Identification of androgen receptors in normal human osteoblast-like cells.

Abstract

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...

Effects of testosterone replacement therapy on cortical and trabecular bone mineral density, vertebral body area and paraspinal muscle area in hypogonadal men

“…In vitro studies reporting the occurrence of androgen receptors (38), androgen metabolism (39) and androgen effects (40) on human osteoblastic cells support this conclusion. A recent study has demonstrated a change in biochemical serum markers of bone metabolism in response to testosterone replacement therapy, suggesting a reduction in bone remodelling (8).…”

Androgen receptor (AR) in osteocytes is important for the maintenance of male skeletal integrity: Evidence from targeted AR disruption in mouse osteocytes

“…AR expression has been shown in a number of bone cells including pluripotent mesenchymal bone marrow stromal cells, osteoblasts, osteoclasts and osteocytes. (6)(7)(8) Osteoblasts are potential targets for androgen action because they contain a high AR expression. (9)(10)(11) Thus far two osteoblastspecific AR knockouts have been generated.…”

Sex Hormones and Osteoporosis in Men

“…Biochemical evidence of hypogonadism is found in about 20% of men with vertebral fractures [2] and up to 50% of men with hip fractures [3,4], often without other clinical features of gonadal failure. In cell culture studies using human osteoblastlike cells from both men and women [5][6][7], specific androgen receptors can be identified by nuclear and cytosolic binding assays (Table 1). Similarly, osteoblasts obtained from fresh femoral bone specimens can also metabolize the circulating androgen androstenedione to testosterone and 5␣-dihydrotestosterone (DHT) in both men and women [8] at rates dependent mainly on androstenedione concentration.…”

“…However, estrogen receptors are at least equally abundant in cultures of these cell lines [5,21] and osteoblasts respond metabolically to estrogens in vitro [22] and in fresh biopsy samples of male bone [23]. Aromatase activity is present in cultured osteoblast cell lines [22], demonstrating that these cells have the capacity to convert testosterone to estradiol (and vice versa).…”