c-Src Activation Plays a Role in Endothelin-dependent Hypertrophy of the Cardiac Myocyte

Activation of the atrial natriuretic peptide (ANP) gene is regarded as one of the earliest and most reliable markers of hypertrophy in the ventricular cardiac myocyte. We have examined the role of the nonreceptor tyrosine kinases in the signaling mechanism(s) leading to hypertrophy using human ANP gene promoter activity as a marker. Endothelin (ET), a well known hypertrophic agonist, increased activity of c-Src, c-Yes, and Fyn within minutes and promoted a selective redistribution of each of these kinases within the cell. Overexpression of c-Src effected a significant increase in activity of a cotransfected human ANP promoter-driven chloramphenicol acetyl transferase reporter, while expression of either c-Yes or Fyn was considerably less effective in this regard. ET-dependent stimulation of the human ANP gene promoter was partially inhibited by co-transfection with dominant negative Ras or dominant negative Src or Csk or by treatment with the potent Src family-selective tyrosine kinase inhibitor PP1, suggesting that the Src family kinases are involved in signaling ET-dependent activation of this promoter. Both ET- and Src-dependent activation of the ANP promoter required the presence of a CArG motif in a serum response element-like structure between −422 and −413 but did not appear to require assembly of a ternary complex for full activity. These findings support a role for Src in the activation of ANP gene expression and suggest that this kinase may contribute in an important way to the signaling mechanisms that activate hypertrophy in the cardiac myocyte. Activation of the atrial natriuretic peptide (ANP) gene is regarded as one of the earliest and most reliable markers of hypertrophy in the ventricular cardiac myocyte. We have examined the role of the nonreceptor tyrosine kinases in the signaling mechanism(s) leading to hypertrophy using human ANP gene promoter activity as a marker. Endothelin (ET), a well known hypertrophic agonist, increased activity of c-Src, c-Yes, and Fyn within minutes and promoted a selective redistribution of each of these kinases within the cell. Overexpression of c-Src effected a significant increase in activity of a cotransfected human ANP promoter-driven chloramphenicol acetyl transferase reporter, while expression of either c-Yes or Fyn was considerably less effective in this regard. ET-dependent stimulation of the human ANP gene promoter was partially inhibited by co-transfection with dominant negative Ras or dominant negative Src or Csk or by treatment with the potent Src family-selective tyrosine kinase inhibitor PP1, suggesting that the Src family kinases are involved in signaling ET-dependent activation of this promoter. Both ET- and Src-dependent activation of the ANP promoter required the presence of a CArG motif in a serum response element-like structure between −422 and −413 but did not appear to require assembly of a ternary complex for full activity. These findings support a role for Src in the activation of ANP gene expression and suggest that this kinase may contribute in an important way to the signaling mechanisms that activate hypertrophy in the cardiac myocyte. Atrial natriuretic peptide (ANP) 1The abbreviations used are: ANP, atrial natriuretic peptide; hANP, human ANP; ET, endothelin; AII, angiotensin II; PHE, phenylephrine; MAP, mitogen-activated protein; SRE, serum response element; TK, thymidine kinase; PBS, phosphate-buffered saline; PIPES, 1,4-piperazinediethanesulfonic acid; SRF, serum response factor; Ets, E26 transformation-specific; CArG: CA-rich G, AP-1, activator protein-1. 1The abbreviations used are: ANP, atrial natriuretic peptide; hANP, human ANP; ET, endothelin; AII, angiotensin II; PHE, phenylephrine; MAP, mitogen-activated protein; SRE, serum response element; TK, thymidine kinase; PBS, phosphate-buffered saline; PIPES, 1,4-piperazinediethanesulfonic acid; SRF, serum response factor; Ets, E26 transformation-specific; CArG: CA-rich G, AP-1, activator protein-1.is a cardiac hormone involved in the regulation of intravascular volume and blood pressure (1Baxter J.D. Lewicki J. Gardner D.G. Bio/Technology. 1988; 6: 529-546Crossref Scopus (71) Google Scholar). Under normal conditions, the ANP gene is expressed almost exclusively in the cardiac atria, although modest levels of expression are also detected in the cardiac ventricle, hypothalamus, aortic arch, and lung (2Gardner D.G. Deschepper C.F. Ganong W.F. Hane S. Fiddes J. Baxter J.D. Lewicki J. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 6697-6701Crossref PubMed Scopus (197) Google Scholar, 3Gardner D.G. Vlasuk G.P. Baxter J.D. Fiddes J.C. Lewicki J.A. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 2175-2179Crossref PubMed Scopus (126) Google Scholar). Ventricular ANP gene expression is elevated during embryonic and early neonatal life but remains low throughout adulthood unless the ventricle is subjected to hemodynamic stress (e.g. volume or pressure overload). In this context, the ventricular myocyte undergoes characteristic biochemical and morphological changes that signal the initiation of hypertrophy. At the level of gene expression, this includes activation of the immediate early genes (proto-oncogenes like c-fos, c-jun, c-myc, and egr-1) followed by reactivation of a fetal gene program (e.g. ANP, β-myosin heavy chain, and α-skeletal actin) and subsequent up-regulation of sarcomeric contractile proteins (e.g. myosin light chain-2 and cardiac actin) (4Chien K.R. Zhu H. Knowlton K.U. Miller-Hance W. van-Bilsen M. O'Brien T.X. Evans S.M. Annu. Rev. Physiol. 1993; 55: 77-95Crossref PubMed Scopus (319) Google Scholar). The high degree of fidelity with which ANP gene expression is activated in this process has led to its identification as one of the earliest and most reliable markers of hypertrophy. While noin vitro system has been shown to mimic hypertrophy in vivo with absolute fidelity, the neonatal rat cardiac myocyte responds to a number of mechanical (e.g. passive stretch) (5Sadoshima J. Jahn L. Takahashi T. Kulik T.J. Izumo S. J. Biol. Chem. 1992; 267: 10551-10560Abstract Full Text PDF PubMed Google Scholar, 6Sadoshima J. Izumo S. EMBO J. 1993; 12: 1681-1692Crossref PubMed Scopus (561) Google Scholar) and biochemical (e.g. endothelin (ET) (7Shubeita H.E. McDonough P.M. Harris A.N. Knowlton K.U. Glembotski C.C. Brown J.H. Chien K.R. J. Biol. Chem. 1990; 265: 20555-20562Abstract Full Text PDF PubMed Google Scholar, 8Irons C.E. Murray S.F. Glembotski C.C. J. Biol. Chem. 1993; 268: 23417-23421Abstract Full Text PDF PubMed Google Scholar), angiotensin II (AII) (9Sadoshima J. Izumo S. Circ. Res. 1993; 73: 413-423Crossref PubMed Scopus (1276) Google Scholar), phenylephrine (PHE) (10Knowlton K.U. Baracchini E. Ross R.S. Harris A.N. Henderson S.A. Evans S.M. Glembotski C.C. Chien K.R. J. Biol. Chem. 1991; 266: 7759-7768Abstract Full Text PDF PubMed Google Scholar), or growth factors (11Parker T.G. Chow K.L. Schwartz R.J. Schneider M.D. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 7066-7070Crossref PubMed Scopus (77) Google Scholar, 12MacLellan W.R. Lee T.-C. Schwartz R.J. Schneider M.D. J. Biol. Chem. 1994; 269: 16754-16760Abstract Full Text PDF PubMed Google Scholar)) stimuli with phenotypic changes that closely parallel those seen with hypertrophy in the whole animal. Endothelin-1, a 21-amino acid vasoconstrictor, is one of the most potent hypertrophic stimuli in the neonatal myocyte system (7Shubeita H.E. McDonough P.M. Harris A.N. Knowlton K.U. Glembotski C.C. Brown J.H. Chien K.R. J. Biol. Chem. 1990; 265: 20555-20562Abstract Full Text PDF PubMed Google Scholar, 13Chien K.R. Knowlton K.U. Zhu H. Chien S. FASEB J. 1991; 5: 3037-3046Crossref PubMed Scopus (688) Google Scholar). ET-1 binds to a specific heterotrimeric G protein-coupled receptor that is linked to a number of well defined intracellular signaling pathways. Activation of phospholipase C, mobilization of intracellular calcium, activation of protein kinase C, and stimulation of MAP kinase activity have each been linked to ET-1 in the neonatal cardiac myocyte (4Chien K.R. Zhu H. Knowlton K.U. Miller-Hance W. van-Bilsen M. O'Brien T.X. Evans S.M. Annu. Rev. Physiol. 1993; 55: 77-95Crossref PubMed Scopus (319) Google Scholar, 7Shubeita H.E. McDonough P.M. Harris A.N. Knowlton K.U. Glembotski C.C. Brown J.H. Chien K.R. J. Biol. Chem. 1990; 265: 20555-20562Abstract Full Text PDF PubMed Google Scholar,14Bogoyevitch M.A. Glennon P.E. Andersson M.B. Clerk A. Lazou A. Marshall C.J. Parker P.J. Sugden P.H. J. Biol. Chem. 1994; 269: 1110-1119Abstract Full Text PDF PubMed Google Scholar). The latter is of particular interest in that MAP kinase has classically been associated with activation of receptors associated with tyrosine kinase activity. Several recent studies indicate that G protein-coupled receptors employ a very unique mechanism to effect this stimulation. This involves a Gβγ subunit-mediated increase in Src-tyrosine kinase activity, which in turn leads to phosphorylation of the Shc adapter protein, activation of the Shc-Grb2-Sos-Ras pathway, and increased MAP kinase activity (15Luttrell L.M. Hawes B.E. van Biesen T. Luttrell D.K. Lansing T.J. Lefkowitz R.J. J. Biol. Chem. 1996; 271: 19443-19450Abstract Full Text Full Text PDF PubMed Scopus (491) Google Scholar, 16Wan Y. Kurosaki T. Huang X.-Y. Nature. 1996; 380: 541-544Crossref PubMed Scopus (256) Google Scholar, 17Lopez-IIasaca M. Crespo P. Pellici G.P. Gutkind S.J. Wetzker R. Science. 1997; 275: 394-397Crossref PubMed Scopus (627) Google Scholar, 18Fooscchi M. Chari S. Dunn M. Sorokin A. EMBO J. 1997; 16: 6439-6451Crossref PubMed Scopus (141) Google Scholar). ET has been shown to stimulate tyrosine kinase activity in a number of systems (19Zachary I. Sinnett-Smith J. Turner C.E. Rozengurt E. J. Biol. Chem. 1993; 268: 22060-22065Abstract Full Text PDF PubMed Google Scholar, 20Simonson M.S. Wang Y. J. Physiol. 1996; Google Scholar, S. P. J. 1997; PubMed Google Scholar) has been linked to activation of c-Src in rat M.S. J. Biol. Chem. PubMed Scopus Google Scholar, M.S. Wang Y. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). In cardiac the is J. A. Res. 1994; PubMed Scopus Google Scholar) have shown that of tyrosine kinase activity activation of ANP, and myosin light chain-2 promoter activity in and Izumo J. Izumo S. EMBO J. 1996; PubMed Scopus Google Scholar) have that a tyrosine kinase closely to in these that this leads to stimulation of Ras and MAP biochemical markers that have been linked to the hypertrophic process J. Izumo S. EMBO J. 1993; 12: 1681-1692Crossref PubMed Scopus (561) Google Scholar, A. J. S. H.E. Chien K.R. J. Biol. Chem. 1993; 268: Full Text PDF PubMed Google Scholar, M.A. Marshall C.J. Sugden P.H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). are the findings of Y. I. T. R. S. I. Y. T. Y. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar), which the of the hypertrophic response tyrosine kinase suggesting that stimulation of MAP kinase by is linked to activation of protein kinase is as to the role of the tyrosine kinases in the signaling associated with the of hypertrophy. the this have the role of c-Src and tyrosine kinases in the signaling the ET receptor to activation of the ANP gene promoter by to the of hypertrophy in the neonatal cardiac myocyte. We have that ET activate c-Src in the myocyte and that this activation is closely to the subsequent activation of ANP gene The the of the human ANP gene linked to for chloramphenicol acetyl transferase the the of the to its The promoter was as J. Gardner D.G. J. Biol. Chem. 1988; Full Text PDF PubMed Google Scholar, Gardner D.G. 1991; 5: PubMed Scopus Google Scholar). was by using an which its and in the serum response of the gene using the for the CArG and and to the and The of the thymidine kinase promoter to to has been Science. PubMed Scopus Google Scholar). The of the gene and for and its CArG by using and its CArG as an which and the and a which and the used to the which of and negative Ras was by W. J. Csk was c-Src and J. A. and the and of c-Src was and Fyn and by c-Src c-Yes and Fyn that the to in the of c-Src was by This is by the is in the the kinase is in an activated H. T. Y. Y. M. J. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). was the peptide which in the of and the was a and was that c-Yes and Fyn was used for those used for of rat ventricular as Gardner D.G. Biol. 1992; 12: PubMed Scopus Google Scholar) and in serum the was to a defined serum In 12: PubMed Scopus Google Scholar). in serum the in of phosphate-buffered of with of expression as in the for was to with using the of using less within a each was in the was to serum Endothelin was The and the for activity and activity as Gardner D.G. Biol. 1992; 12: PubMed Scopus Google Scholar). was ventricular using the of ventricular was by to in and for and to the by for and with a rat ANP with using the in to of the and with to for in and of by with ET by of and with subsequent using in each of and and T. Biol. 1996; 16: PubMed Scopus Google Scholar). for was by by with of to and followed by of with specific of of protein for The the of of and for The with and with and and subjected to or the in vitro kinase for protein or in and to a in those serum was for treatment with a as by the with the in for a with and a with by using the system with in in the presence of for in and treatment with a with and the was c-Src kinase activity, c-Src was by with The of ventricular protein with Src kinase and using of a Src kinase The is Src-dependent phosphorylation of a peptide H. S. Wang J.H. J. Biol. Chem. 1992; 267: Full Text PDF PubMed Google Scholar). the with kinase PIPES, and with kinase of and for by in in and with a of with ET as and with of and either in for or in for and with was in for in in PBS, with and in with in in PBS, for with or in in for and for with a of the with with in was using a was with and of neonatal rat ventricular with ET in a in whole tyrosine kinase activity, by with the of proteins in the was and to of to the of to was increased by treatment with of the to proteins not of of Src in these in the levels of Src protein treatment with ET the was with an that the activated of Src an ET-dependent increase in signal was a of activated Src ET treatment not The ET-dependent activation of Src stimulation of the tyrosine kinase activity associated with this an as well as a Src kinase using a peptide H. S. Wang J.H. J. Biol. Chem. 1992; 267: Full Text PDF PubMed Google Scholar) as the In ET an almost increase in Src kinase activity, which and to levels of This was by of the tyrosine kinase inhibitor during ET stimulation the that of the Src family of tyrosine kinases by ET, examined for the presence of activated Fyn and shown in of with or in the absolute levels of these proteins ET the with that the activated of or a increase in levels of the activated kinases was These and of ET Activation of the Src family tyrosine kinases was by changes in within the cell. of neonatal rat by with In of c-Src a throughout the and a to the of with ET for a redistribution of c-Src in a The redistribution was to ET for the of not with a c-Src a not The levels of Src detected in cardiac in these considerably those seen in cardiac using Fyn an ET-dependent redistribution of the kinase a to in a In the of ET promoted a redistribution the seen in the myocyte to a suggesting a of the of the a that may an effect the assembly of the contractile in the of c-Src, Fyn and in but levels those seen in the ET, to activation of Src M.S. J. Biol. Chem. PubMed Scopus Google Scholar) and myocyte hypertrophy (7Shubeita H.E. McDonough P.M. Harris A.N. Knowlton K.U. Glembotski C.C. Brown J.H. Chien K.R. J. Biol. Chem. 1990; 265: 20555-20562Abstract Full Text PDF PubMed Google Scholar), to Src linked to markers of hypertrophy in this in vitro this a ANP promoter-driven reporter, a that has been shown to to a number of hypertrophic stimuli in vitro (7Shubeita H.E. McDonough P.M. Harris A.N. Knowlton K.U. Glembotski C.C. Brown J.H. Chien K.R. J. Biol. Chem. 1990; 265: 20555-20562Abstract Full Text PDF PubMed Google Scholar, 8Irons C.E. Murray S.F. Glembotski C.C. J. Biol. Chem. 1993; 268: 23417-23421Abstract Full Text PDF PubMed Google Scholar, J. Izumo S. Circ. Res. 1993; 73: 413-423Crossref PubMed Scopus (1276) Google Scholar, K.U. Baracchini E. Ross R.S. Harris A.N. Henderson S.A. Evans S.M. Glembotski C.C. Chien K.R. J. Biol. Chem. 1991; 266: 7759-7768Abstract Full Text PDF PubMed Google Scholar, 12MacLellan W.R. Lee T.-C. Schwartz R.J. Schneider M.D. J. Biol. Chem. 1994; 269: 16754-16760Abstract Full Text PDF PubMed Google Scholar). shown in ET treatment effected a increase in activity, which was by a dominant negative Ras by a dominant negative Src or by a kinase that Src activity phosphorylation of the of the S. M. S.A. EMBO J. 1993; 12: PubMed Scopus Google Scholar). expression of c-Src led to a significant of the promoter and this was by with the dominant negative Ras to a by Csk Overexpression of a effect promoter activation to c-Src In the of significant in the of promoter activation with the Src expression a that in c-Src activity the The by c-Src was with a of by the to for The was with the dominant negative Ras as was is in Fyn or expression led to a modest increase in activity and while a tyrosine kinase expressed in of the was The of activity with Fyn and is not to levels of expression to of the these proteins a of that the used in a level of not the levels of seen with and Fyn is to in promoter activity. the between tyrosine kinase activity and ANP gene of the with a potent and Src family-selective tyrosine kinase inhibitor J.H. Gardner J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar) in a in the ET-dependent increase in ANP levels as well as promoter activity was in the presence of the less selective protein tyrosine kinase not the of each ET and appear to linked to tyrosine kinase in the cardiac myocyte. We to the the promoter. shown in activation of the promoter was with and of the was although not as the was to the presence of a or between and in this promoter. We a number of this and the thymidine kinase gene promoter linked to the of the between and to by was to levels of promoter the of this a to the serum response of the a that has been as a of Src in M.S. J. Biol. Chem. PubMed Scopus Google Scholar). the R. 1986; Full Text PDF PubMed Scopus Google Scholar), the structure of the gene promoter a CArG its with a motif in in a structure to an CArG that also has a in the promoter We examined the role of each of these and in as of the Src within the of and examined for response to a cotransfected expression shown in of the CArG or the in a in promoter activity to of the while of the the for ternary complex assembly the promoter R. 1994; PubMed Scopus Google Scholar), was while of the CArG motif in a significant in the level of by of the or was almost of the in the CArG the between and in the These findings suggest that the CArG motif a dominant role in Src-dependent activation of the promoter. to the of this to the of gene promoter activity, the in of to the shown in ET effected a increase in the activity of and The was by of a the CArG or by a the Ets, and the The findings that an of hypertrophy in the cardiac myocyte a of its activity nonreceptor tyrosine We that ET the activity of c-Src, and in of neonatal rat cardiac ET-dependent of the gene promoter of c-Src, and activation of the promoter by either c-Src or ET the presence of a CArG motif between −422 and −413 in that promoter. This for the that the gene a for c-Src and a role for c-Src in cardiac hypertrophy. G protein-coupled receptors to signal a well defined of (e.g. and phospholipase and while activation of tyrosine kinases was seen as a of the and growth has support for G activation of tyrosine kinase activity Src family kinase within kinase activation by acid has been linked to the of the associated G protein complex (15Luttrell L.M. Hawes B.E. van Biesen T. Luttrell D.K. Lansing T.J. Lefkowitz R.J. J. Biol. Chem. 1996; 271: 19443-19450Abstract Full Text Full Text PDF PubMed Scopus (491) Google Scholar), that the activation involves a and AII, and ET receptors have been shown to stimulate tyrosine kinase activity (19Zachary I. Sinnett-Smith J. Turner C.E. Rozengurt E. J. Biol. Chem. 1993; 268: 22060-22065Abstract Full Text PDF PubMed Google Scholar, M.S. Wang Y. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar, Rozengurt E. J. Biol. 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A. 1993; PubMed Scopus Google Scholar), which Src activity, partially inhibited ET-dependent stimulation of the gene promoter. a recent by T. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar) of c-Src and but not in ventricular support to a role for Src in the hypertrophic studies with that ET of the promoter activation of c-Src M.S. J. Biol. Chem. PubMed Scopus Google Scholar, M.S. Wang Y. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar), a to that In the of the the activation the presence of an CArG motif in the SRE, while the which is required for assembly of the ternary complex of serum response protein or with to an for a response M.S. Wang Y. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar) in the promoter. In the the CArG motif in the promoter also to for the response to Src and The level of promoter by was to the CArG seen with the of the or an the CArG effect by The of the CArG by Src the of a (e.g. a response or a as CArG in the Src The of of the ET the motif in the promoter that of a ternary complex is not a number of studies have shown that growth activation of the promoter the MAP kinase involves ternary complex H. S. Wang J.H. J. Biol. Chem. 1992; 267: Full Text PDF PubMed Google Scholar, R. EMBO J. PubMed Scopus Google Scholar), studies have that stimuli (e.g. and intracellular of heterotrimeric G the R. EMBO J. PubMed Scopus Google Scholar). This is to activation of the family of G proteins and and not with activation of the MAP kinases protein or J. R. Full Text PDF PubMed Scopus Google Scholar). studies have that growth factors may signal the selective phosphorylation of in the J. Biol. 1993; PubMed Scopus Google Scholar), a that signaling the ET-dependent activation of the promoter to to the ternary almost exclusively the CArG motif for the have been with to the regulation of the rat ANP promoter by Murray S.F. Glembotski C.C. Circ. Res. PubMed Scopus Google Scholar). The latter to signal a CArG motif in the promoter of the gene a for ternary complex mechanism hypertrophic stimuli to gene in the cardiac myocyte is Activation of the gene by passive mechanical or an serum response and CArG in the promoter of that gene J. Izumo S. EMBO J. 1996; PubMed Scopus Google Scholar, J. Izumo S. Circ. Res. 1993; 73: PubMed Scopus Google Scholar). and protein kinase of the β-myosin heavy gene the presence of an in the promoter J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar). Activation of the gene by J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), as well as growth W.R. Lee T.-C. Schwartz R.J. Schneider M.D. J. Biol. Chem. 1994; 269: 16754-16760Abstract Full Text PDF PubMed Google Scholar), and CArG motif in the promoter has been linked to activation by growth T.G. Chow Schwartz R.J. Schneider M.D. J. Biol. Chem. 1992; 267: Full Text PDF PubMed Google Scholar). The activation of the rat ANP gene by Murray S.F. Glembotski C.C. Circ. Res. PubMed Scopus Google Scholar) or P.M. Glembotski C.C. J. Biol. Chem. 1997; PubMed Scopus Google Scholar) an CArG motif and a phenylephrine response a structure an Murray S.F. Glembotski C.C. Circ. Res. PubMed Scopus Google Scholar, A. M. EMBO J. 1993; 12: PubMed Scopus Google Scholar), while of the rat natriuretic peptide gene is an between and in that promoter Glembotski C.C. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). is in the used by ventricular to hypertrophic stimuli to the activation of gene CArG motif in the rat ANP promoter has been shown to with its in activity Murray S.F. Glembotski C.C. Circ. Res. PubMed Scopus Google Scholar). of interest to a motif is in the human promoter the of In have shown that the hypertrophic of ET in cardiac in We have also that ET and Src activate the gene promoter a CArG motif in the promoter a for ternary complex these findings suggest a important role for c-Src as a of cardiac hypertrophy. We are to for We are also to J. for We also the and and of and