The present study was designed to investigate a possible role for ovarian steroids in the regulation of rat uterine oxytocin receptor (OTR) mRNA expression before labour. By using a competitive RT-PCR system, we have previously reported that parturition was associated with high levels of uterine OTR mRNA in all the animals examined. On the other hand, near term, some rats showed high OTR mRNA levels while others did not. We therefore examined the changes in OTR mRNA expression before and during prostaglandin F(2)(alpha) (PGF(2)(alpha))-induced parturition; a paradigm adopted to reduce the variation in the onset of parturition. Injection of PGF(2)(alpha) on day 18 of pregnancy significantly increased OTR mRNA expression in all the rats within 24 h of treatment, suggesting that the variation in OTR mRNA levels during spontaneous parturition may be due to the difference in the timing of the onset of parturition. The increase in OTR mRNA was significantly abolished by injection of the anti-oestrogen compound, tamoxifen. The stimulatory action of oestrogen on OTR mRNA expression was then examined in the presence or absence of ovarian factors. Pregnant rats were ovariectomized (OVX) or sham-operated on day 18 of pregnancy and either oestrogen or vehicle was administered 6 h after the surgical operation. Oestrogen increased OTR mRNA significantly in OVX rats 18 h after administration compared with sham-operated animals. Moreover, ovariectomy alone on day 18 of pregnancy increased OTR mRNA expression to a level which reached statistical significance 24 h after the operation. In addition, oestrogen treatment increased OTR mRNA levels in OVX virgin rats in which progesterone tubes were implanted for 1 week and removed 6 h before oestrogen injection. The stimulatory effect of oestrogen was not observed in rats in which the progesterone tubes were implanted for 1 week and not removed. These results suggest that the decline of progesterone is necessary for the expression of the stimulatory effects of oestrogen on uterine OTR mRNA.
T Murata, E Murata, CX Liu, K Narita, K Honda and T Higuchi
K Takahashi, M Ohmichi, M Yoshida, K Hisamoto, S Mabuchi, E Arimoto-Ishida, A Mori, S Tsutsumi, K Tasaka, Y Murata and H Kurachi
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 17beta-estradiol (E(2)) 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 E(2) 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 E(2) 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, p27(kip1) expression. ICI 182,780 abolished the inhibitory effects of E(2) and raloxifene on PDGF-induced pRb phosphorylation. Next, we examined which estrogen receptor (ER) is necessary for these effects of E(2) and raloxifene. Since VSMC express both ERalpha and ERbeta, A10, a rat aortic smooth muscle cell line that expresses ERbeta but not ERalpha, was used. The dose-dependent stimulation of A10 cell proliferation by PDGF was not inhibited by E(2) or raloxifene in contrast to the results obtained in VSMC. Moreover, E(2) and raloxifene significantly inhibited the PDGF-induced cyclin D1 promoter activity in A10 cells transfected with cDNA for ERalpha but not in the parental cells. These results suggested that E(2) 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 E(2) and raloxifene may be mainly mediated by ERalpha.