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...

Although our laboratory has reported that normal human osteoblast-like (hOB) cells contain estrogen receptors, we have failed to find major effects of 17 beta-estradiol (E2) on modulation of proliferation of bone matrix protein production by hOB cells. Because the major effect of E2 in vivo is to decrease bone resorption and because transforming growth factor-beta (TGF-beta) has been reported to decrease osteoclast-mediated bone resorption, we have tested the hypothesis that the effect of E2 on osteoclast activity is, at least in part, indirectly mediated by enhancing production of TGF-beta by osteoblasts. We therefore have extended our studies to examine possible TGF-beta gene expression including the modulation of the release of TGF-beta by E2 in near homogenous populations of hOB cells. TGF-beta protein production was measured using growth inhibition of CCL-64 cells and verified by blocking effects with anti-TGF-beta antibodies. TGF-beta 1 messenger RNA (mRNA) steady state levels were assessed by northern blot analysis and quantitated by densitometric measurement using 18S ribosomal RNA as a reference. There was an E2 dose-dependent increase in TGF-beta protein production within 24 h of challenge with E2. Northern blots from these cells demonstrated a dose-dependent increase in steady state mRNA levels of TGF-beta 1 within 6 h of treatment. PTH was also a potent stimulator of TGF-beta protein and message levels in a dose-dependent manner. Interestingly, coincubation of equimolar concentrations of E2 and PTH (10(-8) M) abrogated the stimulation of TGF-beta 1 mRNA and protein. Decreasing the relative concentration of PTH in this coincubation with E2 increased TGF-beta 1 mRNA and protein levels. These data support the fact that E2 modulates TGF-beta production in osteoblasts. In this manner TGF-beta may mediate E2 inhibition of osteoclast activity.

Silicon in trace amounts enhances bone formation, and the silicon-containing compound zeolite A (ZA) increases eggshell thickness in hens. In the studies reported here, treatment of nearly homogeneous strains of normal human osteoblast-like cells for 48 h with ZA at 0.1-100 micrograms/ml induced a dose-dependent increase (r = 0.35, P < 0.001) in DNA synthesis (n = 31) to 162 +/- 16% (mean +/- SEM) of control and in the proportion of cells in mitosis (n = 4) from 9.1 +/- 1.8 to 27.0 +/- 4.5% (r = 0.69, P < 0.005). ZA treatment also increased alkaline phosphatase activity (P < 0.05) and osteocalcin release (P < 0.05) but did not significantly affect collagen production per individual cell. The mitogenic action of ZA was dependent on cell seeding density over the range of 1250-40,000 cells per cm2, which is consistent with induction of an autocrine factor(s). TGF-beta is a potent mitogen for osteoblasts. ZA treatment increased the steady-state mRNA levels of transforming growth factor beta 1 (TGF-beta 1) and induced the release of the latent form of TGF-beta protein into the conditioned medium within 6 h. We conclude that ZA induces the proliferation and differentiation of cells of the osteoblast lineage.

In humans, glucocorticoids are known to have marked effects on bone metabolism and function, including the significant regulation of osteoblast cells. To aid in the understanding of the mechanism of glucocorticoid action on normal human osteoblasts (hOB), confluent cells were analyzed for the presence of glucocorticoid receptors (GR) as well as for the effects of the glucocorticoid dexamethasone (Dex) on the expression of both the rapid responding nuclear proto-oncogenes and the late responding structural genes for bone matrix proteins. The interactions between Dex and 1,25 dihydroxy vitamin D3 (1,25 D3) on the gene expression in these cells were also examined. Using a functional receptor assay, a mean of 11,600 functional nuclear bound glucocorticoid receptors (range 6,000-22,000) was measured in fifteen separate cell strains. Northern blot analysis with a cDNA probe to the human GR was used to demonstrate the presence of a 7Kb transcript which is a candidate mRNA for GR in these cells. In agreement with previous studies, treatment of the hOB cells with Dex increased the steady state mRNA levels for alkaline phosphatase (AP) but displayed little or no effect on the mRNA levels for osteocalcin (OC) and glyceraldehyde phosphate dehydrogenase (GAPDH). Interestingly, the 1,25 D3 inductions of mRNA levels for OC were blocked by Dex but enhanced for AP. The above effects of Dex on AP and OC gene expression, including the interaction with 1,25 D3, were also shown to occur at the level of protein. The effect of Dex on the mRNA levels of the nuclear proto-oncogenes c-myc, c-fos, and c-jun was also investigated, since the oncoproteins (Fos/Jun) appear to play a role in the delayed glucocorticoid regulation of structural genes. Interestingly, Dex increased the steady state levels of c-myc, c-fos, and c-jun mRNAs in nonproliferating (confluent) hOB cells by 3.5-, 10-, and 2.0-fold, respectively, over control (untreated cells) values within one h of steroid treatment. The Dex-induced mRNA levels were transient and returned to basal values within 24 h of the steroid treatment. A reduced but qualitatively similar pattern of response was found in proliferating hOB cells. The pattern of response of these genes to glucocorticoids in hOB cells mimics the response in avian liver cells but not in reproductive cells. These results support the theory that hOB cells are target cells for glucocorticoids, and that as a primary event glucocorticoids rapidly regulate the expression of the nuclear oncoproteins Fos/Jun in these cells.

Prostaglandin (PG) biosynthesis by cytokine stimulated normal adult human osteoblast-like (hOB) cells was evaluated by thin layer chromatography, high performance liquid chromatography, and specific immunoassays. PGE2 was the predominant PG formed under all incubation conditions tested. Control samples produced measurable amounts of PGE2, and the measured level of this metabolite increased by 22-fold (from 7 to 152 ng/ml) following a 20 h treatment with the combination of TGF beta and tumor necrosis factor-alpha(TNF). The production of 6-keto-PGF1 alpha (the stable metabolite of prostacyclin) and of PGF2 alpha were each increased by about five-fold (from about 0.5 to 2.5 ng/ml) in samples treated with the cytokines. Thus, TGF beta and TNF exerted a regulation of hOB cell PG biosynthesis that was principally directed towards an increased PGE2 biosynthesis, with lesser effects on the production of other PG metabolites. COX-2 mRNA levels were increased within 2 h of cytokine stimulation, reached a maximum at 6-12 h, and levels had appreciably diminished by 24 h after treatment. Both TGF beta and TNF could independently increase COX-2 mRNA levels and PG biosynthesis. However, the increased production of PGE2 resulting from TNF stimulation was blocked by the addition of an interleukin-1 beta (IL-1 beta) neutralizing antibody, suggesting that TNF regulation of hOB cell PG synthesis was secondary to its capacity to increase hOB cell IL-1 beta production. TGF beta regulation of PG production was not affected by the addition of the neutralizing antibody. These studies support the proposition that PGs can be important autocrine/paracrine mediators of bone biology, whose production by hOB cells is responsively regulated by osteotropic cytokines.