 1991; PubMed Scopus Google Scholar, T. A. P.A. M. G. Circulation. 1994; 90: Scholar). the and secretion of ET-1 in endothelial cells by a that is by nitric oxide and and by (15Kourembanas S. McQuillan L.P. Leung G.K. Faller D.V. J. Clin. Invest. 1993; 92: 99-104Crossref PubMed Scopus (422) Google Scholar, 16Hieda H.S. Gomez-Sanchez C.E. Life Sci. 1990; 47: 247-251Crossref PubMed Scopus (96) Google Scholar, 17Kourembanas S. Marsden P.A. McQuillan L.P. Faller D.V. J. Clin. Invest. 1991; 88: 1054-1057Crossref PubMed Scopus (634) Google Scholar). 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Nature. 1988; PubMed Scopus Google Scholar). by cells on the in and through and were as previously I. Bishopric N.H. Discher D.J. Wu X. Webster K.A. Cardiovasc. Res. 1995; 30: 975-984Crossref PubMed Google Scholar). including human ET-1 from and K.A. Nature. 1988; PubMed Scopus Google Scholar) were by to on the were a II and on by and by with as the and cells were by the as previously I. Bishopric N.H. Discher D.J. Wu X. Webster K.A. Cardiovasc. Res. 1995; 30: 975-984Crossref PubMed Google Scholar). were cell were cells were to of of including an luciferase from for were to hypoxia a Expression of luciferase activity, to the as previously I. Bishopric N.H. Discher D.J. Wu X. Webster K.A. Cardiovasc. Res. 1995; 30: 975-984Crossref PubMed Google Scholar). were from of or cells a with or to hypoxia as previously I. Bishopric N.H. Discher D.J. Wu X. Webster K.A. Cardiovasc. Res. 1995; 30: 975-984Crossref PubMed Google J. Discher D.J. Bishopric N.H. Webster K.A. Biochem. Biophys. Res. Commun. PubMed Scopus Google Scholar, X. Bishopric N.H. Discher D.J. Webster K.A. Mol. Cell. Biol. 1996; PubMed Scopus Google Scholar). cell cell with the cells hypoxia to Webster K.A. Circ. Res. 1997; PubMed Scopus Google Scholar). of the used were as the were the as for the were with kinase and Life of were to binding that of nuclear protein in of a of GATA-2, and were from from were for on X. Bishopric N.H. Discher D.J. Webster K.A. Mol. Cell. Biol. 1996; PubMed Scopus Google Scholar). were of and GATA-2 from S. from F. and from of and were by The were and in vitro the were from Life the wild type ET-1 and in the wild type sequence including to to and to were synthesized of AP-1 and GATA-2, and of The were by and of were were by in and two with were with a were to the and the for of vitro were to with binding and with of of the in a of The for and to the were from by with binding and with binding two with binding and were with a in for and on as for The were used to from nuclear In the protein from the were with to the of were for with in and with for in the the were for with a of or and enhanced the sequence of the in the ET-1 promoter base pairs and upstream of the site, including the of factor binding The base shown were and in pairs as and The expression of the wild type promoter and in endothelial cells or is shown in A. The AP-1 and GATA-2 expression to of the wild In with expression of the wild type promoter by hypoxia J. Discher D.J. Bishopric N.H. Webster K.A. Biochem. Biophys. Res. Commun. PubMed Scopus Google Scholar). response eliminated in site activation of the ET-1 promoter by hypoxia in requires GATA-2, AP-1, and NF-1 binding It reported previously that the expression of AP-1 and GATA-2 site in the ET-1 promoter were complemented by overexpressing the factor M.-E. Quertermous T. J. Biol. Chem. 1990; Full Text PDF PubMed Google Scholar). for of the response to hypoxia, wild type and mutated promoter were with HIF-1α, or GATA-2 expression results are shown in Expression of the wild type promoter by and GATA-2 but not by HIF-1α, The fold activation of the wild type promoter by hypoxia not in either case. Expression of the HIF-1 site not by of GATA-2, or HIF-1α, and induction of any The expression of the AP-1 and GATA-2 site complemented by either or GATA-2 in with a report M.-E. Quertermous T. J. Biol. Chem. 1990; Full Text PDF PubMed Google Scholar), and the response to hypoxia to by of GATA-2, or HIF-1α. of did not the hypoxia response of these not results that the binding site is essential but not sufficient for activation of the ET-1 promoter by hypoxia. of the AP-1 and GATA-2 site and by the factors that of these factors be to the HIF-1 complex binding of either AP-1 or GATA-2 site by that these sites may modulate the binding of were to hypoxia the binding of the HIF-1 site flanking shown in to the binding of AP-1, GATA-2, and HIF-1 were from and M.-E. Quertermous T. J. Biol. Chem. 1990; Full Text PDF PubMed Google Scholar, J. Discher D.J. Bishopric N.H. Webster K.A. Biochem. Biophys. Res. Commun. PubMed Scopus Google Scholar). in the the of specific and site sequences or the ET-1 as the hypoxia did not the or binding activity of either AP-1 or is in to cell AP-1 binding is by hypoxia R. T. Kobayashi S. M. 1997; Full Text PDF PubMed Scopus Google Scholar). In from or or cells were with the ET-1 HIF-1 and and or with a the erythropoietin The HIF-1 the and that the ET-1 site is a HIF-1 binding Binding studies with a the ET-1 to are shown in the AP-1 and HIF-1 were confirmed by with specific with an p300/CBP the of in these The on the the of the GATA-2 and the did not a Mutation of the GATA-2 site in the did not the binding of AP-1 or HIF-1 factors. a the promoter activation by did not HIF-1 binding in the by the eliminated the results that the ET-1 the HIF-1 complex the binding of AP-1 GATA-2 is not by hypoxia, and is for binding in used to in the ET-1 complex. used previously by and to in the transcriptional of factors, and p300/CBP in the regulation of the and genes Mol. Cell. Biol. PubMed Scopus Google Scholar). In ET-1 with or were used to in vitro as and the results a ET-1 with factor binding sites or with The in of and in HIF-1 were in the but the with during with the wild type were the factors were or in were the mutated of the AP-1 or GATA-2 sites in the ET-1 did not the binding of GATA-2, or HIF-1 be by and GATA-2 has that these in and are endogenous levels of factors in and not of the AP-1 or GATA-2 binding sites did not the protein to the site is sufficient to In support of of AP-1 and GATA-2 sites the binding of factors The of GATA-2 and by the enhanced HIF-1 and in the binding that GATA-2 and in the of It to the present in binding not endogenous not In support of binding activity, the HIF-1 site HIF-1 binding but additional GATA-2 or The by the level of HIF-1 binding to the be to with AP-1, GATA-2, or from the HIF-1 any of the factors to the that were on specific binding results support for physical or among GATA-2, and HIF-1 to the ET-1 We were to in GATA-2, or HIF-1 not that the a and physical associations with factors as which may mediate complex and of the factors The studies in that AP-1, GATA-2, HIF-1 or to to a response to hypoxia. The protein p300/CBP has been to with AP-1, factors, and Mol. Cell. Biol. PubMed Scopus Google Scholar, T. K. T. S. H. S. J. Biol. Chem. Full Text Full Text PDF Scopus Google Scholar, J. Y. Nature. PubMed Scopus Google Scholar, B.M. U. H. Google Scholar, R. S. M.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: PubMed Scopus Google Scholar). one of AP-1 and GATA-2 here may be to the recruitment of to the ET-1 for the wild type ET-1 or were endothelial cells with a expression of the and expression of the wild type ET-1 promoter but did not change the fold induction by hypoxia with with not may and expression of the ET-1 promoter in endothelial cells. of the expression of the HIF-1 site but did not support induction by in expression of the HIF-1 site hypoxia these overexpression the expression of AP-1 and GATA-2 site by and the hypoxia response to for the induction of AP-1 and GATA-2 site be complemented by AP-1, GATA-2, HIF-1α, or The of by that least one of AP-1 and GATA-2 is the recruitment of to the ET-1 complex. the depletion of the response as with the promoter and sites Mol. Cell. Biol. PubMed Scopus Google Scholar). for by the binding site of Expression of the wild type ET-1 promoter in the of the but the fold induction by hypoxia did not change with not of the expression by but did not change the fold In to the fold induction but not the expression of the in cells We the of expression on the endogenous ET-1 shown in ET-1 were by in with but the by hypoxia. the expression and induction of were and change of to modulate the expression of the ET-1 gene promoter but not the fold induction by hypoxia. results that may the level of expression of the ET-1 through with AP-1 and GATA-2, but the hypoxia response is of hypoxia activation with were used to from and nuclear shown in to the wild type and to with site in binding and nuclear and did not change with binding to the but and hypoxia, that the ET-1 HIF-1 site is a binding site for with AP-1 or to the as and binding to the that to HIF-1 with these results that the ET-1 flanking sites may binding to and the with the GATA-2, and AP-1 sites are in the ET-1 to functions were 50 base were GATA-2 and and HIF-1 and AP-1 sites and the of the shown in of the expression of the but did not the fold induction by hypoxia. We reported previously that the induction of the endogenous ET-1 transcript by hypoxia to endothelial cells with activation in cells or cardiac I. Bishopric N.H. Discher D.J. Wu X. Webster K.A. Cardiovasc. Res. 1995; 30: 975-984Crossref PubMed Google Scholar). see to the regulation of the the wild type ET-1 in and cells. shown in of these cell induction of promoter of the a the ET-1 promoter eliminated the that the the of the ET-1 promoter of the ET-1 factors is GATA-2 has been shown to contribute to gene expression J. Biol. Chem. 2000; Full Text Full Text PDF Scopus Google Scholar, J. Biol. Chem. 273: Full Text Full Text PDF PubMed Scopus Google Scholar). the of factors cells. shown in the hypoxia response by GATA-2 and but not by or the the essential of GATA-2 factors in an complex and least in for the of In report have shown that of the GATA-2, AP-1, or NF-1 sites in the ET-1 promoter eliminated activation of the promoter by hypoxia. The HIF-1 site eliminated the hypoxia response AP-1 and GATA-2 site were complemented by overexpressing AP-1, GATA-2, or p300/CBP factors. results important these factors that not the binding of factors. In is the of roles for factors or NF-1 in the transcriptional activation of a promoter by hypoxia. have not protein binding to the NF-1 site in of the site eliminated and that the NF-1 binding in a to AP-1 and GATA-2 in the complex. binding of in vitro that and GATA-2 with the ET-1 sites were the of these factors and the complex M.-E. Yoshizumi M. Quertermous T. J. Biol. Chem. 1991; 266: Full Text PDF PubMed Google Scholar, M.-E. Quertermous T. J. Biol. Chem. 1990; Full Text PDF PubMed Google Scholar). The of these factors the a the for least one binding The enhanced of AP-1 and GATA-2 by the in the of HIF-1 factors that these factors be by with HIF-1 or factors with results the physical as well as among AP-1, and GATA-2 factors. It is that did not in of these factors the and were not the of binding for of the complex and among factors. activation to ET-1 with GATA-2 or AP-1 by overexpressing the factors or by overexpressing either or of AP-1 and GATA-2 site with the of in the binding of these factors to the ET-1 that a for p300/CBP in the binding of HIF-1 flanking site to the promoter and erythropoietin Mol. Cell. Biol. PubMed Scopus Google Scholar). In these studies of the flanking response element site in the recruitment and activation by hypoxia. of the ET-1 flanking site by overexpression in studies that these the of HIF-1 for an that may be of studies support roles for the ET-1 HIF-1 flanking sites, the of HIF-1 binding and promoting recruitment of The ET-1 from the and in two and binding sites in addition to the HIF-1 complex. binding to the and sites is on hypoxia Mol. Cell. Biol. PubMed Scopus Google see is not the for the ET-1 to the wild type ET-1 and to site but to the and for binding from nuclear that to the ET-1 AP-1 and GATA-2 sites in a is in to the and sites and may for important in the functions of these In the of binding to the ET-1 is with a of on the fold activation of the ET-1 promoter by hypoxia and is in to the and fold activation with and binding Mol. Cell. Biol. PubMed Scopus Google Scholar, R. S. M.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: PubMed Scopus Google Scholar). binding the fold activation of the and genes by hypoxia but not the ET-1 are with a the HIF-1 flanking sites of the ET-1 the of HIF-1 and It is that the wild type ET-1 has a binding site that is of in be that site is any of the flanking sites is We have for the The of ET-1 by the flanking sites to of the In to the wild type ET-1 promoter not to hypoxia in or cells I. Bishopric N.H. Discher D.J. Wu X. Webster K.A. Cardiovasc. Res. 1995; 30: 975-984Crossref PubMed Google Scholar). The response be by overexpressing GATA-2 and HIF-1α. the of these factors in regulating the response of the ET-1 to hypoxia and a flanking site factors modulate of HIF-1 binding through studies have essential of AP-1 to the activation of and vascular growth factor R. T. Kobayashi S. M. 1997; Full Text PDF PubMed Scopus Google Scholar, J. Biol. Chem. 1995; PubMed Scopus Google Scholar, J. A.M. Am. J. Physiol. Google Scholar, H. A. Y. K. T. F. R. M. H. 2000; PubMed Google Scholar, G. A. Biochem. Biophys. Res. Commun. 2000; PubMed Scopus Google Scholar). It that AP-1 may roles in HIF-1 and binding in these In the AP-1 site is from the HIF-1 binding site is the with the ET-1 results that to the HIF-1 site may not be a for the roles of these factors. The ET-1 promoter may be in not AP-1 but GATA-2 and NF-1 factors for a important by multiple site which a of may be to that potent vasoconstrictor is not by hypoxia in the