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Cloning and Functional Analysis of the Promoter for KDR/flk-1, a Receptor for Vascular Endothelial Growth Factor

1995; Elsevier BV; Volume: 270; Issue: 39 Linguagem: Inglês

10.1074/jbc.270.39.23111

1083-351X

Cam Patterson, Mark A. Perrella, Chung-Ming Hsieh, Masao Yoshizumi, Mu-En Lee, Edgar Haber,

Nitric Oxide and Endothelin Effects

KDR/flk-1 is one of two receptors for vascular endothelial growth factor, a potent angiogenic peptide. KDR/flk-1 is an early marker for endothelial cell progenitors, and its expression is restricted to endothelial cells in vivo. To investigate the molecular mechanisms regulating expression of KDR/flk-1, we cloned and characterized the promoter of the human KDR/flk-1 gene. The transcription start site was localized by primer extension and ribonuclease protection to a nucleotide 303 base pairs (bp) 5′ of the initiation methionine codon. The 5′-flanking sequence is rich in G and C residues and contains five Sp1 elements but no TATA consensus sequence. By reporter gene transfection experiments, we found that ~4 kilobases of KDR/flk-1 5′-flanking sequence directed high level luciferase activity in bovine aortic endothelial cells; further deletion analysis revealed positive regulatory elements between bp −225 to −164, −95 to −77, −77 to −60, and +105 to +127. Mutation of an atypical GATA sequence between bp +105 and +127 did not affect promoter activity, suggesting that GATA elements are not essential for the high level promoter activity of this gene. Consistent with endothelial cell-restricted expression of KDR/flk-1 mRNA, we found that the 4-kilobase flanking sequence directed high level promoter activity in endothelial cells but not in other cell types. To our knowledge this is the first report characterizing the KDR/flk-1 promoter. Understanding the KDR/flk-1 promoter will allow us to investigate endothelial cell-specific gene regulation and to uncover methods for targeting gene delivery specifically to endothelial cells. KDR/flk-1 is one of two receptors for vascular endothelial growth factor, a potent angiogenic peptide. KDR/flk-1 is an early marker for endothelial cell progenitors, and its expression is restricted to endothelial cells in vivo. To investigate the molecular mechanisms regulating expression of KDR/flk-1, we cloned and characterized the promoter of the human KDR/flk-1 gene. The transcription start site was localized by primer extension and ribonuclease protection to a nucleotide 303 base pairs (bp) 5′ of the initiation methionine codon. The 5′-flanking sequence is rich in G and C residues and contains five Sp1 elements but no TATA consensus sequence. By reporter gene transfection experiments, we found that ~4 kilobases of KDR/flk-1 5′-flanking sequence directed high level luciferase activity in bovine aortic endothelial cells; further deletion analysis revealed positive regulatory elements between bp −225 to −164, −95 to −77, −77 to −60, and +105 to +127. Mutation of an atypical GATA sequence between bp +105 and +127 did not affect promoter activity, suggesting that GATA elements are not essential for the high level promoter activity of this gene. Consistent with endothelial cell-restricted expression of KDR/flk-1 mRNA, we found that the 4-kilobase flanking sequence directed high level promoter activity in endothelial cells but not in other cell types. To our knowledge this is the first report characterizing the KDR/flk-1 promoter. Understanding the KDR/flk-1 promoter will allow us to investigate endothelial cell-specific gene regulation and to uncover methods for targeting gene delivery specifically to endothelial cells. INTRODUCTIONVascular endothelial growth factor (VEGF) 1The abbreviations used are: VEGFvascular endothelial growth factorbpbase pair(s)kbkilobase(s)HUVEChuman umbilical vein endothelial cell(s)PCRpolymerase chain reactionBAECbovine aortic endothelial cell(s). is a potent and specific endothelial cell mitogen(1Connolly D.T. Heuvelman D.M. Nelson R. Olander J.V. Eppley B.L. Delfino J.J. Leingruber R.M. Feder J. J. Clin. Invest. 1989; 84: 1470-1478Google Scholar, 2Leung D.W. Cachianes G. Kuang W.-J. Goeddel D.V. Ferrara N. Science. 1989; 246: 1306-1309Google Scholar). Through interactions with its receptors KDR/flk-1 and flt1, VEGF has critical roles in the growth and maintenance of vascular endothelial cells and in the development of new blood vessels in physiologic and pathologic states(3Aiello L.P. Avery R.L. Arrigg P.G. Keyt B.A. Jampel H.D. Shah S.T. Pasquale L.R. Thieme H. Iwamoto M.A. Park J.E. Nguyen H.V. Aiello L.M. Ferrara N. King G.L. N. Engl. J. Med. 1994; 331: 1480-1487Google Scholar, 4Shweiki D. Itin A. Soffer D. Keshet E. Nature. 1992; 359: 843-845Google Scholar, 5Berkman R. Merrill M. Reinhold W. J. Clin. Invest. 1993; 91: 153-159Google Scholar). The patterns of embryonic expression of VEGF suggest that it is crucial for differentiation of endothelial cells from hemangioblasts and for development of blood vessels at all stages of growth(6Jakeman L.B. Armanini M. Phillips H.S. Ferrara N. Endocrinology. 1993; 133: 848-859Google Scholar, 7Breier G. Albrecht U. Sterrer S. Risau W. Development. 1992; 114: 521-532Google Scholar). Among many potentially angiogenic factors, VEGF is the only one whose pattern of expression, secretion, and activity suggests a specific angiogenic function in normal development(8Klagsbrun M. Soker S. Curr. Biol. 1993; 3: 699-702Google Scholar).High affinity receptors for VEGF are found only on endothelial cells, and VEGF binding has been demonstrated on macro- and microvascular endothelial cells and in quiescent and proliferating endothelial cells, suggesting that these receptors are important for both growth and maintenance of all endothelial cells(6Jakeman L.B. Armanini M. Phillips H.S. Ferrara N. Endocrinology. 1993; 133: 848-859Google Scholar, 9Jakeman L.B. Winer J. Bennett G.L. Altar A. Ferrara N. J. Clin. Invest. 1992; 89: 244-253Google Scholar). The tyrosine kinases KDR/flk-1 and flt1 have been identified as candidate VEGF receptors by affinity cross-linking and competition binding assays(10de Vries C. Escobedo J.A. Ueno H. Houck K. Ferrara N. Williams L.T. Science. 1992; 255: 989-991Google Scholar, 11Millauer B. Wizigmann-Voos S. Schnurch H. Martinez R. Moller N.P.H. Risau W. Ullrich A. Cell. 1993; 72: 835-846Google Scholar, 12Terman B.I. Dougher-Vermazen M. Carrion M.E. Dimitrov D. Armellino D.C. Gospodarowicz D. Bohlen P. Biochem. Biophys. Res. Commun. 1992; 187: 1579-1586Google Scholar). These two receptor tyrosine kinases contain seven similar extracellular immunoglobulin domains and a conserved intracellular tyrosine kinase domain interrupted by a kinase insert(10de Vries C. Escobedo J.A. Ueno H. Houck K. Ferrara N. Williams L.T. Science. 1992; 255: 989-991Google Scholar, 13Matthews W. Jordan C.T. Gavin M. Jenkins N.A. Copeland N.G. Lemischka I.R. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9026-9030Google Scholar, 14Terman B. Carrion M. Kovacs E. Rasmussen B. Eddy R. Shows T. Oncogene. 1991; 6: 1677-1683Google Scholar); they are expressed specifically by endothelial cells in vivo(11Millauer B. Wizigmann-Voos S. Schnurch H. Martinez R. Moller N.P.H. Risau W. Ullrich A. Cell. 1993; 72: 835-846Google Scholar, 15Peters K. de Vries C. Williams L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8915-8919Google Scholar, 16Quinn T.P. Peters K.G. de Vries C. Ferrara N. Williams L.T. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7533-7537Google Scholar, 17Yamaguchi T.P. Dumont D.J. Conion R.A. Breitman M.L. Rossant J. Development. 1993; 118: 489-498Google Scholar). In situ hybridization in the developing mouse has demonstrated that KDR/flk-1 is expressed in endothelial cells at all stages of development, as well as in the blood islands in which endothelial cell precursors first appear(11Millauer B. Wizigmann-Voos S. Schnurch H. Martinez R. Moller N.P.H. Risau W. Ullrich A. Cell. 1993; 72: 835-846Google Scholar), and that KDR/flk-1 specifies endothelial cell precursors at their earliest stages of development(17Yamaguchi T.P. Dumont D.J. Conion R.A. Breitman M.L. Rossant J. Development. 1993; 118: 489-498Google Scholar).The vascular endothelium is critical for physiologic responses including thrombosis and thrombolysis, lymphocyte and macrophage homing, modulation of the immune response, and regulation of vascular tone. The endothelium is also intimately involved in the pathogenesis of vascular diseases such as atherosclerosis(18Ross R. Nature. 1993; 362: 801-809Google Scholar). Although a number of genes expressed in the endothelium have been characterized(19Collins T. Williams A. Johnston G.I. Kim J. Eddy R. Shows T. Gimbrone Jr., M.A. Bevilacqua M.P. J. Biol. Chem. 1991; 266: 2466-2473Google Scholar, 20Iademarco M.F. McQuillan J.J. Rosen G.D. Dean D.C. J. Biol. Chem. 1992; 267: 16323-16329Google Scholar, 21Jahroudi N. Lynch D.C. Mol. Cell. Biol. 1994; 14: 999-1008Google Scholar, 22Lee M.E. Bloch K.D. Clifford J.A. Quertermous T. J. Biol. Chem. 1990; 265: 10446-10450Google Scholar), expression of these genes is either not limited to vascular endothelium (e.g. the genes encoding von Willebrand factor, endothelin-1, vascular cell adhesion molecule-1, platelet/endothelial cell adhesion molecule-1) or is restricted to specific subpopulations of endothelial cells (e.g. the gene for endothelial-leukocyte adhesion molecule-1). (A fragment of the promoter for Tek/Tie2, another developmentally regulated endothelial cell receptor tyrosine kinase, has recently been shown to direct transgene expression in subpopulations of endothelial cells during mouse embryonic development but not in endothelial cells of adult mice(23Schlaeger T.M. Qin Y. Fujiwara Y. Magram J. Sato T.N. Development. 1995; 121: 1089-1098Google Scholar). This suggests that the Tek/Tie2 promoter fragment used in this study is sufficient to direct gene expression to subpopulations of endothelial cells during specific periods of development, although functional elements within this promoter have not yet been identified.) In contrast with cells derived from the skeletal muscle and hematopoietic lineages, little is known about the mechanisms of specification and differentiation of endothelial cells. To understand the molecular mechanisms regulating cell type specificity and activation of the differentiation pathway in endothelial cells, we studied the control of KDR/flk-1 transcription.As a first step we cloned and characterized the promoter for the human KDR/flk-1 gene. We report here the sequence of the 5′-flanking region of the gene and identify a single transcription start site located 303 bp 5′ of the initiation methionine codon. Four kilobases of KDR/flk-1 5′-flanking sequence were found to have promoter activity similar to that of the potent SV40 promoter/enhancer in reporter gene transfection experiments in endothelial cells. Deletion analysis in endothelial cells showed the presence of positive regulatory elements in regions from bp −225 to −164, −95 to −77, −77 to −60, and +105 to +127. We found that KDR/flk-1 mRNA was expressed specifically in endothelial cells in culture and that 4 kb of the KDR/flk-1 5′-flanking sequence had cell type-specific promoter activity in transient transfection assays.DISCUSSIONAs a receptor for VEGF, KDR/flk-1 plays an essential role in angiogenesis and endothelial cell growth, and it is among the earliest markers of endothelial cell differentiation during development. Moreover, in situ analysis and immunocytochemistry have shown that KDR/flk-1 expression is restricted to endothelial cells in vivo; presumably this restricted pattern of expression determines the pattern of VEGF activity. Despite the importance of the KDR/flk-1 gene in endothelial cell growth, the mechanisms that regulate and restrict its expression are not known. We report for the first time the cloning and characterization of the human and mouse KDR/flk-1 promoters, and we identify regions containing positive regulatory elements within the 5′-flanking region of the human gene.Analysis of the human KDR/flk-1 5′-flanking region reveals that the transcription start site is located 303 bp 5′ of the methionine initiation codon. Like constitutive endothelial nitric oxide synthase (40Marsden P.A. Heng H.H.Q. Scherer S.W. Stewart R.J. Hall A.V. Shi X.-M. Tsui L.-C. Schappert K.T. J. Biol. Chem. 1993; 268: 17478-17488Google Scholar), another gene expressed in endothelial cells, KDR/flk-1 lacks a TATA box, is rich in G and C residues, and has numerous putative binding sites for Sp1, a ubiquitous nuclear protein that can initiate transcription of TATA-less genes(41Pugh B. Tjian R. Cell. 1990; 61: 1187-1197Google Scholar). We identified by deletion analysis three sequences within the 5′-flanking region of the KDR/flk-1 gene which appear to contain elements important for its expression in endothelial cells. Deletion of sequences between bp −225 and −164 reduced activity to 63% that of the full-length promoter, deletion between −95 and −77 further reduced promoter activity to 20%, and deletion from −77 to −60 reduced promoter activity to less than 5%. Because potential binding sites for Sp1, AP-2, NFκB, and E-box proteins located within these three positive regulatory elements in the human KDR/flk-1 gene are also present in the mouse 5′-flanking sequence, they may represent functional binding domains. AP-2 is a developmentally regulated trans-acting factor (42Mitchell P. Timmons P. Hebert J. Rigby P. Tjian R. Genes & Dev. 1991; 5: 105-119Google Scholar) without a demonstrated role in endothelial cell gene regulation. NFκB, however, trans-activates the inducible expression of vascular cell adhesion molecule-1 and tissue factor in endothelial cells (20Iademarco M.F. McQuillan J.J. Rosen G.D. Dean D.C. J. Biol. Chem. 1992; 267: 16323-16329Google Scholar, 43Moll T. Czyz M. Holzmuller H. Hofer-Warbinek R. Wagner E. Winkler H. Bach F. Hofer E. J. Biol. Chem. 1995; 270: 3849-3857Google Scholar) and is known to be a mediator of tissue-specific gene regulation(33Lenardo M. Baltimore D. Cell. 1989; 58: 227-229Google Scholar). Nuclear proteins that bind the E-box motif include the basic helix-loop-helix family of trans-acting factors. E-box-binding proteins have not been clearly associated with endothelial cell gene expression, although members of this family are critical for proper maturation of many cell types, including skeletal muscle and B lymphocytes(36Buskin J.N. Hauschka S.D. Mol. Cell. Biol. 1989; 9: 2627-2640Google Scholar, 44Murre C. McCaw P. Vaessin H. Caudy M. Jan L. Jan Y. Cabrera C. Buskin J. Hauschka S. Lassar A. Weintraub H. Baltimore D. Cell. 1989; 58: 537-544Google Scholar). Further experiments will be necessary to determine if these or other unidentified nuclear proteins specifically trans-activate the KDR/flk-1 gene.Four zinc finger-containing transcription factors in the GATA protein family bind to the consensus sequence (A/T)GATA(A/G) and regulate cell type-specific gene expression in many cell lineages(45Orkin S. Blood. 1992; 80: 575-581Google Scholar); among these GATA-2 has been most closely linked to endothelial cell gene expression. GATA-2 functions as an enhancer of endothelin-1 gene expression (39Lee M.E. Temizer D.H. Clifford J.A. Quertermous T. J. Biol. Chem. 1991; 266: 16188-16192Google Scholar) and acts to restrict expression of von Willebrand factor to endothelial cells(21Jahroudi N. Lynch D.C. Mol. Cell. Biol. 1994; 14: 999-1008Google Scholar). We observed that the human KDR/flk-1 5′-flanking region has two potential GATA-binding sequences, at positions −759 and +107, and that loss of the element located at −759 had no effect on expression of KDR/flk-1 in endothelial cells. The potential GATA element at position +107 is in a region of the first exon which we have identified as a powerful positive regulatory element. Although this GATA sequence (GGATAT) differs from the GATA-binding sequences of endothelin-1 and von Willebrand factor and from the consensus GATA sequence (A/T)GATA(A/G), we speculated that it might be the functional motif in the region between +105 and +127 because the functional GATA site in the von Willebrand factor gene is located similarly in the first exon and because a similar GATA element is found in the first exon of the mouse KDR/flk-1 gene. To our surprise, mutation of 3 bp in this element (GATA to GTCG), which had been observed to prevent trans-activation of the GATA cis-acting element in the endothelin-1 promoter,2 had no significant effect on KDR/flk-1 promoter activity (Fig. 5). Thus, our deletion analysis and mutagenesis studies do not support a functional role for the two GATA sequences in the human promoter in its high level activity in endothelial cells. These observations are consistent with the finding that early stages of endothelial cell development are normal in mice deficient in GATA-2 (46Tsai F. Keller G. Kuo F. Weiss M. Chen J. Rosenblatt M. Alt F. Orkin S. Nature. 1994; 371: 221-226Google Scholar) and suggest that other transcription factors are necessary for expression of the human KDR/flk-1 gene.We demonstrate in this study that expression of KDR/flk-1 is restricted to endothelial cells in culture, as it is in vivo. Moreover, we show that the activity of the KDR/flk-1 promoter in endothelial cells is similar to that of the potent SV40 promoter/enhancer and that this high level activity is specific to endothelial cells: activity in other cell types is markedly diminished. We do not yet understand why we observed low but detectable promoter activity in transient transfection assays of cell types that do not express the KDR/flk-1 gene in vivo; however, this situation is not unique among cell type-specific genes(47Goldhamer D. Faerman A. Shani M. Emerson C. Science. 1992; 256: 538-542Google Scholar). It is possible that other silencer elements outside of the 15.5-kb 5′-flanking region are necessary to block promoter activity completely in nonendothelial cells. Alternatively, the context of the promoter in relation to normal chromatin structure may be essential for precise regulation of the gene. An example of this type of regulation can be found in the control of MyoD expression. MyoD, like KDR/flk-1, is developmentally regulated, and it marks skeletal muscle precursors at an early stage(48Sassoon D. Lyons G. Wright W. Lin V. Lassar A. Weintraub H. Buckingham M. Nature. 1989; 341: 303-307Google Scholar). The MyoD 5′-flanking region contains an enhancer element that increases MyoD expression in many cell types in culture, even though MyoD expression is specific to skeletal muscle in vivo(47Goldhamer D. Faerman A. Shani M. Emerson C. Science. 1992; 256: 538-542Google Scholar). In contrast, transgenic constructs containing the MyoD enhancer are skeletal muscle-specific, implying that chromatin structure modifies the activity of this enhancer and regulates cell type specificity. Our results suggest that tissue-specific regulation of KDR/flk-1 involves a complex interaction between known, widely distributed nuclear factors and other, unknown elements. Therefore a complete explanation of the mechanisms of endothelial cell-specific expression of KDR/flk-1 may require integration of in vivo and in vitro observations.Identification of the regulatory mechanisms responsible for KDR/flk-1 expression is likely to provide important information about the specification and differentiation of endothelial cells early in embryogenesis. Moreover, knowledge about DNA elements that restrict gene expression to endothelial cells may be useful for deciphering the function of proteins in this cell type and, potentially, for directing or preventing expression of genes specifically in endothelial cells INTRODUCTIONVascular endothelial growth factor (VEGF) 1The abbreviations used are: VEGFvascular endothelial growth factorbpbase pair(s)kbkilobase(s)HUVEChuman umbilical vein endothelial cell(s)PCRpolymerase chain reactionBAECbovine aortic endothelial cell(s). is a potent and specific endothelial cell mitogen(1Connolly D.T. Heuvelman D.M. Nelson R. Olander J.V. Eppley B.L. Delfino J.J. Leingruber R.M. Feder J. J. Clin. Invest. 1989; 84: 1470-1478Google Scholar, 2Leung D.W. Cachianes G. Kuang W.-J. Goeddel D.V. Ferrara N. Science. 1989; 246: 1306-1309Google Scholar). Through interactions with its receptors KDR/flk-1 and flt1, VEGF has critical roles in the growth and maintenance of vascular endothelial cells and in the development of new blood vessels in physiologic and pathologic states(3Aiello L.P. Avery R.L. Arrigg P.G. Keyt B.A. Jampel H.D. Shah S.T. Pasquale L.R. Thieme H. Iwamoto M.A. Park J.E. Nguyen H.V. Aiello L.M. Ferrara N. King G.L. N. Engl. J. Med. 1994; 331: 1480-1487Google Scholar, 4Shweiki D. Itin A. Soffer D. Keshet E. Nature. 1992; 359: 843-845Google Scholar, 5Berkman R. Merrill M. Reinhold W. J. Clin. Invest. 1993; 91: 153-159Google Scholar). The patterns of embryonic expression of VEGF suggest that it is crucial for differentiation of endothelial cells from hemangioblasts and for development of blood vessels at all stages of growth(6Jakeman L.B. Armanini M. Phillips H.S. Ferrara N. Endocrinology. 1993; 133: 848-859Google Scholar, 7Breier G. Albrecht U. Sterrer S. Risau W. Development. 1992; 114: 521-532Google Scholar). Among many potentially angiogenic factors, VEGF is the only one whose pattern of expression, secretion, and activity suggests a specific angiogenic function in normal development(8Klagsbrun M. Soker S. Curr. Biol. 1993; 3: 699-702Google Scholar).High affinity receptors for VEGF are found only on endothelial cells, and VEGF binding has been demonstrated on macro- and microvascular endothelial cells and in quiescent and proliferating endothelial cells, suggesting that these receptors are important for both growth and maintenance of all endothelial cells(6Jakeman L.B. Armanini M. Phillips H.S. Ferrara N. Endocrinology. 1993; 133: 848-859Google Scholar, 9Jakeman L.B. Winer J. Bennett G.L. Altar A. Ferrara N. J. Clin. Invest. 1992; 89: 244-253Google Scholar). The tyrosine kinases KDR/flk-1 and flt1 have been identified as candidate VEGF receptors by affinity cross-linking and competition binding assays(10de Vries C. Escobedo J.A. Ueno H. Houck K. Ferrara N. Williams L.T. Science. 1992; 255: 989-991Google Scholar, 11Millauer B. Wizigmann-Voos S. Schnurch H. Martinez R. Moller N.P.H. Risau W. Ullrich A. Cell. 1993; 72: 835-846Google Scholar, 12Terman B.I. Dougher-Vermazen M. Carrion M.E. Dimitrov D. Armellino D.C. Gospodarowicz D. Bohlen P. Biochem. Biophys. Res. Commun. 1992; 187: 1579-1586Google Scholar). These two receptor tyrosine kinases contain seven similar extracellular immunoglobulin domains and a conserved intracellular tyrosine kinase domain interrupted by a kinase insert(10de Vries C. Escobedo J.A. Ueno H. Houck K. Ferrara N. Williams L.T. Science. 1992; 255: 989-991Google Scholar, 13Matthews W. Jordan C.T. Gavin M. Jenkins N.A. Copeland N.G. Lemischka I.R. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9026-9030Google Scholar, 14Terman B. Carrion M. Kovacs E. Rasmussen B. Eddy R. Shows T. Oncogene. 1991; 6: 1677-1683Google Scholar); they are expressed specifically by endothelial cells in vivo(11Millauer B. Wizigmann-Voos S. Schnurch H. Martinez R. Moller N.P.H. Risau W. Ullrich A. Cell. 1993; 72: 835-846Google Scholar, 15Peters K. de Vries C. Williams L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8915-8919Google Scholar, 16Quinn T.P. Peters K.G. de Vries C. Ferrara N. Williams L.T. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7533-7537Google Scholar, 17Yamaguchi T.P. Dumont D.J. Conion R.A. Breitman M.L. Rossant J. Development. 1993; 118: 489-498Google Scholar). In situ hybridization in the developing mouse has demonstrated that KDR/flk-1 is expressed in endothelial cells at all stages of development, as well as in the blood islands in which endothelial cell precursors first appear(11Millauer B. Wizigmann-Voos S. Schnurch H. Martinez R. Moller N.P.H. Risau W. Ullrich A. Cell. 1993; 72: 835-846Google Scholar), and that KDR/flk-1 specifies endothelial cell precursors at their earliest stages of development(17Yamaguchi T.P. Dumont D.J. Conion R.A. Breitman M.L. Rossant J. Development. 1993; 118: 489-498Google Scholar).The vascular endothelium is critical for physiologic responses including thrombosis and thrombolysis, lymphocyte and macrophage homing, modulation of the immune response, and regulation of vascular tone. The endothelium is also intimately involved in the pathogenesis of vascular diseases such as atherosclerosis(18Ross R. Nature. 1993; 362: 801-809Google Scholar). Although a number of genes expressed in the endothelium have been characterized(19Collins T. Williams A. Johnston G.I. Kim J. Eddy R. Shows T. Gimbrone Jr., M.A. Bevilacqua M.P. J. Biol. Chem. 1991; 266: 2466-2473Google Scholar, 20Iademarco M.F. McQuillan J.J. Rosen G.D. Dean D.C. J. Biol. Chem. 1992; 267: 16323-16329Google Scholar, 21Jahroudi N. Lynch D.C. Mol. Cell. Biol. 1994; 14: 999-1008Google Scholar, 22Lee M.E. Bloch K.D. Clifford J.A. Quertermous T. J. Biol. Chem. 1990; 265: 10446-10450Google Scholar), expression of these genes is either not limited to vascular endothelium (e.g. the genes encoding von Willebrand factor, endothelin-1, vascular cell adhesion molecule-1, platelet/endothelial cell adhesion molecule-1) or is restricted to specific subpopulations of endothelial cells (e.g. the gene for endothelial-leukocyte adhesion molecule-1). (A fragment of the promoter for Tek/Tie2, another developmentally regulated endothelial cell receptor tyrosine kinase, has recently been shown to direct transgene expression in subpopulations of endothelial cells during mouse embryonic development but not in endothelial cells of adult mice(23Schlaeger T.M. Qin Y. Fujiwara Y. Magram J. Sato T.N. Development. 1995; 121: 1089-1098Google Scholar). This suggests that the Tek/Tie2 promoter fragment used in this study is sufficient to direct gene expression to subpopulations of endothelial cells during specific periods of development, although functional elements within this promoter have not yet been identified.) In contrast with cells derived from the skeletal muscle and hematopoietic lineages, little is known about the mechanisms of specification and differentiation of endothelial cells. To understand the molecular mechanisms regulating cell type specificity and activation of the differentiation pathway in endothelial cells, we studied the control of KDR/flk-1 transcription.As a first step we cloned and characterized the promoter for the human KDR/flk-1 gene. We report here the sequence of the 5′-flanking region of the gene and identify a single transcription start site located 303 bp 5′ of the initiation methionine codon. Four kilobases of KDR/flk-1 5′-flanking sequence were found to have promoter activity similar to that of the potent SV40 promoter/enhancer in reporter gene transfection experiments in endothelial cells. Deletion analysis in endothelial cells showed the presence of positive regulatory elements in regions from bp −225 to −164, −95 to −77, −77 to −60, and +105 to +127. We found that KDR/flk-1 mRNA was expressed specifically in endothelial cells in culture and that 4 kb of the KDR/flk-1 5′-flanking sequence had cell type-specific promoter activity in transient transfection assays.