Focal Adhesion Kinase Signaling Regulates Cardiogenesis of Embryonic Stem Cells

The signaling steps that induce cardiac differentiation in embryonic stem (ES) cells are incompletely understood. We examined the effect of adhesion signaling including Src and focal adhesion kinase (FAK) on cardiogenesis in mouse ES cells using α-myosin heavy chain promoter-driven enhanced green fluorescent protein or luciferase as reporters. Cardiac transcription factors including Nkx2.5 and Tbx5 mRNA were first expressed at day 4 in hanging drop embryoid bodies, and adhesion of embryoid bodies to surfaces at or before that day strongly inhibited differentiation of ES cells to cardiomyocytes. Since adhesion signaling could suppress cardiogenesis through Src kinases, embryoid bodies were exposed to the small molecule PP2, known as a Src family kinase inhibitor. PP2 during embryoid body adhesion dramatically increased cardiomyocyte differentiation and decreased mRNA expression of neuronal cellular adhesion molecule and α-fetoprotein, neuroectodermal, and endodermal markers, respectively. Surprisingly, although there was an interaction between Src and FAK in cardiogenesis, the procardiogenic effect of PP2 appeared incompletely explained by Src kinase inhibition, since another Src family kinase inhibitor, SU6656, failed to induce cardiogenesis. Instead, PP2 specifically inhibited adhesion-induced FAK phosphorylation. In ES cells stably expressing FAK-related nonkinase, which functions as a dominant negative FAK, cell migration from embryoid bodies was inhibited, whereas α-myosin heavy chain expression and myosin-stained cardiomyocytes were increased, suggesting that reducing cell motility may contribute to cardiogenesis. These data indicate that FAK is a key regulator of cardiogenesis in mouse ES cells and that FAK signaling within embryoid bodies can direct stem cell lineage commitment. The signaling steps that induce cardiac differentiation in embryonic stem (ES) cells are incompletely understood. We examined the effect of adhesion signaling including Src and focal adhesion kinase (FAK) on cardiogenesis in mouse ES cells using α-myosin heavy chain promoter-driven enhanced green fluorescent protein or luciferase as reporters. Cardiac transcription factors including Nkx2.5 and Tbx5 mRNA were first expressed at day 4 in hanging drop embryoid bodies, and adhesion of embryoid bodies to surfaces at or before that day strongly inhibited differentiation of ES cells to cardiomyocytes. Since adhesion signaling could suppress cardiogenesis through Src kinases, embryoid bodies were exposed to the small molecule PP2, known as a Src family kinase inhibitor. PP2 during embryoid body adhesion dramatically increased cardiomyocyte differentiation and decreased mRNA expression of neuronal cellular adhesion molecule and α-fetoprotein, neuroectodermal, and endodermal markers, respectively. Surprisingly, although there was an interaction between Src and FAK in cardiogenesis, the procardiogenic effect of PP2 appeared incompletely explained by Src kinase inhibition, since another Src family kinase inhibitor, SU6656, failed to induce cardiogenesis. Instead, PP2 specifically inhibited adhesion-induced FAK phosphorylation. In ES cells stably expressing FAK-related nonkinase, which functions as a dominant negative FAK, cell migration from embryoid bodies was inhibited, whereas α-myosin heavy chain expression and myosin-stained cardiomyocytes were increased, suggesting that reducing cell motility may contribute to cardiogenesis. These data indicate that FAK is a key regulator of cardiogenesis in mouse ES cells and that FAK signaling within embryoid bodies can direct stem cell lineage commitment. Embryonic stem (ES) 3The abbreviations used are:ESembryonic stemFAKfocal adhesion kinaseMHCmyosin heavy chainGFPgreen fluorescent proteinEGFPenhanced GFPBrdUrd(+)-5-bromo-2′-deoxyuridineFRNKFAK-related nonkinaseNCAMneuronal cellular adhesion moleculePECAM-1platelet endothelial cell adhesion molecule-1α-FPα-fetoproteinPDGFplatelet-derived growth factorERKextracellular signal-regulated kinase cells are totipotent cells derived from the inner cell mass of the preimplantation embryo (1Boheler K.R. Czyz J. Tweedie D. Yang H.T. Anisimov S.V. Wobus A.M. Circ. Res. 2002; 91: 189-201Crossref PubMed Scopus (609) Google Scholar, 2Sachinidis A. Fleischmann B.K. Kolossov E. Wartenberg M. Sauer H. Hescheler J. Cardiovasc. Res. 2003; 58: 278-291Crossref PubMed Scopus (196) Google Scholar, 3Gepstein L. Circ. 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Therefore, we explored adhesion signaling pathways, including Src and FAK, in the differentiation of mouse ES cells to cardiomyocytes. Here we report that embryoid body adhesion before the initiation of the cardiac differentiation program inhibits cardiogenesis. We explored signaling pathways downstream of including the Src tyrosine kinase We that of ES cells to PP2, a Src family kinase inhibitor, during embryoid body adhesion dramatically promotes cardiogenesis, as by in and cardiac expression and Surprisingly, we that the effect of PP2 was incompletely explained by Src kinase inhibition, and PP2 effect in by of adhesion-induced FAK In ES cells stably expressing FAK-related nonkinase, which functions as a dominant negative FAK, cell migration was inhibited, whereas cardiogenesis was These indicate that FAK signaling is a key negative regulator of cardiogenesis in mouse ES and FAK can promote cardiogenesis. and ES II, and the were from factor was from The was from The and luciferase were from and were from Science. The was from and to were from The mouse to cardiac and myosin were from the The to and were from Cell The to FAK and the of FAK were from and were from and were from and the mouse to were from was from the PP2, SU6656, and were from was from ES Cell and ES cells were on cells in with and were induce differentiation, cells were first and as hanging for embryoid body as T. Lord B. Schulze P.C. Fryer R.M. Sarang S.S. Gullans S.R. Lee R.T. Circulation. 2003; 107: 1912-1916Crossref PubMed Scopus (405) Google Scholar). with ES factor was and other embryoid body cells were on In cardiomyocytes within embryoid bodies were at and promoter-driven was and in T. Lord B. Schulze P.C. Fryer R.M. Sarang S.S. Gullans S.R. Lee R.T. Circulation. 2003; 107: 1912-1916Crossref PubMed Scopus (405) Google Scholar). the and were with and and the luciferase of and were to luciferase expression The of the was from with and and into the luciferase expression the expression the to of mouse was by from mouse The used were as to and The was into the with the and was by ES cells were with and for with as T. Lord B. Schulze P.C. Fryer R.M. Sarang S.S. Gullans S.R. Lee R.T. Circulation. 2003; 107: 1912-1916Crossref PubMed Scopus (405) Google Scholar). and was from ES cells with and of was with using the and the as T. Lord B. Schulze P.C. Fryer R.M. Sarang S.S. Gullans S.R. Lee R.T. Circulation. 2003; 107: 1912-1916Crossref PubMed Scopus (405) Google Scholar). of were as to and and and and and and and and and and and of Tbx5 and the is as for 4 and for for for by for a was by that the between the of and were within a and there were and for Tbx5 and respectively. bodies of cells were at day and luciferase were using the luciferase embryoid bodies were with with and with for cells were for with the to myosin and for with were with for and was as T. Lord B. Schulze P.C. Fryer R.M. Sarang S.S. Gullans S.R. Lee R.T. Circulation. 2003; 107: 1912-1916Crossref PubMed Scopus (405) Google Scholar). ES cells or embryoid bodies were with and with and the protein of cell were by were to in were with and with or and enhanced the to the of FAK was used as the cell were with protein and the of protein were with to FAK by with protein for were three with in and to Cell was as C. D.A. J. 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In cells from day 5 embryoid bodies, the of cells was decreased with PP2 as well as In the of was increased, whereas the cell were decreased with both at day 4 and 5 the other by cell migration was decreased both in and but not in or cells These data indicate that FAK cell migration and promotes cardiogenesis in embryoid In the we promoter-driven or luciferase as for cardiac differentiation from embryoid bodies of mouse ES cells. We first that embryoid body adhesion before the initiation of inhibits cardiogenesis and that of PP2, a known as an Src family kinase inhibitor, during embryoid body adhesion promotes cardiogenesis. we that effect of PP2 by Src kinase the procardiogenic effect is at to of adhesion-induced FAK In embryoid bodies, transcription factors and mRNA to after the induction by the that the of ES cell differentiation to cardiomyocytes that of cardiogenesis in the embryoid body adhesion before expression of these transcription cardiogenesis was strongly inhibited, suggesting that signals with the initial cardiac differentiation we the of integrin signaling on cardiogenesis, we that PP2, an of Src kinase that is an downstream kinase in integrin dramatically promotes cardiogenesis in ES cells. PP2 not induce or cell within embryoid bodies, since protein not The effect of PP2 was not in ES cell embryoid body ES cell can transcription factor expression and induce endoderm differentiation T. M. S. N. 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These of in differentiation in FAK knock-out ES and embryoid bodies were to which is the cardiogenesis in In mRNA or protein expression was not in their of FAK can to in cell or and is possible that cellular to or to in to cell commitment. et al. Lee White J.M. R. R.M. J. Biol. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar) reported that PP2 differentiation by of adhesion-induced FAK phosphorylation. In cell migration was inhibited both in cells and that of cell but not cell may promote cardiogenesis in ES cells. FAK effect by of of the downstream of FAK, since signaling is reported to mouse ES cell H. M.J. J. Biol. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar) as well as initial cardiac differentiation of mouse embryonic cells by signaling H. H. T. T. H. I. Circ. Res. 2005; PubMed Scopus Google Scholar). although their model of embryonic cells was from embryoid body that of signaling after day 4 inhibited differentiation into as we in the of FAK These could to of cardiomyocytes from ES cells and that FAK signaling could promote cardiogenesis of stem cells. to cardiomyocyte by PP2, downstream of FAK, and the mechanisms of the initial embryoid body may The mouse to cardiac and myosin by S. J. J. J. and D. A. were from the the of the of and by the of of with