Cyclin D1, p16, and retinoblastoma gene regulate mitogenic signaling of endothelin in rat mesangial cells

To elucidate the mechanisms by which endothelin (ET)-1 induces proliferation of mesangial cells, we investigated the involvement of the first gap phase of the cell cycle (G1) cyclin, cyclin-dependent kinase 4 (CDK4) activity, and the retinoblastoma gene product (pRb) in ET-l-stimulated cell cycle progression. In the present study, ET-1 stimulated CDK4 activity and cell cycle progression via ET A-type receptors and induced cyclin D1 mRNA and protein expression in rat mesangial cells. We also found that ET-1 stimulation of mesangial cell proliferation was inhibited by antisense oligonucleotides directed against cyclin D1 and by overexpression of a nonphosphorylatable form of pRb. To investigate the functional roles of p16(INK4) and p21(cip1) in ET-l-stimulated mesangial cell proliferation, we used adenovirus-mediated gene transfer. Endothelin-l-stimulated [H-3]-thymidine incorporation, CDK4 kinase activity, and the percent of cells in S phase were found to be significantly inhibited by overexpression of p16(INK4) and slightly inhibited by overexpression of p21(cip1). Thus, ET-1 induced cyclin D1 expression and stimulated CDK4 activity and cell cycle progression via the A-type receptor in rat mesangial cells. These effects were regulated by the expression of cyclin D1, p16(INK4), p21(cip1), and phosphorylatable form of pRb. The results of the present study provide the basis for further investigation of basic and therapeutic approaches towards mesangial proliferative diseases.