Cardiac Hypertrophy Induced by Mitogen-activated Protein Kinase Kinase 7, a Specific Activator for c-Jun NH 2 -terminal Kinase in Ventricular Muscle Cells* (Received for publication, December 4, 1997, and in revised form, January 12, 1998) Yibin Wang‡, Bing Su§, Valerie P. Sah¶, Joan Heller Brown¶, Jiahuai Hani, and Kenneth R. Chien‡** From the Departments of ‡Medicine and ¶Pharmacology, University of California at San Diego, La Jolla, California 92093, the §Department of Immunology, University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030, and the iDepartment of Immunology, The Scripps Research Institute, La Jolla, California 92037 Activation of stress-activated protein kinases, includ- ing the p38 and the c-Jun NH 2 -terminal kinases (JNK), have been associated with the onset of cardiac hyper- trophy and cell death in response to hemodynamic over- load and ischemia/reperfusion injury. Upon infection of cultured neonatal rat cardiac myocytes with recombi- nant adenoviral vectors expressing a wild type and a constitutively active mutant of MKK7 (or JNKK2), JNK was specifically activated without affecting other mito- gen-activated protein kinases, including extracellular signal-regulated protein kinases and p38. Specific acti- vation of the JNK pathway in cardiac myocytes induced characteristic features of hypertrophy, including an in- crease in cell size, elevated expression of atrial natri- uretic factor, and induction of sarcomere organization. In contrast, co-activation of both JNK (by MKK7) and p38 (by MKK3 or MKK6) in cardiomyocytes led to an induction of cytopathic responses and suppression of hypertrophic responses. These data provide the first direct evidence that activation of JNK alone is sufficient to induce characteristic features of cardiac hypertro- phy, thereby supporting an active role for the JNK path- way in the development of cardiac hypertrophy. The cytopathic response, as a result of co-activation of both JNK and p38, may contribute to the loss of contractile function and viability of cardiomyocytes following he- modynamic overload and cardiac ischemia/reperfusion injury. Cardiac hypertrophy and heart failure are multi-step patho- physiological processes involving extracellular stimuli, such as mechanical stress, cytokines and growth factors, and intracel- lular signaling pathways, including mitogen-activated protein (MAP) 1 kinase pathways (1–5). The MAP kinase signaling pathways consist of three major phosphorylation cascades, i.e. the extracellular signal-regulated protein kinases (ERK), the c-Jun NH 2 -terminal kinases (JNK), and the p38 MAP kinases (6, 7). JNK and p38 pathways are collectively termed stress- activated protein kinases because they are activated by various stress-related stimuli (8). It has been shown that p38 MAP kinase activity is activated in hypertrophied 2 and ischemic hearts (10, 11). In neonatal cardiomyocytes, activation of the p38 pathway by an upstream activator MKK6 induces charac- teristic features of hypertrophy (12, 13). In contrast, activation of p38 activity by another upstream activator, MKK3, gives rise to the mixed responses of hypertrophy and apoptosis, possibly mediated by different p38 isoforms (12). These studies support the hypothesis that p38 may mediate the hypertrophic process in vivo and contribute to the myocyte apoptosis that is observed in post-ischemic hearts as well as end stage failing hearts (14). JNK is the first MAP kinase that is activated in mouse heart by pressure-overload, 3 and is also activated in Ras induced hypertrophied transgenic hearts (15). JNK activation is also associated with ischemia/reperfusion (10, 11). It can be induced by an a-adrenergic agonist, phenylephrine (PE), and is re- quired for the induction of atrial natriuretic factor (ANF) pro- moter activity by PE in cardiac myocytes (15). In a separate study, activated MEKK-1, an upstream activator of JNK, has been shown to be able to activate some of the characteristic features of hypertrophy in neonatal cardiomyocytes (13). How- ever, because PE treatment and the activated MEKK-1 also activate other MAP kinases, including ERK and p38, the spe- cific function of JNK in cardiac hypertrophy is still not yet clear. Recently, several groups have reported the cloning of a new MAP kinase kinase, MKK7 (also named JNKK-2), which is able to specifically activate the JNK pathway without affecting ERK and p38 activities (16 –18). 4 In this study, we examine the effects of MKK7-mediated JNK activation on neonatal cardiac myocytes. Our results show that specific activation of the JNK pathway in cardiac myocytes is sufficient to induce character- istic features of hypertrophy. Furthermore, co-activation of JNK and p38 pathways in cardiac myocytes leads to cytopathic responses in cardiac myocytes and induction of cell death. These results support the hypothesis that both JNK and p38 play pivotal roles in the development of cardiac hypertrophy and that combined effects of JNK and p38 may contribute to the pathophysiological process of heart failure. EXPERIMENTAL PROCEDURES Culture of Neonatal Rat Cardiac Myocytes—Ventricular myocytes from 1–2-day-old Sprague-Dawley rats were prepared using a Percoll gradient method as described previously (19). The cells were plated in * This work was supported in part by grants from the American Heart Association and National Institutes of Health (to K. R. C.), by American Heart Association Grant-in-Aid 95007690 and National In- stitutes of Health Grants GM51417 and AI41637 (to J. H.), and by National Institutes of Health Grants HL28143 and HL46345 (to J. H. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ** To whom correspondence should be addressed: Dept. of Medicine and Center for Molecular Genetics, Mail Box 0613-C, 9500 Gilman Dr., University of California at San Diego, La Jolla, CA 92093. Tel.: 619- 534-4801; Fax: 619-534-8081; E-mail: kchien@ucsd.edu. 1 The abbreviations used are: MAP, mitogen-activated protein ki- nase; ERK, extracellular signal-regulated protein kinase; JNK, c-Jun NH 2 -terminal kinase; PE, phenylephrine; ANF, atrial natriuretic fac- tor; Adv, adenovirus; GFP, green fluorescent protein. 2 J. J. Hunter, M. Shimizu, J. Brown, V. P. Sah, K. Gottshall, C. Milano, R. Lefkowiz, J. H. Brown, and K. R. Chien, submitted for publication. 3 P. V. Sah and J. H. Brown, unpublished results. 4 J. Yang, L. New, Y. Jiang, J. Han, and B. Su, unpublished results. Communication THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 273, No. 10, Issue of March 6, pp. 5423–5426, 1998 © 1998 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. This paper is available on line at http://www.jbc.org 5423 by guest on May 20, 2020 http://www.jbc.org/ Downloaded from by guest on May 20, 2020 http://www.jbc.org/ Downloaded from by guest on May 20, 2020 http://www.jbc.org/ Downloaded from