Effects of estrogen on collagen synthesis by cultured human osteoblasts depend on the rate of cellular differentiation

Estrogen is known to act on osteoblasts according to their stage of differentiation and estrogen receptor (ER) isoform expression. The aim of this study was to determine when type I collagen (COL1) synthesis by cultured low-passage, human bone-derived osteoblasts (hOBs) is upregulated in response to estrogen. Cell lines from female donors aged 1 and 66 years were cultured for 11 days on collagen in growth medium supplemented with human serum, hydrocortisone, and beta-glycerophosphate. Young-donor hOBs grew more quickly than old-donor hOBs and did not mineralize. Old-donor hOBs formed mineralized nodules 5 days after reaching confluence. Changes in mRNA levels with time for ERs, type I collagen, and alkaline phosphatase reflected the faster differentiation of the old-donor cells. The ERbeta/ERalpha ratio fell threefold in young-donor hOBs but rose 300-fold in old-donor hOBs. Increased ERbeta/ERalpha ratios prevented ligand-dependent downregulation of ERalpha transcription, resulting in reduced proliferation in old-donor hOBs. Upregulation of COL1 mRNA expression in response to estrogen was confined to intermediate stages of differentiation, resulting in significant increases in COL1 mRNA by estradiol only in young-donor cells. Since the young and old-donor hOBs were cultured under identical conditions, our results indicate that the response of hOBs to estrogen is largely dependent on intracellular mechanisms that control the timing of cellular differentiation.