Transcriptional Regulation of Uterine Vascular Endothelial Growth Factor during Early Gestation in a Carnivore Model, Mustela vison

Vascular endothelial growth factor (VEGF) is an essential angiogenic signaling element that acts through its two tyrosine kinase receptors, inducing both proliferation of endothelial cells and vascular permeability. Given the importance of vasculogenesis and angiogenesis to early pregnancy, it is of interest to understand the mechanisms regulating vascular development at this stage. We previously demonstrated that VEGF and receptors are up-regulated during embryo implantation in an unique animal model, the mink, a species displaying obligate embryonic diapause. Herein we examined the role of prostaglandin E2 (PGE2) as a regulator of VEGF during early pregnancy and established the mechanisms of this regulation. We demonstrate that activated embryos secrete PGE2 and that expression of PGE synthase protein in the uterus is dependent upon direct contact with invading trophoblast cells during implantation. Using mink uterine stromal cells transfected with mink VEGF promoter driving the luciferase reporter gene, we show that PGE2 induces promoter transactivation and that this response can be eliminated by blockade of protein kinase A. Treatment with antagonists to PGE2 receptors EP2 and EP4 eliminated the PGE2-induced response in transfected cells. Deletional studies of the promoter revealed that a region of 99 bp upstream of the transcription start site is required for PGE2-induced transactivation. Mutation of an AP2/Sp1 cluster, found within the 99 bp, completely eliminated the PGE2 response. Furthermore, chromatin immunoprecipitation assays confirmed binding of the AP2 and Sp1 transcription factors to the endogenous mink VEGF promoter in uterine cells. PGE2 stimulated acetylation of histone H3 associated with the promoter region containing the AP2/Sp1 cluster. Taken together, these results demonstrate that PGE2 plays an important role in regulating uterine and thus placental vascular development, acting through its receptors EP2 and EP4, provoking protein kinase A activation of AP2 and Sp1 as well as acetylation of histone H3 to transactivate the VEGF promoter. Vascular endothelial growth factor (VEGF) is an essential angiogenic signaling element that acts through its two tyrosine kinase receptors, inducing both proliferation of endothelial cells and vascular permeability. Given the importance of vasculogenesis and angiogenesis to early pregnancy, it is of interest to understand the mechanisms regulating vascular development at this stage. We previously demonstrated that VEGF and receptors are up-regulated during embryo implantation in an unique animal model, the mink, a species displaying obligate embryonic diapause. Herein we examined the role of prostaglandin E2 (PGE2) as a regulator of VEGF during early pregnancy and established the mechanisms of this regulation. We demonstrate that activated embryos secrete PGE2 and that expression of PGE synthase protein in the uterus is dependent upon direct contact with invading trophoblast cells during implantation. Using mink uterine stromal cells transfected with mink VEGF promoter driving the luciferase reporter gene, we show that PGE2 induces promoter transactivation and that this response can be eliminated by blockade of protein kinase A. Treatment with antagonists to PGE2 receptors EP2 and EP4 eliminated the PGE2-induced response in transfected cells. Deletional studies of the promoter revealed that a region of 99 bp upstream of the transcription start site is required for PGE2-induced transactivation. Mutation of an AP2/Sp1 cluster, found within the 99 bp, completely eliminated the PGE2 response. Furthermore, chromatin immunoprecipitation assays confirmed binding of the AP2 and Sp1 transcription factors to the endogenous mink VEGF promoter in uterine cells. PGE2 stimulated acetylation of histone H3 associated with the promoter region containing the AP2/Sp1 cluster. Taken together, these results demonstrate that PGE2 plays an important role in regulating uterine and thus placental vascular development, acting through its receptors EP2 and EP4, provoking protein kinase A activation of AP2 and Sp1 as well as acetylation of histone H3 to transactivate the VEGF promoter. Prostaglandin E2 (PGE2) 2The abbreviations used are: PGE, prostaglandin E; COX, cyclooxygenase; VEGF, vascular endothelial growth factor; PKA, protein kinase A; ChIP, chromatin immunoprecipitation; RACE, rapid amplification of cDNA ends. is a prostanoid synthesized through the cyclooxygenase pathway characterized by the initial step of formation of prostaglandin H2 from arachidonic acid, catalyzed by the cyclooxygenases 1 and 2 (COX-1 and -2). Formation of PGE2 follows formation of prostaglandin H2 from arachidonic acid and is dependent on the presence of prostaglandin E synthase (PGE synthase). Two isoforms of PGE synthase have been identified; one is a cytosolic form that acts mostly on COX-1-derived prostaglandin H2. The second is a microsomal form, preferentially coupled with the inducible COX-2 induction of PGE2 generation (1Murakami M. Naraba H. Tanioka T. Semmyo N. Nakatani Y. Kojima F. Ikeda T. Fueki M. Ueno A. Oh S. Kudo I. J. Biol. Chem. 2000; 275: 32783-32792Abstract Full Text Full Text PDF PubMed Scopus (856) Google Scholar). PGE2 exerts its effects following binding to specific receptors containing seven transmembrane domains (2Narumiya S. Sugimoto Y. Ushikubi F. Physiol. Rev. 1999; 79: 1193-1226Crossref PubMed Scopus (0) Google Scholar). Four receptor subtypes have been identified to date: EP1, -2, -3, and -4, each activating different intracellular pathways. Knock-out models for each subtype have been investigated, and mice deficient for EP2 presented impaired ovulation and fertilization (3Tilley S.L. Audoly L.P. Hicks E.H. Kim H.S. Flannery P.J. Coffman T.M. Koller B.H. J. Clin. Invest. 1999; 103: 1539-1545Crossref PubMed Scopus (215) Google Scholar). The role of prostaglandins in reproductive processes has been extensively investigated. COX-2-deficient mice have impaired ovulation, fertilization, implantation, and decidualization (4Lim H. Paria B.C. Das S.K. Dinchuk J.E. Langenbach R. Trzaskos J.M. Dey S.K. Cell. 1997; 91: 197-208Abstract Full Text Full Text PDF PubMed Scopus (1262) Google Scholar). PGE2 is luteoprotective (5Arosh J.A. Banu S.K. Chapdelaine P. Madore E. Sirois J. Fortier M.A. Endocrinology. 2004; 145: 2551-2560Crossref PubMed Scopus (187) Google Scholar); it also plays a role in regulation of immune responses at the site of embryo attachment (6Lala P.K. Am. J. Reprod. Immunol. 1989; 20: 147-152Crossref PubMed Scopus (18) Google Scholar), in ovulation (7Matsumoto H. Ma W. Smalley W. Trzaskos J. Breyer R.M. Dey S.K. Biol. Reprod. 2001; 64: 1557-1565Crossref PubMed Scopus (133) Google Scholar), and in the decidualization process in rats (8Johnston M.E. Kennedy T.G. Biol. Reprod. 1984; 31: 959-966Crossref PubMed Scopus (11) Google Scholar, 9Kennedy T.G. Doktorcik P.E. Prostaglandins. 1988; 35: 207-219Crossref PubMed Scopus (24) Google Scholar). Recently, Wang et al. (10Wang X. Su Y. Deb K. Raposo M. Morrow J.D. Reese J. Paria B.C. J. Biol. Chem. 2004; 279: 30579-30587Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar) identified PGE2 as the major prostaglandin at implantation sites in hamsters, and expression of microsomal PGE synthase was correlated with expression of COX-2. In mice, both microsomal PGE synthase mRNA and protein were localized in the subluminal stroma surrounding the implanting blastocyst (11Ni H. Sun T. Ding N.Z. Ma X.H. Yang Z.M. Biol. Reprod. 2002; 67: 351-358Crossref PubMed Scopus (86) Google Scholar). The authors suggested embryonic induction of local PGE synthase, since expression of the enzyme was absent in pseudopregnant females as well as at interimplantation sites in the uterus. Early pregnancy in mammals is associated with morphological and functional changes in uterine cells, accompanied by vascular remodeling. These changes are required for both placental and embryonic development (12Breier G. Albrecht U. Sterrer S. Risau W. Development. 1992; 114: 521-532Crossref PubMed Google Scholar, 13Klauber N. Rohan R.M. Flynn E. D'Amato R.J. Nat. Med. 1997; 3: 443-446Crossref PubMed Scopus (179) Google Scholar, 14Risau W. Nature. 1997; 386: 671-674Crossref PubMed Scopus (4846) Google Scholar, 15Smith S.K. Hum. Reprod. Update. 1998; 4: 509-519Crossref PubMed Scopus (193) Google Scholar). Vascular endothelial growth factor (VEGF) is the major regulator of angiogenesis (16Ferrara N. Davis-Smyth T. Endocr. Rev. 1997; 18: 4-25Crossref PubMed Scopus (3668) Google Scholar) and is an important factor in regulation of the events of early implantation and establishment of the placenta (17Matsumoto H. Ma W.G. Daikoku T. Zhao X. Paria B.C. Das S.K. Trzaskos J.M. Dey S.K. J. Biol. Chem. 2002; 277: 29260-29267Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). VEGF is a homodimeric glycoprotein of 40-45 kDa, and, while best known for its potent endothelial cell-specific mitogenic activity, it also plays a role in increasing vascular permeability (18Ferrara N. Henzel W.J. Biochem. Biophys. Res. Commun. 1989; 161: 851-858Crossref PubMed Scopus (2011) Google Scholar, 19Gospodarowicz D. Abraham J.A. Schilling J. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 7311-7315Crossref PubMed Scopus (555) Google Scholar, 20Keck P.J. Hauser S.D. Krivi G. Sanzo K. Warren T. Feder J. Connolly D.T. Science. 1989; 246: 1309-1312Crossref PubMed Scopus (1801) Google Scholar, 21Leung D.W. Cachianes G. Kuang W.J. Goeddel D.V. Ferrara N. Science. 1989; 246: 1306-1309Crossref PubMed Scopus (4450) Google Scholar). Prostaglandins are among the factors reported to regulate VEGF (22Gately S. Cancer Metastasis Rev. 2000; 19: 19-27Crossref PubMed Scopus (345) Google Scholar). VEGF effects on angiogenesis are dependent upon binding to tyrosine kinase receptors, Flt-1 (Fms-like tyrosine kinase, also known as VEGFR-1) and KDR (kinase domain region, also known as VEGFR-2) (23Jussila L. Alitalo K. Physiol. Rev. 2002; 82: PubMed Scopus Google Scholar). We have previously demonstrated that VEGF and its receptors are up-regulated during of in the mink uterus J. S. Biol. Reprod. PubMed Scopus Google Scholar). Kennedy T.G. Biol. Reprod. 20: PubMed Scopus Google Scholar) that PGE2 is a regulator of vascular permeability at implantation sites in PGE2 has been to VEGF in a of cells R.M. J. Immunol. 2004; PubMed Scopus Google Scholar), cells R. Cancer Res. Google Scholar), endothelial cells R. S. H. Biochem. Biophys. Res. Commun. 2001; PubMed Scopus Google Scholar), cells G. D. Y. Biochem. Biophys. Res. Commun. PubMed Scopus Google Scholar), cells J.D. Yang Y. J. Wang X.H. Google Scholar), and cells T. Prostaglandins PubMed Scopus Google Scholar). are of PGE2 of VEGF and are to uterine In the we have established the of expression of PGE synthase in the uterus during early pregnancy and the role of PGE2 in implantation in an unique We the that local PGE2 of the VEGF as well as on the of this and the of were by the in with of the of of the and were and on a were during the 2 of to We and have that obligate embryonic and embryo activation and implantation in the mink W. J. Sci. PubMed Scopus Google Scholar, W. Biol. Reprod. PubMed Scopus Google Scholar, L. D. J. Reprod. Scholar), and a of of 1 was 1 following and for on the the of by the presence of uterine and of trophoblast was in females by two of were at the early of implantation from females at and following the as well as from pseudopregnant we have previously K. A. D.W. J. Reprod. Scholar), the in and in pseudopregnant and were and at induction of the were in and at mink uterine stromal A. J. Biol. Reprod. PubMed Scopus Google Scholar) was used for the in were in with containing of and of A from an of the mink A. and D. was also used for was in with and the as A by was also cells were in the as the mink cells and for were by of the uterine of females in and following initial embryo activation J.A. Biol. Reprod. 2004; PubMed Scopus (37) Google Scholar) with containing of were in of with for in the presence of mink uterine cells. of embryo were The for PGE2 was to et al. Sirois J. Biol. Reprod. 1999; PubMed Scopus Google Scholar). from was used with of with PGE2 and of and with prostaglandin prostaglandin and was and the of from to of and were in with of was an following the of the was by at and 1 of was used for transcription with the transcription to the for PGE and in the of and were used to for PGE synthase and for PGE receptors of the were and a cDNA was a following the and were with a and by for et were in a of were in a and with of used to PGE synthase and the in a and of the VEGF and region of the mink VEGF was by the from a from mink The for The were a for was by et was and The transcription start site of the VEGF was the for promoter of the of we the to the site of transcription from of the mink for amplification are in for and in a of PGE in were used to demonstrate expression of PGE synthase during the early implantation were and with in was for 1 in and were at with PGE synthase in A second was for 1 to PGE were was used as of the mink VEGF was a were from the by and for Mutation of the AP2 and Sp1 sites was the used for were the and to and to was to and in were and were in in this was used to the were transfected with of the containing the mink VEGF promoter for to the of was following the of and were 1 following were with the luciferase to the results for of the of promoter the of of PGE2 from to for the role of the protein kinase A were with and 1 and for the at a of 1 to PGE2 of the PGE2 receptors were antagonists for PGE2 receptors EP2 and EP4 were to transfected cells 1 to with was the luciferase and was with a assays were as by and 1999; 19: PubMed Scopus Google Scholar) with uterine stromal cells were in a and with PGE2 for to cells were for and protein were by the of to the at a of for at were in in of and and with a at 2 with at and was used to the chromatin used for the of was from of the was by with of for at histone and were and at The was and with in the following 2 2 1 1 The histone were by at for by was and with the was with an of The used for the are in from the of the were as were on a and was used for luciferase this was the of and the of in were of were by the of and the A of was of during the and activation were in the presence of mink uterine cells to to in to secrete of The embryos following activation by and that were in growth J.A. Biol. Reprod. 2004; PubMed Scopus (37) Google Scholar) of PGE2 in with uterine cells. cells to this of PGE and mRNA in the from implantation and interimplantation sites as well as from pseudopregnant females mRNA for PGE The of mRNA for this enzyme was in from the implantation of both embryonic and uterine at following implantation with uterine receptor expression was the receptors of the EP2 and EP4 subtypes were at and with among the The receptor mRNA was at of the sites of PGE in the the expression of the PGE synthase at implantation sites we were in it was of uterine embryonic We found the PGE synthase protein to be in the of In the PGE synthase was localized in the stromal surrounding the implanting embryo was in the uterine to the invading from interimplantation sites confirmed that in that cells in these direct contact with embryonic this protein E and and of the of the VEGF upstream of the was identified by of the The transcription site at bp upstream of the was by of the promoter of the and confirmed by the of different mink two of uterine and one of The VEGF promoter region in the mink a to the and and of the mink promoter identified response element previously identified in the and VEGF and among with the and was an important region was found in the promoter region, bp upstream of the transcription start PGE2 VEGF the reporter luciferase by the mink VEGF promoter in mink uterine stromal cells. We that PGE2 was of inducing a induction in transcription of the reporter gene, in response to the of and and a induction to the of in a of induction were at the different and of PGE2 Furthermore, we of and response to prostaglandin in the form of induction of VEGF The results that mink VEGF promoter is activated by PGE2 in mink cells as well as in the cells PGE2 of VEGF stromal cells transfected with a mink VEGF promoter driving the luciferase were with the and 1 of the VEGF promoter with following with PGE2 the of the pathway in PGE2-induced VEGF transfected cells were with the in of the activation by PGE2 blockade of the PGE2 response in mink cells EP2 and EP4 the PGE2-induced of VEGF that PGE2 VEGF through a pathway we to that the response in VEGF transcription was dependent upon binding to the receptors previously known to in to the receptors EP1, and EP4 were to the transfected stromal cells 1 to with binding to the for EP2 also We an to for the of this The on the transcription of the reporter by the mink VEGF promoter The both EP2 and receptors the transactivation of the VEGF promoter A was following with the EP4 of promoter activation was the cells were with antagonists for EP2 and EP4 AP2 and Sp1 PGE2-induced in the VEGF promoter to the of the promoter following PGE2 The two containing and bp, in of induction following PGE2 The 99 bp upstream of the transcription site in induction by different from the two of response to PGE2 was bp upstream of the and a of bp completely eliminated promoter the 99 bp upstream of the an AP2 and an Sp1 binding site were by We a to these sites to these transcription Given and a was to with both demonstrated that of these sites completely eliminated the PGE2-induced promoter transactivation of the mink VEGF promoter. of the promoter driving the luciferase reporter were transfected mink uterine stromal cells. Treatment of the of PGE2 for The of the AP2 and Sp1 sites are by of the AP2 and Sp1 sites the PGE2-induced VEGF promoter transactivation. stromal cells were transfected with containing AP2 and Sp1 sites of these response cells were for with and promoter was by luciferase The of in AP2 and Sp1 with the VEGF in to the of the AP2/Sp1 and of PGE2-induced promoter we to with PGE2 the binding of these two transcription factors to the endogenous mink VEGF promoter in the uterine stromal by cells were for to the of of PGE2 cells were and with the AP2 and Sp1 was and the was by the promoter region the transcription start the binding of both transcription factors to the VEGF promoter region in uterine cells was by with PGE2 A induction of AP2 binding to the VEGF promoter region was following PGE2 PGE2 the binding of Sp1 to the VEGF promoter. PGE2 PGE2 also plays a role in of we a on cells with an histone H3 on stromal cells were for and were with PGE2 for were and was and the was by the promoter region the transcription start Treatment with PGE2 was in inducing acetylation of histone H3 in with The region by to the promoter region containing the binding sites for AP2 and to be in PGE2 transactivation of the mink VEGF promoter A and and angiogenesis is to of A. N. Y. Acad. Sci. PubMed Scopus Google Scholar), the in to the implanting and VEGF has been demonstrated to be one of the major angiogenic factors inducing proliferation and of endothelial cells as well as permeability in in a of and (16Ferrara N. Davis-Smyth T. Endocr. Rev. 1997; 18: 4-25Crossref PubMed Scopus (3668) Google Scholar). The early of Kennedy T.G. Biol. Reprod. PubMed Scopus Google Scholar) demonstrated the importance of prostaglandins in the process of embryo implantation in and in the form of of implantation in mice of the COX-2 (4Lim H. Paria B.C. Das S.K. Dinchuk J.E. Langenbach R. Trzaskos J.M. Dey S.K. Cell. 1997; 91: 197-208Abstract Full Text Full Text PDF PubMed Scopus (1262) Google Scholar). We established the of COX-2 expression by both the trophoblast and uterine stroma at the site of implantation in the unique we in the the mink Sirois J. A. Endocrinology. 1998; PubMed Scopus Google Scholar). by et al. (17Matsumoto H. Ma W.G. Daikoku T. Zhao X. Paria B.C. Das S.K. Trzaskos J.M. Dey S.K. J. Biol. Chem. 2002; 277: 29260-29267Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar) on the COX-2-deficient have a functional prostaglandin and VEGF expression in implantation. We previously demonstrated in the mink VEGF mRNA and protein are up-regulated the of implantation J. S. Biol. Reprod. PubMed Scopus Google Scholar), an important role for this growth factor in this species and the for this In the we the to PGE2 as an important regulator of VEGF transcription during the in we show that the mink embryo that has from is an important of we have demonstrated the presence of the PGE synthase, the enzyme for of PGE2 from prostaglandin and have that it is in the uterus during early of pregnancy, a second of mRNA studies that PGE synthase is dependent on the presence of the invading we have the expression of PGE synthase in the stroma surrounding the invading and have demonstrated its from the interimplantation with a in mice PGE synthase mRNA and protein in the stroma surrounding the implanting blastocyst (11Ni H. Sun T. Ding N.Z. Ma X.H. Yang Z.M. Biol. Reprod. 2002; 67: 351-358Crossref PubMed Scopus (86) Google Scholar). The PGE synthase expression we to with expression of is localized at the sites of trophoblast in the of the uterine during early implantation Sirois J. A. Endocrinology. 1998; PubMed Scopus Google Scholar). the presence of the for PGE2 in a to for PGE2 by the PGE In the we were in of VEGF and the mechanisms in this regulation. PGE2 was its effects on VEGF expression in the of PGE2 by the and the presence of PGE synthase at the site of implantation. a in mink uterine stromal cells with the mink VEGF promoter driving the reporter we show that PGE2 induces expression of VEGF as PGE2 can through different receptors, each its second (2Narumiya S. Sugimoto Y. Ushikubi F. Physiol. Rev. 1999; 79: 1193-1226Crossref PubMed Scopus (0) Google Scholar). acts through activation of the and by inducing a in intracellular M. S. M. Endocrinology. 2001; PubMed Scopus Google Scholar), EP2 and EP4 both responses through activation of the and both have been in PGE2-induced regulation of VEGF T. Prostaglandins PubMed Scopus Google Scholar, S. 2004; 18: PubMed Scopus Google Scholar, D. D. L. J. A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). isoforms have been identified to and can through different signaling associated with and activation (2Narumiya S. Sugimoto Y. Ushikubi F. Physiol. Rev. 1999; 79: 1193-1226Crossref PubMed Scopus (0) Google Scholar), the of for this receptor subtype in uterine and embryonic that it is in early implantation processes We demonstrated the presence of EP2 and EP4 mRNA in the mink uterine during early of implantation and as well as in the pseudopregnant in expression and thus regulation of these receptors was that regulation of the PGE2 of the receptors is for the effects in VEGF The that these two receptors responses that the in VEGF transcription is of protein P. J. Biochem. Biol. 1998; PubMed Scopus Google Scholar), we were to within the promoter region of the mink VEGF gene, responses transcription factors to binding were are We to that the AP2 and Sp1 are the of the PGE2 induction of the VEGF promoter. Mutation of these for AP2 and Sp1 was to of transcription induction by the of promoter The of has to that PGE2 binding of these two factors to the promoter region of with the studies of cells of the have Sp1 of in the induction of VEGF by PGE2 D. D. L. J. A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). the AP2/Sp1 has previously been to be in regulation of VEGF J. F. J. G. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, P. R. J. L. R. M. S. K. Biochem. J. PubMed Scopus Google Scholar). The AP2/Sp1 is also in regulation of gene, following of G. J.D. Endocrinology. 2004; 145: PubMed Scopus Google Scholar). The region we show transactivation in a region of the mink VEGF from to with in a region to regulate VEGF promoter activation by growth through the AP2 site in J. J. 1997; PubMed Scopus Google Scholar). has also been demonstrated by through AP2/Sp1 in P. R. J. L. R. M. S. K. Biochem. J. PubMed Scopus Google Scholar). protein kinase VEGF expression through AP2/Sp1 binding in J. F. J. G. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar), and growth factor through Sp1 in G. A. A. D. G. 1997; PubMed Scopus (187) Google Scholar). Taken together, the results in and and with the mink VEGF promoter for the that this promoter region, with the transcription factors AP2 and plays a role in the regulation of VEGF In to of chromatin are also required in to transactivation of promoter these histone acetylation has been associated with chromatin and of transcription factors to promoter Sci. 1998; PubMed Scopus Google Scholar). In the mink model, PGE2 acetylation of H3 localized to the promoter region, the containing the response for AP2 and that PGE2 induces activation of the transcription factors in with induction of chromatin to promoter binding and transactivation. In the the that PGE2 of embryonic and the expression of VEGF at implantation sites in the uterus of the in this unique of is on the presence of local prostaglandin for establishment of the required for of early We for animal for and for

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