Ras/ERK1 pathway regulation of p27KIP1-mediated G1-phase cell-cycle arrest in cordycepin-induced inhibition of the proliferation of vascular smooth muscle cells.

Cordycepin, the main constituent of Cordyceps militaris, demonstrated an anti-atherogenic effect in experimental animals. However, the effects of cordycepin on cell-cycle regulation and the signaling pathway in vascular smooth muscle cells (VSMC) remain largely unknown; therefore, unexpected roles of cordycepin-induced inhibition in VSMC growth were investigated. Mechanisms in cordycepin-treated VSMC were examined via an MTT assay, a thymidine uptake experiment, FACS analysis, immunoblot analysis, kinase assay, immunoprecipitation assay, and transient transfection assays. Cordycepin inhibited cell growth, induced G1-phase cell-cycle arrest, down-regulated cyclins and cyclin-dependent kinase (CDK) expression, and up-regulated p27KIP1 expression in VSMC. Cordycepin induced activation of JNK, p38MAPK and ERK1/2. Blocking of the ERK function using either ERK1/2-specific inhibitor U0126 or a small interfering RNA (si-ERK1) reversed p27KIP1 expression, inhibition of cell growth, and decreased cell-cycle proteins in cordycepin-treated VSMC. Ras activation was increased by cordycepin. Transfection of cells with dominant negative Ras (RasN17) mutant genes rescued cordycepin-induced ERK1/2 activity, increased p27KIP1 expression, inhibited cell proliferation, and reduced cell cycle proteins. In conclusion, our findings indicate that Ras/ERK1 pathways participate in p27KIP1-mediated G1-phase cell-cycle arrest induced by cordycepin via a decrease in cyclin/CDK complexes in VSMC.