Both estrogen and raloxifene cause G1 arrest of vascular smooth muscle cells

Abstract

The proliferation of vascular smooth muscle cells (VSMC) is a crucial pathophysiological process in the development of atherosclerosis. Although estrogen is known to inhibit the proliferation of VSMC, the mechanism responsible for this effect remains to be elucidated. In addition, the effect of raloxifene on VSMC remains unknown. We have shown here that 17β-estradiol (E2) and raloxifene significantly inhibited the platelet-derived growth factor (PDGF -stimulated proliferation of cultured human VSMC. Flow cytometry demonstrated that PDGF-stimulated S-phase progression of the cell cycle in VSMC was also suppressed by E2 or raloxifene. We found that PDGF-induced phosphorylation of retinoblastoma protein (pRb), whose hyperphosphorylation is a hallmark of the G1-S transition in the cell cycle, was significantly inhibited by E2 and raloxifene. These effects were associated with a decrease in cyclin D1 expression, without a change in cyclin-dependent kinase 4 or cyclin-dependent kinase inhibitor, p27kip1 expression. ICI 182,780 abolished the inhibitory effects of E2 and raloxifene on PDGF-induced pRb phosphorylation. Next, we examined which estrogen receptor (ER) is necessary for these effects of E2 and raloxifene. Since VSMC express both ERα, and ERβ, A10, a rat aortic smooth muscle cell line that expresses ERβ but not ERα, was used. The dose-dependent stimulation of A10 cell proliferation by PDGF was not inhibited by E2 or raloxifene in contrast to the results obtained in VSMC. Moreover, E2 and raloxifene significantly inhibited the PDGF-induced cyclin D1 promoter activity in A 10 cells transfected with cDNA for ERα but not in the parental cells. These results suggested that E2 and raloxifene exert an antiproliferative effect in VSMC treated with PDGF, at least in part through inhibition of pRb phosphorylation, and that the inhibitory effects of E2 and raloxifene may be mainly mediated by ERα.