In seven strains of cultured normal human osteoblast-like cells, a mean of 1615 molecules of tritium-labeled 17 beta-estradiol per cell nucleus could be bound to specific nuclear sites. The nuclear binding of the labeled steroid was temperature-dependent, steroid-specific, saturable, and cell type-specific. These are characteristics of biologically active estrogen receptors. Pretreatment with 10 nanomolar estradiol in vitro increased the specific nuclear binding of progesterone in four of six cell strains, indicating an induction of functional progesterone receptors. RNA blot analysis demonstrated the presence of messenger RNA for the human estrogen receptor. The data suggest that estrogen acts directly on human bone cells through a classical estrogen receptor-mediated mechanism.
The sex steroids, androgens and estrogens, are major regulators of bone metabolism. However, whether these hormones act on bone cells through direct or Androgens and estrogens are major regulators of bone metabolism in males and females, respectively (1). Both hormones interact with growth hormone in control of the adolescent growth spurt (2-4). After growth is completed, androgens and estrogens maintain bone mass in the adult. In women, menopause causes accelerated bone loss that can be prevented by estrogen administration (5,6). Treatment of postmenopausal women with androgen derivatives, synthetic anabolic steroids, produces beneficial effects on bone metabolism (7, 8). In males, hypogonadism is associated with bone loss, which is stabilized by testosterone administration (9).Because previous attempts to demonstrate receptors in bone cells of experimental animals (10, 11) or humans (12) have been unsuccessful, the prevailing view has been that the action of sex steroids on skeletal tissue is mediated indirectly. Recent data, however, suggest that estrogen has a direct action. Both specific nuclear binding of estradiol and the presence of estrogen receptor mRNA were demonstrated in normal human osteoblast-like cell strains (13) (17,18). In addition, a cDNA probe for the human androgen receptor (19) was used to determine whether human osteoblasts contained androgen receptor mRNA. METHODSCell Culture. Human bone cells were cultured from fresh samples of trabecular bone obtained during orthopedic procedures, by a procedure modified from that developed by Robey and Termine (20). These cultured cells had the typical characteristics of the osteoblast phenotype (13, 21). In brief, residual fibrous tissue was carefully dissected from the bone samples, which were then washed and minced in phosphatebuffered saline and digested with crude bacterial collagenase (GIBCO) at 1 mg/ml in Dulbecco's modified Eagle's medium (DMEM, GIBCO) for 2 hr at 370C in a shaking water bath. These fragments were then cultured in a phenol red-free medium (GIBCO) approximately equivalent to a Ca2+-free 1:1 (vol/vol) mixture of Ham's F-12 and DMEM supplemented with 10% heat-inactivated fetal bovine serum and penicillin/streptomycin. Steroid receptor assays were routinely performed after the second passage of cells in 175-cm2 culture flasks (20-30 population doublings). Two to 3 months of culture were required to obtain the 4 million cells needed for each assay in replicate.In experiments to assess nuclear binding of radiolabeled steroids, the medium was replaced for 48 hr by the same medium containing 20% fetal bovine serum and 2 mM Ca2'.Twenty-four hours before receptor assay, the medium was changed to serum-free medium containing 2 mM Ca2 .Nuclear Binding Assay. The specific nuclear binding of tritiated steroids in these cells was measured by a nuclear binding assay as described in detail previously and modified for tissue culture cells (13,17,18). In brief, human osteoblastlike cells at confluence were removed by trypsinization and...
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