ESTROGEN RECEPTOR-ALPHA IS DEVELOPMENTALLY-REGULATED DURING OSTEOBLAST DIFFERENTIATION AND CONTRIBUTES LO SELECTIVE RESPONSIVENESS OF GENE-EXPRESSION

Estrogen responsiveness of bone is a fundamental regulatory mechanism operative in skeletal homeostasis. We examined the expression of estro gen receptor-alpha (ER) messenger RNA (mRNA) in cultured rat calvarial -derived osteoblasts during progressive development of the osteoblast phenotype. Levels of ER message were compared with the expression of t raditional osteoblastic markers that have been mapped throughout the d ifferentiation process of these cells. ER transcripts, measured using semiquantitative BT-PCR analysis, were expressed at low levels in earl y stage proliferating osteoblasts and increased at confluence upon ini tial expression of bone cell phenotypic genes. A 23-fold up-regulation of ER mRNA expression coincided with the initiation of alkaline phosp hatase activity (day 8). ER mRNA levels progressively increased 70-fol d, reaching a maximum level on days 22-25 in fully differentiated oste oblasts when osteocalcin expression peaked, but declined precipitously by day 32 in osteocytic cells. Analysis of RNA isolated directly from rat calvaria confirmed these in vitro results and demonstrated that E R message levels become more abundant postnatally as bone becomes more mineralized. We also examined the responsiveness of osteoblasts to 17 beta-estradiol (17 beta-E-2) at two periods of maturation: the nodule -forming stage (day 14) and the late mineralization stage (day 30). Es tradiol suppressed the levels of alkaline phosphatase, osteocalcin, os teonectin, and ER mRNAs on day 14, but up-regulated these messages on day 30. in contrast, 17 beta-E-2 treatment regulated the steady state levels of transforming growth factor-beta 1 and type I procollagen mRN As only in the late mineralization stage, whereas histone H4 message w as unaffected by the steroid at either stage of differentiation. Thus, the observed developmental expression of ER mRNA correlates with prog ressive osteoblast differentiation and may be a contributing factor to differential regulation of bone cell gene expression by 17 beta-E-2.