Isoenzyme-specific protein kinase C and c-Jun N-terminal kinase activation by electrically stimulated contraction of neonatal rat ventricular myocytes.

:Previous studies from our laboratory and others indicate that contraction-induced mechanical loading of cultured neonatal rat ventricular myocytes produces many of the phenotypic changes associated with cardiomyocyte hypertrophy in vivo, and that these changes occur via the activation of serine-threonine protein kinases. These may include the extracellular regulated protein kinases (ERK1 and ERK2), the c-Jun N-terminal kinases (JNK1, JNK2, and JNK3), and one or more isoenzymes of protein kinase C. In this study, we assessed whether one or more of these kinases are activated by stimulated contraction, and whether activation was isoenzyme-specific. Low-density, quiescent cultures of neonatal rat ventricular myocytes were maintained in serum-free medium, or electrically stimulated to contract (3 Hz) for up to 48 h. ERK and JNK activation was assessed by Western blotting with polyclonal antibodies specific for the phosphorylated forms of both kinases. PKC activation was analysed by subcellular fractionation, detergent extraction, and Western blotting using isoenzyme-specific monoclonal antibodies. Stimulated contractile activity produced myocyte hypertrophy, as indicated by increased cell size, a 15+/-5% increase in total protein/DNA ratio, and induction of ANF and beta MHC gene transcription. Electrical pacing did not cause ERK1/2 or JNK1 activation, but increased JNK2 and JNK3 phosphorylation by;two-fold. Subcellular fractionation revealed a time-dependent increase in PKC delta, and to a much lesser extent PKC xi, in a Triton X-100-soluble membrane fraction within 5 min of the onset of stimulated contraction. PKC alpha was not activated by electrical pacing. These results indicate that contraction-induced mechanical loading acutely activates some but not all of the specific isoenzymes of JNKs and PKCs in cardiomyocytes.

Strait JB ，Samarel AM 《JOURNAL OF MOLECULAR AND CELLULAR CARDIOLOGY》

Pro-inflammatory cytokines stimulate mitogen-activated protein kinase subfamilies, increase phosphorylation of c-Jun and ATF2 and upregulate c-Jun protein in neonatal rat ventricular myocytes.

Pro-inflammatory cytokines may be important in the pathophysiological responses of the heart. We investigated the activation of the three mitogen-activated protein kinase (MAPK) subfamilies ¿c-Jun N-terminal kinases (JNKs), p38-MAPKs and extracellularly-responsive kinases (ERKs) by interleukin-1 beta (IL-1 beta) or tumour necrosis factor alpha (TNF alpha) in primary cultures of myocytes isolated from neonatal rat ventricles. Both cytokines stimulated a rapid (maximal within 10 min) increase in JNK activity. Although activation of JNKs by IL-1 beta was transient returning to control values within 1 h, the response to TNF alpha was sustained. IL-1 beta and TNF alpha also stimulated p38-MAPK phosphorylation, but the response to IL-1 beta was consistently greater than TNF alpha. Both cytokines activated ERKs, but to a lesser degree than that induced by phorbol esters. The transcription factors, c-Jun and ATF2, are phosphorylated by the MAPKs and are implicated in the upregulation of c-Jun. IL-1 beta and TNF alpha stimulated the phosphorylation of c-Jun and ATF2. However, IL-1 beta induced a greater increase in c-Jun protein. Inhibitors of protein kinase C (PKC) (Ro318220, GF109203X) and the ERK cascade (PD98059) attenuated the increase in c-Jun induced by IL-1 beta, but LY294002 (an inhibitor of phosphatidylinositol 3' kinase) and SB203580 (an inhibitor of p38-MAPK, which also inhibits certain JNK isoforms) had no effect. These data illustrate that some of the pathological effects of IL-1 beta and TNF alpha may be mediated through the MAPK cascades, and that the ERK cascade, rather than JNKs or p38-MAPKs, are implicated in the upregulation of c-Jun by IL-1 beta.

Clerk A ，Harrison JG ，Long CS ，Sugden PH ... -  《JOURNAL OF MOLECULAR AND CELLULAR CARDIOLOGY》

PKC mediates cyclic stretch-induced cardiac hypertrophy through Rho family GTPases and mitogen-activated protein kinases in cardiomyocytes.

Signaling events, including Rho GTPases and protein kinase C (PKC), are involved in cardiac hypertrophy. However, the mechanisms by which these pathways cooperate during the hypertrophic process remain unclear. Using an in vitro cyclic stretch model with neonatal rat cardiomyocytes, we demonstrated that stretch-induced activation of RhoA, Rac1/Cdc42, and phosphorylation of Rho-guanine nucleotide dissociation inhibitor (GDI) were prevented by inhibition or depletion of PKC, using chelerythrine and phorbol 12-myristate 13-acetate, indicating that phorbol ester-sensitive PKC isozymes may be upstream regulators of Rho GTPases. Using adenoviral-mediated gene transfer of wild-type (WT) and dominant-negative (DN) mutants of PKCalpha and delta, we found that stretch-induced activation of Rho GTPases and phosphorylation of Rho-GDI were mainly regulated by PKCalpha. PKCdelta was involved in regulation of the activation of Rac1. Stretch-induced increases in [(3)H]-leucine incorporation, myofibrillar reorganization and cell size, were blocked by inhibition of Rho GTPases, or overexpression of DN PKCalpha and delta, suggesting that PKCalpha and delta are both required in stretch-induced hypertrophy, through Rho GTPases-mediated signaling pathways. The mechanism, whereby PKC and Rho GTPases regulate hypertrophy, was associated with mitogen-activated protein (MAP) kinases. Stretch-stimulated phosphorylation of MEK1/ERK1/2 and MKK4/JNK was inhibited by overexpression of DN PKCalpha and delta, and that of MKK3/p38 inhibited by DN PKCdelta. The phosphorylation of ERK and JNK induced by overexpression of WT PKCalpha, and the phosphorylation of p38 induced by WT PKCdelta, were regulated by Rho GTPases. This study represents the first evidence that PKCalpha and delta are important regulators in mediating activation of Rho GTPases and MAP kinases, in the cyclic stretch-induced hypertrophic process.

Pan J ，Singh US ，Takahashi T ，Oka Y ，Palm-Leis A ，Herbelin BS ，Baker KM ... -  《JOURNAL OF CELLULAR PHYSIOLOGY》

Endothelin-1-induced cardiac hypertrophy is inhibited by activation of peroxisome proliferator-activated receptor-alpha partly via blockade of c-Jun NH2-terminal kinase pathway.

Irukayama-Tomobe Y ，Miyauchi T ，Sakai S ，Kasuya Y ，Ogata T ，Takanashi M ，Iemitsu M ，Sudo T ，Goto K ，Yamaguchi I ... -  《-》

Activation of extracellular signal-regulated kinase during silibinin-protected, isoproterenol-induced apoptosis in rat cardiac myocytes is tyrosine kinase pathway-mediated and protein kinase C-dependent.

Zhou B ，Wu LJ ，Tashiro S ，Onodera S ，Uchiumi F ，Ikejima T ... -  《ACTA PHARMACOLOGICA SINICA》