Mechanical loading activates mitogen-activated protein kinase and S6 peptide kinase in cultured rat cardiac myocytes.
Mechanical loading activates mitogen-activated protein kinase and S6 peptide kinase in cultured rat cardiac myocytes.
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DOI：
10.1016/s0021-9258(19)50309-x
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发表时间：
1993-06
期刊：
影响因子：
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通讯作者：
T. Yamazaki;K. Tobe;E. Hoh;K. Maemura;Toshikazu Kaida;I. Komuro;H. Tamemoto;T. Kadowaki;R. Nagai;Yoshio Yazakis
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文献类型：
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作者：
T. Yamazaki;K. Tobe;E. Hoh;K. Maemura;Toshikazu Kaida;I. Komuro;H. Tamemoto;T. Kadowaki;R. Nagai;Yoshio Yazakis
超负荷心肌细胞增加细胞大小（肥大）的分子机制仍然未知。我们先前已经表明，机械负荷增加了蛋白质合成和原癌基因c-fos mRNA的表达（Komuro，I.，Kaida，T.，Shibazaki，Y.，Kurabayashi，M.，Katoh，Y.哦，E.，Takaku，F.，Yazaki，Y.（1990）J.Biol.Chem.265，3595-3598; Komuro，I.，Katoh，Y.，Kaida，T.，Shibazaki，Y.，Kurabayashi，M.，哦，E.，Takaku，F.，Yazaki，Y.（1991）J.Biol.Chem.266，1265-1268）。已知多种生长因子均可激活丝裂原活化蛋白激酶（MAP）和S6激酶。为了阐明MAP激酶和S6激酶是否与机械负荷诱导的心肌肥大的细胞内信号传导相关，我们在可变形培养皿中培养新生大鼠心肌细胞，并通过拉伸贴壁的心肌细胞来施加体外机械负荷。结果表明：（1）牵张10 min时激活的髓鞘碱性蛋白（MBP）激酶活性最高，30 min时恢复到对照组水平; 2）在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳后，在含MBP的凝胶中进行激酶测定，结果显示，牵张诱导的MBP激酶活性主要在抗MAP激酶抗体的免疫沉淀组分中迁移到42 kDa处，表明牵张主要增加心肌细胞中42 kDa的MAP激酶活性;（3）牵张可诱导心肌细胞MAP激酶磷酸化; 4）当通过用100 nM 12-O-十四酰基-佛波醇-13-乙酸酯预孵育肌细胞24小时或用2nM星形孢菌素预孵育肌细胞30分钟来耗尽蛋白激酶C时，与没有蛋白激酶C耗尽的肌细胞中的激酶活性相比，牵张诱导的MBP激酶活性降低约60-70%; 5）尽管通过将肌细胞与50 μ M tyrphostin或20 μ M染料木黄酮预孵育30分钟来耗尽受体酪氨酸激酶，牵张诱导的MBP激酶活性没有变化; 6）牵张诱导的MBP激酶活性部分依赖于跨肌膜Ca 2+内流; 7）肌细胞牵张也增加了抗S6激酶II抗体免疫沉淀物中S6肽（RRLSSLRA）激酶活性。结果表明，牵张可使MAP激酶和S6肽激酶活性升高，这可能是牵张诱导特异性基因表达和蛋白质合成增加的重要机制。
The molecular mechanisms by which overloaded cardiac myocytes increase the cell size (hypertrophy) remain unknown. We have previously shown that mechanical loading increased the protein synthesis and the expression of proto-oncogene c-fos mRNA (Komuro, I., Kaida, T., Shibazaki, Y., Kurabayashi, M., Katoh, Y. Hoh, E., Takaku, F., and Yazaki, Y. (1990) J. Biol. Chem. 265, 3595-3598; Komuro, I., Katoh, Y., Kaida, T., Shibazaki, Y., Kurabayashi, M., Hoh, E., Takaku, F., and Yazaki, Y. (1991) J. Biol. Chem. 266, 1265-1268). It has been known that both mitogen-activated protein (MAP) kinase and S6 kinase can be activated by many kinds of growth factors. To clarify whether MAP kinase(s) and S6 kinase(s) are associated with the intracellular signaling of cardiac hypertrophy induced by mechanical loading, we cultured neonatal rat cardiac myocytes in deformable dishes and imposed an in vitro mechanical loading by stretching the adherent myocytes. In this study, we demonstrated that 1) myocyte stretching maximally activated a kinase activity toward myelin basic protein (MBP) at 10 min after stretching, and the kinase activity returned to the control level at 30 min after stretching; 2) kinase assays in MBP-containing gel, after sodium dodecyl sulfate-polyacrylamide gel electrophoresis, revealed that stretch-induced MBP kinase activity mainly migrated at 42 kDa in the immunoprecipitated fraction of anti-MAP kinase antibody, suggesting that the stretching mainly increased the 42-kDa MAP kinase activity in cardiac myocytes; 3) phosphorylation of MAP kinase was induced after stretching cardiac myocytes; 4) when protein kinase C was depleted by preincubating myocytes with 100 nM 12-O-tetradecanoyl-phorbol-13-acetate for 24 h or 2 nM staurosporine for 30 min, stretch-induced MBP kinase activity was decreased by approximately 60-70% as compared with the kinase activity in myocytes without protein kinase C depletion; 5) although the receptor tyrosine kinases were depleted by preincubating myocytes with 50 microM tyrphostin or 20 microM genistein for 30 min, there was no change in the stretch-induced MBP kinase activity; 6) stretch-induced MBP kinase activity was partially dependent on transsarcolemmal influx of Ca2+; 7) myocyte stretching also increased S6 peptide (RRLSSLRA) kinase activity in the anti-S6 kinase II antibody immunoprecipitates. From these results, we conclude that myocyte stretching increases the activities of MAP kinase and S6 peptide kinase, which may play an important role in the induction of the specific genes and the increase in the protein synthesis.