Rho/ROCK Pathway Contributes to the Activation of Extracellular Signal-regulated Kinase/GATA-4 during Myocardial Cell Hypertrophy

The low molecular weight GTPase Rho mediates a variety of cytoskeleton-dependent cell functions and stretch- and Gq protein-induced hypertrophic responses in cardiac myocytes. Although ROCK, one of Rho's effectors, has been suggested to mediate hypertrophic signals, the relationship of Rho/ROCK with downstream signals is unknown. A zinc finger transcription factor, GATA-4, is activated by extracellular signal-regulated kinase 1/2 and is required for the up-regulation of the endothelin-1 gene during myocardial cell hypertrophy. However, it is unknown whether Rho/ROCK signals are linked to downstream GATA-4. By transient transfection assays using a dominant-negative mutant and an activated derivative of ROCK-I, we showed that ROCK-I participates in GATA-4-dependent endothelin-1 transcription. Inhibition of the Rho/ROCK pathway by Y-27632, a selective inhibitor of ROCK, suppressed phenylephrine-stimulated phosphorylation of extracellular signal-regulated kinase 1/2 and increased the DNA binding activity of cardiac GATA-4. Interestingly, latrunculin B, which inhibits actin polymerization, also prevents phenylephrine-induced responses. These findings demonstrate that the Rho/ROCK pathway is linked to downstream GATA-4 via the activation of extracellular signal-regulated kinases during myocardial cell hypertrophy. The results also suggest that changes in actin dynamics provide a convergence point for Rho/ROCK to the downstream signals during this process. The low molecular weight GTPase Rho mediates a variety of cytoskeleton-dependent cell functions and stretch- and Gq protein-induced hypertrophic responses in cardiac myocytes. Although ROCK, one of Rho's effectors, has been suggested to mediate hypertrophic signals, the relationship of Rho/ROCK with downstream signals is unknown. A zinc finger transcription factor, GATA-4, is activated by extracellular signal-regulated kinase 1/2 and is required for the up-regulation of the endothelin-1 gene during myocardial cell hypertrophy. However, it is unknown whether Rho/ROCK signals are linked to downstream GATA-4. By transient transfection assays using a dominant-negative mutant and an activated derivative of ROCK-I, we showed that ROCK-I participates in GATA-4-dependent endothelin-1 transcription. Inhibition of the Rho/ROCK pathway by Y-27632, a selective inhibitor of ROCK, suppressed phenylephrine-stimulated phosphorylation of extracellular signal-regulated kinase 1/2 and increased the DNA binding activity of cardiac GATA-4. Interestingly, latrunculin B, which inhibits actin polymerization, also prevents phenylephrine-induced responses. These findings demonstrate that the Rho/ROCK pathway is linked to downstream GATA-4 via the activation of extracellular signal-regulated kinases during myocardial cell hypertrophy. The results also suggest that changes in actin dynamics provide a convergence point for Rho/ROCK to the downstream signals during this process. myosin heavy chain endothelin-1 phenylephrine luciferase chloramphenicol acetyltransferase electrophoretic mobility shift assay extracellular signal-regulated kinase atrial natriuretic factor glyceraldehyde-3-phosphate dehydrogenase Cardiac myocytes within the adult heart are terminally differentiated and do not undergo cell division. In response to stimuli that affect the mechanical load on the heart or in response to various neurohumoral factors, the heart adapts through the activation of a hypertrophic response in individual cardiac muscle cells. This response is characterized by an increase in myocyte size, accumulation of contractile proteins within individual cardiac cells, and activation of embryonic gene marker expression (for reviews, see Refs. 1Chien K.R. Knowlton K.U. Zhu H. Chien S. FASEB J. 1991; 5: 3037-3046Crossref PubMed Scopus (708) Google Scholar, 2MacLellan W.R. Hawker J. Schneider M.D. Marks A.R. Taubman M.B. Molecular Biology of Cardiovascular Disease. Marcel Dekker, Inc., New York1997: 327-378Google Scholar, 3Copper G. Annu. Rev. Med. 1997; 48: 13-23Crossref PubMed Scopus (99) Google Scholar). For example, genes such as β-myosin heavy chain (MHC)1 and atrial natriuretic factor become highly expressed within ventricular myocytes (4Izumo S. Lompre A. Matsuoka R. Koren G. Schwarz K. Nadal-Ginard B. Mahdavi V. J. Clin. Invest. 1987; 79: 970-977Crossref PubMed Scopus (387) Google Scholar, 5Lee R.T. Bloch K.D. Pfeffer J.M. Pfeffer M.A. Neer E.J. Seidman C.E. J. Clin. Invest. 1988; 81: 431-434Crossref PubMed Scopus (99) Google Scholar, 6Izumo S. Nadal-Ginard B. Mahdavi V. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 339-343Crossref PubMed Scopus (772) Google Scholar). Studies focused on elucidating the mechanisms of transcriptional regulation of these genes have identified a group of DNA-binding factors that might mediate the nuclear response to hypertrophic stimuli. These factors include the GATA family of zinc finger transcription factors, which mediate transcriptional activation of the genes for β-MHC and angiotensin II type 1a receptor during pressure overload-induced hypertrophy in vivo (7Hasegawa K. Lee S.J. Jobe S.M. Markham B.E. Kitsis R.N. Circulation. 1997; 96: 3943-3953Crossref PubMed Scopus (154) Google Scholar, 8Herzig T.C. Jobe S.M. Aoki H. Molkentin J.D. Cowley A.W. Izumo S. Markham B.E. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7543-7548Crossref PubMed Scopus (180) Google Scholar, 9Morimoto T. Hasegawa K. Kaburagi S. Kakita T. Masutani H. Kitsis R.N. Matsumori A. Sasayama S. J. Biol. Chem. 1999; 274: 12811-12818Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). GATA factors are also required for the transcriptional activation of the endothelin-1 (ET-1) gene during the transition from compensation to heart failure (10Morimoto T. Hasegawa K. Kaburagi S. Kakita T. Wada H. Yanazume T. Sasayama S. J. Biol. Chem. 2000; 275: 13721-13726Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). During the hypertrophic process, cardiac GATA-4 is directly or indirectly phosphorylated by extracellular signal-regulated kinase (ERK)1/2, which increases the DNA binding ability of GATA-4 (10Morimoto T. Hasegawa K. Kaburagi S. Kakita T. Wada H. Yanazume T. Sasayama S. J. Biol. Chem. 2000; 275: 13721-13726Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). However, the relationship between ERKs/GATA-4 and upstream signaling pathways is unknown. One of the important upstream signaling molecules that mediate hypertrophic responses is the Ras-like small GTPase Rho. Rho plays a critical role in a variety of cytoskeleton-dependent cell functions including actin polymerization, F-actin bundling, myosin-based contractility, focal adhesion formation, and cytokinesis in other cell types (11Leung T. Chen X.Q. Manser E. Lim L. Mol. Cell. Biol. 1996; 16: 5313-5327Crossref PubMed Google Scholar, 12Amano M. Chihara K. Kimura K. Fukata Y. Nakamura N. Matsuura Y. Kaibuchi K. Science. 1997; 275: 1308-1311Crossref PubMed Scopus (956) Google Scholar, 13Uehata M. Ishizaki T. Satoh H. Ono T. Kawahara T. Morishita T. Tamakawa H. Yamagami K. Inui J. M. S. 1997; PubMed Scopus Google Scholar, A. Cell. Full Text PDF PubMed Scopus Google Scholar, N. A. Biol. 1997; PubMed Scopus Google Scholar). In cardiac activated Rho has on and and atrial natriuretic factor expression M. Y. Chien K.R. J. Biol. Chem. 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Kaburagi S. Kakita T. Masutani H. Kitsis R.N. Matsumori A. Sasayama S. J. Biol. Chem. 1999; 274: 12811-12818Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, T. Hasegawa K. Kaburagi S. Kakita T. Wada H. Yanazume T. Sasayama S. J. Biol. Chem. 2000; 275: 13721-13726Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, T. Hasegawa K. T. Kaburagi S. Wada H. Sasayama S. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). to GATA from The of the of these as and also a that the in the as a by and by as T. Hasegawa K. Kaburagi S. Kakita T. Masutani H. Kitsis R.N. Matsumori A. Sasayama S. J. Biol. Chem. 1999; 274: 12811-12818Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, T. Hasegawa K. Kaburagi S. Kakita T. Wada H. Yanazume T. Sasayama S. J. Biol. Chem. 2000; 275: 13721-13726Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, T. Hasegawa K. T. Kaburagi S. Wada H. Sasayama S. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). For a of in the binding by on in is and with and with for by in and for with and and with an that phosphorylated and of For as by a to a Inc., The and of by by using on the via from The in with For of the and the The of the from as a for are as using or of with with a to phenylephrine of myocardial cell hypertrophy K.R. Knowlton K.U. Zhu H. Chien S. FASEB J. 1991; 5: 3037-3046Crossref PubMed Scopus (708) Google Scholar, 2MacLellan W.R. Hawker J. Schneider M.D. Marks A.R. Taubman M.B. Molecular Biology of Cardiovascular Disease. Marcel Dekker, Inc., New York1997: 327-378Google Scholar, 3Copper G. Annu. Rev. Med. 1997; 48: 13-23Crossref PubMed Scopus (99) Google Scholar, T. Hasegawa K. Kaburagi S. Kakita T. Wada H. Yanazume T. Sasayama S. J. Biol. 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A of such in B, the myocardial cell in with in with also the increase of the cell by endothelin-1 and hypertrophic However, not affect the myocardial cell in the in cardiac and that the of is not to the of on the expression of cardiac genes β-MHC and atrial natriuretic factor, up-regulation is a marker for myocardial cell hypertrophy. in the of β-MHC and atrial natriuretic factor gene expression by However, not affect the expression of these The expression of the and expressed gene not by or these demonstrate that a of the hypertrophic of the expression of and ventricular cardiac myocytes with or in the or of for from these myocytes with an to β-MHC with a and with a a B, The in are the from The up-regulation of the expression of the during myocardial cell hypertrophy is in the of and that transcriptional activation of the during hypertrophy an GATA within this (10Morimoto T. Hasegawa K. Kaburagi S. Kakita T. Wada H. Yanazume T. Sasayama S. J. Biol. Chem. 2000; 275: 13721-13726Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). the of on activation of of by the cardiac myocytes. A small of to for transfection of these with increased the expression from by with cells, which is in with (10Morimoto T. Hasegawa K. Kaburagi S. Kakita T. Wada H. Yanazume T. Sasayama S. J. Biol. Chem. 2000; 275: 13721-13726Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). the increase activity with with not affect the activity in cardiac myocytes with These findings suggest that this signaling in cardiac myocytes. that expression of GATA-4 or the activity in a (10Morimoto T. Hasegawa K. Kaburagi S. Kakita T. Wada H. Yanazume T. Sasayama S. J. Biol. Chem. 2000; 275: 13721-13726Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). whether ROCK-I participates in we in cells, which GATA factors (10Morimoto T. Hasegawa K. Kaburagi S. Kakita T. Wada H. Yanazume T. Sasayama S. J. Biol. 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Hasegawa K. Kaburagi S. Kakita T. Wada H. Yanazume T. Sasayama S. J. Biol. Chem. 2000; 275: 13721-13726Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). the between Rho/ROCK and we whether ventricular myocytes or with or or for and the myocytes of by using an that the of these activated of with in cardiac myocytes and cells. In cardiac activation in a and in cardiac In the phosphorylation of in and in of activation by might whether the DNA binding activity of GATA-4 in cardiac from cardiac myocytes with in the or the of or with as a These with a the GATA in that a as by the that it by an of GATA not by the of an the GATA with a that the an of the with GATA-4, we The by not by The of the GATA-4 increased in nuclear from myocytes B, with from the increase in the GATA binding activity by B, In binding not by These using of and to A. J. R. Cell. 1999; Full Text Full Text PDF PubMed Scopus Google suggest that kinase is one of of and that actin a convergence point for signaling to the we whether actin dynamics a role in through For this we the of latrunculin B, which on ventricular myocytes with or or latrunculin for and the myocytes of by as in latrunculin activation in a and it not affect the phosphorylation the and we whether latrunculin the DNA binding activity of cardiac GATA-4. from cardiac myocytes with in the or the of latrunculin or with as a These to using the GATA as a in the increase in the GATA binding activity by latrunculin binding by latrunculin latrunculin prevents activation of and increases the DNA binding activity of cardiac increase in the cardiac GATA binding Cardiac myocytes with or of latrunculin for and with or for as of from these with a the GATA in A and with that the in we whether in actin dynamics to the of myocardial cell hypertrophy. cardiac myocytes with or in the or of latrunculin for These to with an in cardiac myocytes with increases in cell and as with cells. actin and in cardiac myocytes with latrunculin in with in B, latrunculin the increase of the cell by or However, latrunculin not affect the myocardial cell in cardiac myocytes. These demonstrate that latrunculin the hypertrophic Rho is a of the Rho family of small proteins and The activation of Rho the of and focal in various cell types including cardiac myocytes (11Leung T. Chen X.Q. Manser E. Lim L. Mol. Cell. Biol. 1996; 16: 5313-5327Crossref PubMed Google Scholar, 12Amano M. Chihara K. Kimura K. Fukata Y. Nakamura N. Matsuura Y. Kaibuchi K. Science. 1997; 275: 1308-1311Crossref PubMed Scopus (956) Google Scholar, 13Uehata M. Ishizaki T. Satoh H. Ono T. Kawahara T. Morishita T. Tamakawa H. Yamagami K. Inui J. M. S. 1997; PubMed Scopus Google Scholar, A. Cell. Full Text PDF PubMed Scopus Google Scholar, N. A. Biol. 1997; PubMed Scopus Google Scholar). of that Rho mediates hypertrophic signals in cardiac myocytes M. Y. Chien K.R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, T. T. S. E. K. H. M. 1997; 81: PubMed Scopus Google Scholar, K. Y. M. E. Y. N. N. T. R. N. Y. K. 1999; PubMed Scopus Google Scholar, A. J. Mol. Cell. 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PubMed Scopus Google Scholar). and kinase to mediate hypertrophic responses through signals activated by hypertrophic stimuli the and a of transcription The zinc finger transcription factor GATA-4 is one of the of hypertrophic signals that mediate transcriptional activation of genes angiotensin II type natriuretic factor, and endothelin-1 in cardiac myocytes (7Hasegawa K. Lee S.J. Jobe S.M. Markham B.E. Kitsis R.N. Circulation. 1997; 96: 3943-3953Crossref PubMed Scopus (154) Google Scholar, 8Herzig T.C. Jobe S.M. Aoki H. Molkentin J.D. Cowley A.W. Izumo S. Markham B.E. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7543-7548Crossref PubMed Scopus (180) Google Scholar, 9Morimoto T. Hasegawa K. Kaburagi S. Kakita T. Masutani H. Kitsis R.N. Matsumori A. Sasayama S. J. Biol. Chem. 1999; 274: 12811-12818Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, T. Hasegawa K. Kaburagi S. Kakita T. Wada H. Yanazume T. Sasayama S. J. Biol. 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These findings suggest that the Rho/ROCK pathway to the activation of during hypertrophic signaling in cardiac myocytes. The as to signals are to of kinase have been These include myosin the myosin binding of myosin chain and kinase K. M. M. Chihara K. Fukata Y. M. B. J. T. K. A. Kaibuchi K. Science. 1996; PubMed Scopus Google Scholar, M. M. Kimura K. Fukata Y. Chihara K. T. Matsuura Y. Kaibuchi K. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, K. K. M. Ishizaki T. S. K. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar, K. M. Nakamura N. T. M. T. H. R. M. Kaibuchi K. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, M. Ishizaki T. S. N. A. A. T. K. K. S. Science. 1999; PubMed Scopus Google Scholar). A that actin a convergence point for signaling to the A. J. R. 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