Applied Pressure Enhances Cell Proliferation through Mitogen-activated Protein Kinase Activation in Mesangial Cells*

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 μm), 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 μm) 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.