Differential Regulation of Transforming Growth Factor β Signaling Pathways by Notch in Human Endothelial Cells

Notch and transforming growth factor β (TGFβ) play critical roles in endothelial-to-mesenchymal transition (EndMT), a process that is essential for heart development. Previously, we have shown that Notch and TGFβ signaling synergistically induce Snail expression in endothelial cells, which is required for EndMT in cardiac cushion morphogenesis. Here, we report that Notch activation modulates TGFβ signaling pathways in a receptor-activated Smad (R-Smad)-specific manner. Notch activation inhibits TGFβ/Smad1 and TGFβ/Smad2 signaling pathways by decreasing the expression of Smad1 and Smad2 and their target genes. In contrast, Notch increases SMAD3 mRNA expression and protein half-life and regulates the expression of TGFβ/Smad3 target genes in a gene-specific manner. Inhibition of Notch in the cardiac cushion of mouse embryonic hearts reduces Smad3 expression. Notch and TGFβ synergistically up-regulate a subset of genes by recruiting Smad3 to both Smad and CSL binding sites and cooperatively inducing histone H4 acetylation. This is the first evidence that Notch activation affects R-Smad expression and that cooperative induction of histone acetylation at specific promoters underlies the selective synergy between Notch and TGFβ signaling pathways. Notch and transforming growth factor β (TGFβ) play critical roles in endothelial-to-mesenchymal transition (EndMT), a process that is essential for heart development. Previously, we have shown that Notch and TGFβ signaling synergistically induce Snail expression in endothelial cells, which is required for EndMT in cardiac cushion morphogenesis. Here, we report that Notch activation modulates TGFβ signaling pathways in a receptor-activated Smad (R-Smad)-specific manner. Notch activation inhibits TGFβ/Smad1 and TGFβ/Smad2 signaling pathways by decreasing the expression of Smad1 and Smad2 and their target genes. In contrast, Notch increases SMAD3 mRNA expression and protein half-life and regulates the expression of TGFβ/Smad3 target genes in a gene-specific manner. Inhibition of Notch in the cardiac cushion of mouse embryonic hearts reduces Smad3 expression. Notch and TGFβ synergistically up-regulate a subset of genes by recruiting Smad3 to both Smad and CSL binding sites and cooperatively inducing histone H4 acetylation. This is the first evidence that Notch activation affects R-Smad expression and that cooperative induction of histone acetylation at specific promoters underlies the selective synergy between Notch and TGFβ signaling pathways. During heart development, a subset of endocardial cells undergoes endothelial-to-mesenchymal transition (EndMT) 4The abbreviations used are: EndMTendothelial-to-mesenchymal transitionNICDNotch intracellular domainH3K4Me3trimethylated histone 3 on lysine 4TGFtransforming growth factorR-Smadreceptor-activated SmadSBESmad binding elementHAhemagglutininHMEChuman microvascular endothelial cell(s)qRTquantitative reverse transcriptionqPCRquantitative PCRdnMAML1dominant negative MAML1Enembryonic day nDAPI4′,6-diamidino-2-phenylindoleChIPchromatin immunoprecipitationGAPDHglyceraldehyde-3-phosphate dehydrogenase. and migrates into the cardiac cushion to initiate valve formation (1.Armstrong E.J. Bischoff J. Circ. Res. 2004; 95: 459-470Crossref PubMed Scopus (524) Google Scholar). EndMT is regulated by multiple signaling pathways, including TGFβ and Notch (1.Armstrong E.J. Bischoff J. Circ. Res. 2004; 95: 459-470Crossref PubMed Scopus (524) Google Scholar). Although both pathways play critical roles in cardiovascular development (2.Niessen K. Karsan A. Circ. Res. 2008; 102: 1169-1181Crossref PubMed Scopus (194) Google Scholar, 3.ten Dijke P. Arthur H.M. Nat. Rev. Mol. Cell Biol. 2007; 8: 857-869Crossref PubMed Scopus (615) Google Scholar, 4.High F.A. Epstein J.A. Nat. Rev. Genet. 2008; 9: 49-61Crossref PubMed Scopus (245) Google Scholar), their functional interaction in endothelial cells remains to be fully investigated. We have previously shown that Notch and TGFβ synergistically induce expression of SNAIL and HEY1 in endothelial cells (5.Niessen K. Fu Y. Chang L. Hoodless P.A. McFadden D. Karsan A. J. Cell Biol. 2008; 182: 315-325Crossref PubMed Scopus (265) Google Scholar), both of which play roles in cardiac cushion development (6.Timmerman L.A. Grego-Bessa J. Raya A. Bertran E. Pérez-Pomares J.M. Díez J. Aranda S. Palomo S. McCormick F. Izpisúa-Belmonte J.C. de la Pompa J.L. Genes Dev. 2004; 18: 99-115Crossref PubMed Scopus (756) Google Scholar, 7.Fischer A. Schumacher N. Maier M. Sendtner M. Gessler M. Genes Dev. 2004; 18: 901-911Crossref PubMed Scopus (528) Google Scholar), suggesting functional integration between Notch and TGFβ signaling pathways in endothelial cells during heart development. endothelial-to-mesenchymal transition Notch intracellular domain trimethylated histone 3 on lysine 4 transforming growth factor receptor-activated Smad Smad binding element hemagglutinin human microvascular endothelial cell(s) quantitative reverse transcription quantitative PCR dominant negative MAML1 embryonic day n 4′,6-diamidino-2-phenylindole chromatin immunoprecipitation glyceraldehyde-3-phosphate dehydrogenase. TGFβ is a multifunctional growth factor that is involved in many biological processes, including proliferation, differentiation, and apoptosis (8.Massagué J. Nat. Rev. Mol. Cell Biol. 2000; 1: 169-178Crossref PubMed Scopus (1653) Google Scholar, 9.Attisano L. Wrana J.L. Curr. Opin. Cell Biol. 2000; 12: 235-243Crossref PubMed Scopus (479) Google Scholar). The TGFβ signal is transmitted through specific transmembrane type I and type II serine/threonine kinase receptors. Upon TGFβ binding, the constitutively active TGFβ type II receptor recruits and phosphorylates TGFβ type I receptor, and the latter phosphorylates receptor-activated Smads (R-Smads), including Smad1, Smad2, Smad3, Smad5, and Smad8. The phosphorylated R-Smads then form a complex with a common Smad, Smad4, and translocate into the nucleus to regulate target gene expression through interaction with other cofactors (10.Massagué J. Seoane J. Wotton D. Genes Dev. 2005; 19: 2783-2810Crossref PubMed Scopus (1927) Google Scholar). In endothelial cells, TGFβ binds two distinct type I receptors, ALK1 (activin receptor-like kinase 1) and ALK5, to activate ALK1/Smad1/5/8 and ALK5/Smad2/3 signaling pathways. These two pathways regulate different genes and exert opposing biological functions in endothelial cells (11.Goumans M.J. Lebrin F. Valdimarsdottir G. Trends Cardiovasc. Med. 2003; 13: 301-307Crossref PubMed Scopus (281) Google Scholar, 12.Goumans M.J. Valdimarsdottir G. Itoh S. Rosendahl A. Sideras P. ten Dijke P. EMBO J. 2002; 21: 1743-1753Crossref PubMed Scopus (932) Google Scholar). The evolutionarily conserved Notch signaling pathway determines cell fate by regulating multiple cellular processes, including proliferation, differentiation, and apoptosis (13.Artavanis-Tsakonas S. Rand M.D. Lake R.J. Science. 1999; 284: 770-776Crossref PubMed Scopus (4918) Google Scholar, 14.Bray S. Furriols M. Curr. Biol. 2001; 11: R217-R221Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). In mammals, four Notch receptors (Notch1–Notch4) and five ligands (Dll1 (Delta-like 1), Dll3, Dll4, Jagged1, and Jagged2) have been identified. Notch signaling is initiated by ligand binding, which triggers proteolytic cleavage of the transmembrane receptor and release of the Notch intracellular domain (NICD). Translocation of NICD into the nucleus results in association with the DNA-binding protein CSL and recruitment of coactivators, such as MAML (Mastermind-like) to initiate transcription (15.Wu L. Sun T. Kobayashi K. Gao P. Griffin J.D. Mol. Cell Biol. 2002; 22: 7688-7700Crossref PubMed Scopus (209) Google Scholar, 16.Nam Y. Weng A.P. Aster J.C. Blacklow S.C. J. Biol. Chem. 2003; 278: 21232-21239Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar, 17.Nam Y. Sliz P. Song L. Aster J.C. Blacklow S.C. Cell. 2006; 124: 973-983Abstract Full Text Full Text PDF PubMed Scopus (344) Google Scholar). Cross-talk between the Notch and TGFβ pathways has not been studied in endothelial cells, where both the Smad1/5/8 and Smad2/3 pathways can be activated in the same cell via ALK1 and ALK5 receptors, respectively (11.Goumans M.J. Lebrin F. Valdimarsdottir G. Trends Cardiovasc. Med. 2003; 13: 301-307Crossref PubMed Scopus (281) Google Scholar, 12.Goumans M.J. Valdimarsdottir G. Itoh S. Rosendahl A. Sideras P. ten Dijke P. EMBO J. 2002; 21: 1743-1753Crossref PubMed Scopus (932) Google Scholar). Both synergy and antagonism between Notch and TGFβ signaling have been reported in other cell types, and the interaction between Notch and TGFβ signaling appears to be cell type- and context-dependent (18.Blokzijl A. Dahlqvist C. Reissmann E. Falk A. Moliner A. Lendahl U. Ibáñez C.F. J. Cell Biol. 2003; 163: 723-728Crossref PubMed Scopus (301) Google Scholar, 19.Zavadil J. Cermak L. Soto-Nieves N. Böttinger E.P. EMBO J. 2004; 23: 1155-1165Crossref PubMed Scopus (633) Google Scholar, 20.Niimi H. Pardali K. Vanlandewijck M. Heldin C.H. Moustakas A. J. Cell Biol. 2007; 176: 695-707Crossref PubMed Scopus (121) Google Scholar, 21.Sun Y. Lowther W. Kato K. Bianco C. Kenney N. L. D. M. S. D. 2005; PubMed Scopus Google Scholar, S. K. K. Y. M. S. M. K. H. S. 2005; PubMed Scopus Google Scholar, Y. H. S. J. Cell 2007; PubMed Scopus Google Scholar, S. H. J. Biol. Chem. 2008; Full Text Full Text PDF PubMed Scopus Google Scholar). in Notch signaling activated by of the constitutively active In the we have to the functional of TGFβ and Notch activation at in the (Delta-like is the Notch ligand in endothelial cells C. U. J. A. D. 2001; PubMed Scopus Google Scholar), and activation of Notch in cardiovascular development K. T. T. Genes Dev. 2004; 18: PubMed Scopus Google Scholar). Here, we report for the first that in endothelial cells, Notch activation by NICD expression of cells regulates TGFβ and signaling pathways by the expression of Notch activation the expression of Smad1 and Smad2 and their target Notch increases expression of Smad3 and regulates target genes in a gene-specific manner. Notch not increases SMAD3 mRNA Smad3 protein In of Notch signaling by the expression of dominant negative MAML1 reduces Smad3 expression in the cardiac cushion of mouse embryonic we that activation of TGFβ and Notch signaling recruits Smad3 to both Smad binding and CSL binding sites and cooperatively increases histone H4 acetylation at promoters in which of both pathways a for the induction of a subset of TGFβ and Notch target and the of interaction in and Smad2 and and Cell and and mouse H4 The human microvascular endothelial cell microvascular endothelial cells for and the cell and cells as previously (5.Niessen K. Fu Y. Chang L. Hoodless P.A. McFadden D. Karsan A. J. Cell Biol. 2008; 182: 315-325Crossref PubMed Scopus (265) Google Scholar). and used for of of used for of and TGFβ in and then with of human for cell as previously Y. H. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). and cells and a on for and at for The as the The in and with at 4 for and at for The as the in cell and the protein and used for the and II reverse in the of the on of the for in negative MAML1 in and be Chang and A. with T. T. D. T. T. K. 2005; 102: PubMed Scopus Google of L. and in the expression of Notch in endothelial manner. Inhibition of Notch by expression initiated at embryonic day by of the of the cardiac cushion at by cells with and cell of Smad3 protein expression in the cardiac cushion cells, Notch by expression at and Smad3 protein expression at by Smad3 protein expression and to cell by the in the same the interaction between Smad3 and cells with cell and cell with at 4 The immunoprecipitation then with protein Smad3 protein for at 4 with the with of and to for The for with NICD with of and cell at the Smad3 protein by The of Smad3 with as the of the with with for for Smad3 chromatin immunoprecipitation and for H4 and trimethylated histone 3 on lysine 4 then and the and of chromatin used for with and as a negative of the CSL binding sites Smad3 the the of the genes H4 and and the and in 3 and cells signal through two TGFβ type I receptor pathways, ALK1/Smad1/5/8 and ALK5/Smad2/3 (11.Goumans M.J. Lebrin F. Valdimarsdottir G. Trends Cardiovasc. Med. 2003; 13: 301-307Crossref PubMed Scopus (281) Google Scholar, 12.Goumans M.J. Valdimarsdottir G. Itoh S. Rosendahl A. Sideras P. ten Dijke P. EMBO J. 2002; 21: 1743-1753Crossref PubMed Scopus (932) Google Scholar). the interaction between Notch and we the expression of R-Smads in with activated that NICD Smad1 and Smad2 protein expression by and Smad3 protein and with on protein that NICD mRNA of and by and SMAD3 mRNA and not mRNA TGFβ the of which then form protein with and translocate to the NICD affects the expression of we that of R-Smads and be shown in NICD and and with in R-Smad protein expression. The of Notch activation on of Smad1, Smad2, and Smad3 by for Smad1, Smad2, and Smad3 in and TGFβ NICD affects the protein and of we the functional of NICD on the expression of TGFβ target genes. that activated Notch the and of target genes and and two and P. J. 2006; PubMed Scopus Google Scholar, A. K. S. M. J. A. 2003; 22: PubMed Scopus Google In to of Smad1 and Smad2 NICD the target genes and P. J. 2006; PubMed Scopus Google Scholar, S. Itoh S. D. ten Dijke P. S. J.M. EMBO J. PubMed Scopus Google Scholar, H. W. S. S. Y. J. J. Biol. Chem. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). of Notch and TGFβ expression of and with TGFβ suggesting a common signaling pathway through induction of In contrast, a on the expression of TGFβ/Smad3 target gene H. T. Y. S. M. K. M. Circ. Res. 2001; PubMed Scopus Google Scholar), and HEY1 between NICD and TGFβ to the we have previously for Snail (5.Niessen K. Fu Y. Chang L. Hoodless P.A. McFadden D. Karsan A. J. Cell Biol. 2008; 182: 315-325Crossref PubMed Scopus (265) Google Scholar). These results that the of Notch activation on the expression of TGFβ/Smad3 is Notch activation have as NICD on TGFβ signaling pathways, we with Dll4, the Notch ligand in endothelial cells C. U. J. A. D. 2001; PubMed Scopus Google Scholar), and cells with at a In with the NICD that Notch activation and mRNA expression by and and SMAD3 mRNA expression not mRNA expression with mRNA Smad1 and Smad2 protein and Smad3 protein by Notch activation and of Smad1 and Smad2 and that of Smad3 by Notch activation and that the of the R-Smads to is that the in R-Smad and a of R-Smad to Notch activation the and mRNA of the target genes and and target genes and the mRNA of and with Notch activation with TGFβ inducing and HEY1 expression as as on two Notch and that the we in of and Notch activation in we the cells into and TGFβ and mRNA expression of R-Smads and TGFβ target genes in two by The results that the of on TGFβ signaling is to Notch the in the Notch in to Notch activation has on Smad1 and Smad2 pathways a on Smad3 with induction of a subset of Smad3 target genes. subset of endocardial cells EndMT at in the which is regulated in by Notch and TGFβ pathways (1.Armstrong E.J. Bischoff J. Circ. Res. 2004; 95: 459-470Crossref PubMed Scopus (524) Google Scholar, K. Fu Y. Chang L. Hoodless P.A. McFadden D. Karsan A. J. Cell Biol. 2008; 182: 315-325Crossref PubMed Scopus (265) Google Scholar). Notch affects the expression of Smad3 during EndMT in we Smad3 expression in cardiac of embryonic hearts the endothelial Chang and A. the cells the Notch through into the cells of the cardiac cushion J.A. A. T. A. P. Dev. 2006; PubMed Scopus Google Scholar). Chang and A. We first the of Notch on of the cardiac Notch by inducing expression of at and cardiac cushion cells by and at Inhibition of Notch by for and in a of cell in cardiac cushion by and with that of endocardial Notch EndMT and We then Smad3 expression in cardiac cushion Notch by inducing expression of at and Smad3 protein expression in cardiac cushion cells at by Smad3 in the cardiac cushion cells and in Smad3 protein expression of cell Notch for with Notch activation regulates Smad3 expression in a in of to that Notch regulates Smad3 at the Smad3 protein with the mRNA by NICD and with by and with TGFβ SMAD3 mRNA to a as that by Notch activation and the of Smad3 protein in cells with active Notch and not in cells and NICD Smad3 we with NICD with to protein and Smad3 protein expression by shown in of Smad3 protein in cells in the cells, suggesting that NICD increases Smad3 protein In NICD not the of Smad1 and Smad2 we expression of NICD the protein of Smad3 expression is by the the of cells with with of NICD that NICD protein of the expression of is not by the Smad3 the of protein by NICD can be to at a Although we the interaction between NICD and Smad3 by in cells (18.Blokzijl A. Dahlqvist C. Reissmann E. Falk A. Moliner A. Lendahl U. Ibáñez C.F. J. Cell Biol. 2003; 163: 723-728Crossref PubMed Scopus (301) Google Scholar, 21.Sun Y. Lowther W. Kato K. Bianco C. Kenney N. L. D. M. S. D. 2005; PubMed Scopus Google Scholar), the of Smad3 by Notch is through interaction between Smad3 and NICD remains to be NICD has been shown to with Smad1 and Smad2 Y. Lowther W. Kato K. Bianco C. Kenney N. L. D. M. S. D. 2005; PubMed Scopus Google Scholar, F. Itoh S. M.J. Valdimarsdottir G. T. Y. L. Kato M. ten Dijke P. EMBO J. 2004; 23: PubMed Scopus Google Scholar). the synergy between Notch and TGFβ we with ALK5 kinase a to Notch activation to that the of and Snail by ALK5 Notch ALK5 not the induction of Smad3 by Smad3 that inhibits the signaling of the of Notch on Smad3 protein In contrast, of Notch signaling by the induction of Smad3 expression by and Smad3 to the by TGFβ These the functional integration between Notch and TGFβ signaling pathways and the of Notch on a subset of genes. The that a subset of TGFβ/Smad3 target genes is synergistically by the of Notch activation and TGFβ and to the the selective synergy between Notch activation and TGFβ We as a TGFβ target gene that is by not by Dll4, and is not synergistically by the and HEY1 as TGFβ and Notch target genes that synergistically by TGFβ and Smad3 is by Notch we first the activation of TGFβ and Notch pathways affects the of of Smad3 on the promoters of target genes. have been in S. Itoh S. D. ten Dijke P. S. J.M. EMBO J. PubMed Scopus Google Scholar), H. T. Y. S. M. K. M. Circ. Res. 2001; PubMed Scopus Google Scholar), and HEY1 J. Cermak L. Soto-Nieves N. Böttinger E.P. EMBO J. 2004; 23: 1155-1165Crossref PubMed Scopus (633) Google and CSL binding sites have been in HEY1 Gessler M. Res. 2000; PubMed Scopus Google and promoters not in the to 3 of the the of Smad3 on and CSL binding sites in we with with for and Smad3 on CSL binding sites in and HEY1 promoters a Smad3 by the CSL binding Smad3 results that the of Smad3 on the in the by TGFβ and not by Notch activation with the mRNA expression Smad3 on the in the not by Notch suggesting that induction of mRNA expression by both TGFβ and Notch pathways is not through the of Smad3 on the in In contrast, Smad3 on the CSL binding in the by Notch activation which to the induction of mRNA expression by both pathways Smad3 on both and CSL binding sites in the HEY1 both TGFβ and Notch pathways activated which is with expression that the expression of HEY1 mRNA both pathways activated These results that the of CSL binding sites in promoters is the factor that the recruitment of Smad3 to not to CSL binding sites by TGFβ and Notch and the selective synergy between the and Notch pathways. We histone by TGFβ and Notch the of specific target genes. both R-Smads and NICD with histone S. J. J. Heldin C.H. ten Dijke P. Res. 2000; PubMed Google Scholar, K. Lendahl U. Mol. Cell Biol. 2002; 22: PubMed Scopus Google and Smad3 is to both and CSL binding sites in the promoters of and HEY1 by TGFβ and Notch we TGFβ and Notch signaling pathways cooperatively induce histone acetylation in target genes. with of and for and histone H4 acetylation by H4 by to the of target genes. shown in histone H4 acetylation in the and of by TGFβ and not by Dll4, and with the expression the of both not histone H4 acetylation. In contrast, and in with the expression the of and TGFβ in induction of histone H4 acetylation in the of and HEY1 genes with signaling with the recruitment of Smad3 to both and CSL binding sites in and HEY1 promoters that TGFβ and histone acetylation in the and of genes. TGFβ and affects which is histone for active genes G. Fu Y. S. C. W. Weng Nat. Genet. 2007; PubMed Scopus Google Scholar), histone and that TGFβ in and not in HEY1 and genes on in of genes histone H4 not of histone is involved in the of specific Smad3 and TGFβ In we report that Notch activation the expression of TGFβ and signaling pathways in endothelial and pathways activate different target gene expression and play opposing roles in endothelial cells (11.Goumans M.J. Lebrin F. Valdimarsdottir G. Trends Cardiovasc. Med. 2003; 13: 301-307Crossref PubMed Scopus (281) Google Scholar, 12.Goumans M.J. Valdimarsdottir G. Itoh S. Rosendahl A. Sideras P. ten Dijke P. EMBO J. 2002; 21: 1743-1753Crossref PubMed Scopus (932) Google Scholar). In ALK1 phosphorylates and and of endothelial cells, ALK5 phosphorylates Smad2/3 and inhibits and of endothelial Smad2 and Smad3 have been shown to have both and distinct roles in regulating the expression of TGFβ target genes and TGFβ functions in a cellular context-dependent P. J. 2006; PubMed Scopus Google Scholar, E. J. D. M. C. E.P. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, J.A. J. Cell 2007; PubMed Scopus Google Scholar). of the of TGFβ signaling pathways in cellular can regulate distinct functional M. C. C. J. Cell 2005; PubMed Scopus Google Scholar, F. M.J. L. Valdimarsdottir G. M. C. Arthur H.M. ten Dijke P. EMBO J. 2004; 23: PubMed Scopus Google Scholar). Here, we the first evidence that Notch activation not modulates the between and signaling pathways, the the pathways. These a by which Notch activation modulates TGFβ signaling pathways in in endothelial TGFβ signaling is regulated at multiple including R-Smad expression. has been shown that affects TGFβ signaling by Smad2 protein mRNA expression in endothelial cells, by the and of Smad2 protein A. F. C. M. J.M. L. C. J. Cell 2007; PubMed Scopus Google Scholar). Here, we that Notch activation not increases Smad3 mRNA Smad3 protein as that Notch activation regulates Smad3 expression at both and is the ligand that Notch signaling in endothelial cells C. U. J. A. D. 2001; PubMed Scopus Google Scholar). that Notch activation has as that of NICD on TGFβ/Smad1 and TGFβ/Smad2 pathways. the of Notch activation on TGFβ/Smad3 target gene expression is NICD and The between Notch activation and of NICD on expression that the of Notch activation on TGFβ signaling both NICD and Notch activation a with TGFβ signaling in the of a subset of Smad3 target genes. genes that synergistically by Notch activation and TGFβ including and have been shown to play critical roles in embryonic development (5.Niessen K. Fu Y. Chang L. Hoodless P.A. McFadden D. Karsan A. J. Cell Biol. 2008; 182: 315-325Crossref PubMed Scopus (265) Google Scholar, 7.Fischer A. Schumacher N. Maier M. Sendtner M. Gessler M. Genes Dev. 2004; 18: 901-911Crossref PubMed Scopus (528) Google Scholar, J. A. Curr. Biol. 2002; 12: Full Text Full Text PDF PubMed Scopus Google Scholar, A. C. E. P. Gessler M. Circ. Res. 2007; PubMed Scopus Google Scholar, T. 2006; PubMed Scopus Google Scholar). In of of and and embryonic heart A. Schumacher N. Maier M. Sendtner M. Gessler M. Genes Dev. 2004; 18: 901-911Crossref PubMed Scopus (528) Google Scholar, J. A. Curr. Biol. 2002; 12: Full Text Full Text PDF PubMed Scopus Google Scholar, A. C. E. P. Gessler M. Circ. Res. 2007; PubMed Scopus Google Scholar), and of Snail cardiovascular T. 2006; PubMed Scopus Google Scholar). we have that a TGFβ/Smad3 target in cells H. T. Y. S. M. K. M. Circ. Res. 2001; PubMed Scopus Google Scholar), synergistically by Notch activation and TGFβ signaling in endothelial the association between the of expression and the of a heart has been in a M. E. G. C. C. A. F. 2008; PubMed Scopus Google Scholar). we that of Notch by the of in EndMT and reduces Smad3 expression in cardiac cushion cells of mouse embryonic and of Notch on a distinct signaling pathway the cardiac in (6.Timmerman L.A. Grego-Bessa J. Raya A. Bertran E. Pérez-Pomares J.M. Díez J. Aranda S. Palomo S. McCormick F. Izpisúa-Belmonte J.C. de la Pompa J.L. Genes Dev. 2004; 18: 99-115Crossref PubMed Scopus (756) Google Scholar). Although between Notch and TGFβ signaling (18.Blokzijl A. Dahlqvist C. Reissmann E. Falk A. Moliner A. Lendahl U. Ibáñez C.F. J. Cell Biol. 2003; 163: 723-728Crossref PubMed Scopus (301) Google and between Notch and F. Itoh S. M.J. Valdimarsdottir G. T. Y. L. Kato M. ten Dijke P. EMBO J. 2004; 23: PubMed Scopus Google have been previously the the synergy has not been In we that Smad3 on in the is not by Notch suggesting that induction of by both TGFβ and Notch signaling is not by Smad3 on the of we that Notch activation increases Smad3 on the CSL in the Smad3 on both and CSL sites in the HEY1 is both TGFβ and Notch pathways in with expression of HEY1 by TGFβ and Notch signaling as with signal In contrast, Smad3 of in the is not by Notch and a CSL binding is not in the to 3 the In with Smad3 on both and CSL we that TGFβ and Notch activation histone H4 acetylation in genes both and CSL sites and not in not CSL binding Notch activation is required for Smad3 of CSL binding sites in the and HEY1 promoters and the cooperative induction of histone acetylation the of NICD play a critical in recruiting Smad3 to CSL binding through the interaction between NICD and Smad3, as we have shown both Smad3 and NICD histone to their transcription S. J. J. Heldin C.H. ten Dijke P. Res. 2000; PubMed Google Scholar, K. Lendahl U. Mol. Cell Biol. 2002; 22: PubMed Scopus Google Scholar), formation of the complex histone Smad3 NICD and of histone acetylation and active gene expression. In of we that TGFβ and Notch activation histone acetylation of and HEY1 not the induction of a subset of target such as and by TGFβ and Notch activation is by Smad3 on both and CSL binding sites in their promoters and histone acetylation. In we for the first that Notch signaling affects the expression of R-Smads and modulates the between different signaling pathways in endothelial We that the induction of a subset of genes by Notch and TGFβ signaling is to the recruitment of Smad3 to both and CSL sites in the of target genes and the cooperative induction of histone acetylation. on the antagonism and synergy between Notch and TGFβ signaling pathways on the the functional interaction between two pathways. with

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