| Research Abstract |
Estrogen plays important roles in maintaining bone density and protecting against osteoporosis, but the underlying mechanisms of estrogen action via estrogen receptors (ERs) in bone remain to be clarified. In the present study, we have generated rats harboring a dominant negative ERalpha, which inhibits the actions of not only ERalpha but also ERbeta. We observed bone mineral density (BMD) of the transgenic female rats, after ovariectomized, remained decreased even if 17beta-estradiol (E2) was administrated, whereas, in contrast, the decrease of littermate BMD was completely prevented by E2, suggesting that the prevention from the ovariectomy-induced bone loss by estrogen is mediated by ER pathways. We then isolated primary osteoblasts derived from transgenic rats and from their wild-type littermates. Utilizing cDNA microarray analysis, we found that mRNA level of cyclin D2 was lower in the osteoblasts from the transgenic rats. The protein levels of D-type cyclins including cyclin D2 and cyclin D3 but not cyclin D1 were elevated in wild-type osteoblasts with 17beta-estradiol treatment, resulting in the activation of cyclin-dependent kinases 4 and 6 (Cdk4/6) activities and the promotion of cell growth. Besides, we identified estrogen responsive pathways including Efp and EBAG9 pathways. Especially, we have revealed the molecular mechanism of Efp action as ubiquitin ligase in the cell cycle control. Functional analyzes of these genes in osteoporosis are underway. We have also shown associations between BMD and each SNP in human genes involved in the bone metabolism such as VDP, IL-6, TNFalpa, Klotho, BNP, TNFR1, LRP5/6.
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