Kawata Y, Mizukami Y, Fujii Z, Sakumura T, Yoshida K, Matsuzaki M
Department of Internal Medicine, Yamaguchi University School of Medicine, 1144 Kogushi, Ube, Yamaguchi 755-8505, Japan.
J Biol Chem. 1998 Jul 3;273(27):16905-12. doi: 10.1074/jbc.273.27.16905.
Progressive renal diseases lead to prolonged glomerular hypertension, which induces the proliferation of mesangial cells. This proliferation is thought to be involved in the development of renal injury. Here we investigate mitogen-activated protein kinase (MAPK) activation and cell proliferation in mesangial cells under conditions of high pressure. After pressure-load, the phosphorylation level of MAPK (at Tyr-204) increases rapidly with a peak at 1 min, although the amount of MAPK remains almost constant during pressure-load. To confirm the activation of MAPK, we carried out an immunoprecipitation-kinase assay. MAPK activity during pressure-load shows kinetics similar to that of the tyrosine phosphorylation. In contrast, c-Jun N-terminal kinase 1 (JNK1) phosphorylation falls below basal levels in response to high pressure. Immunocytochemical observations show phosphorylated MAPK in the nucleus at 10 min. The expression of c-Fos, a nuclear transcription factor, is induced by high pressure, and the induction is significantly inhibited by PD98059 (50 microM), an upstream MAPK/extracellular signal-regulated kinase kinase (MEK) inhibitor of MAPK. The expression of the c-Jun that is induced by JNK1 activation remains unchanged during pressure-load. MAPK phosphorylation and cell proliferation by applied pressure are significantly inhibited by genistein, a tyrosine kinase inhibitor in a dose-dependent manner, but not by protein kinase C inhibitors, chelerythrine and GF109203X. Genistein also blocks pressure-induced tyrosine phosphorylation of proteins with molecular masses of 35, 53, and 180 kDa. To clarify the physiological role in MAPK activation under high pressure conditions, we transfected antisense MAPK DNA into mesangial cells. The antisense DNA (2 microM) inhibited MAPK expression by 80% compared with expression in the presence of sense or scrambled DNA, and significantly blocked pressure-induced cell proliferation. Treatment of cells with MEK inhibitor also produced a similar result. MEK inhibitor strongly suppresses DNA synthesis induced by pressure-load. Cyclin D1 expression is significantly increased under high pressure conditions, and the increase is blocked by treatment with MEK inhibitor. These findings show that pressure-load, a novel activator of MAPK, induces the activation of tyrosine kinases, and enhances the proliferation of mesangial cells, probably through cyclin D1 expression.
进行性肾脏疾病会导致肾小球高血压持续存在，进而诱导系膜细胞增殖。这种增殖被认为与肾损伤的发展有关。在此，我们研究了在高压条件下系膜细胞中丝裂原活化蛋白激酶（MAPK）的激活及细胞增殖情况。压力负荷后，MAPK（酪氨酸204位点）的磷酸化水平迅速升高，在1分钟时达到峰值，尽管在压力负荷期间MAPK的总量几乎保持不变。为了证实MAPK的激活，我们进行了免疫沉淀激酶测定。压力负荷期间的MAPK活性显示出与酪氨酸磷酸化相似的动力学。相反，c-Jun氨基末端激酶1（JNK1）的磷酸化在高压作用下降至基础水平以下。免疫细胞化学观察显示，10分钟时细胞核中有磷酸化的MAPK。核转录因子c-Fos的表达由高压诱导，并且这种诱导被PD98059（50微摩尔）显著抑制，PD98059是MAPK的上游MAPK/细胞外信号调节激酶激酶（MEK）抑制剂。由JNK1激活诱导的c-Jun表达在压力负荷期间保持不变。染料木黄酮（一种酪氨酸激酶抑制剂）以剂量依赖的方式显著抑制施加压力引起的MAPK磷酸化和细胞增殖，但蛋白激酶C抑制剂白屈菜红碱和GF109203X则无此作用。染料木黄酮还能阻断压力诱导的分子量为35、53和180 kDa的蛋白质的酪氨酸磷酸化。为了阐明在高压条件下MAPK激活中的生理作用，我们将反义MAPK DNA转染到系膜细胞中。与有义或随机序列DNA存在时的表达相比，反义DNA（2微摩尔）使MAPK表达抑制了80%，并显著阻断了压力诱导的细胞增殖。用MEK抑制剂处理细胞也产生了类似的结果。MEK抑制剂强烈抑制压力负荷诱导的DNA合成。细胞周期蛋白D1的表达在高压条件下显著增加，并且这种增加被MEK抑制剂处理所阻断。这些发现表明，压力负荷作为一种新的MAPK激活剂，诱导酪氨酸激酶的激活，并可能通过细胞周期蛋白D1的表达增强系膜细胞的增殖。
