There are obvious sexual differences in adult skeletal morphology which for the most part are related to differences in size. Higher androgen serum levels in males exert potent osteoanabolic effects and therefore may contribute to this sexual dimorphism of the skeleton. The presence of androgen receptors (AR) in bone cells is a prerequisite for a direct osteoanabolic action of androgens. To investigate the possibility that, in addition to gender-related differences in androgen serum levels, there are also gender-related differences in the osteoblastic expression pattern of the androgen receptor, we examined AR mRNA expression, androgen binding sites, and mitogenic responses to the androgen dihydrotestosterone (DHT) in human osteoblastic cell (HOC) populations. HOCs were isolated from bone biopsy specimens derived from different skeletal sites of healthy adult males and females (2-69 years old). We found that male and female HOCs of all examined ages express similar AR mRNA levels and similar numbers of androgen binding sites. Using whole-cell-binding assays, we observed 3129-8417 androgen binding sites per femoral HOC with apparent KDs of 1.45-2.83 nM depending on the age of the investigated HOC population. Mandibular and cortical HOC of both sexes expressed higher AR mRNA levels, significantly more androgen binding sites per cell, and exhibited significantly greater mitogenic responses to DHT than iliac crest-derived and trabecular HOC of the same skeletal system and the same skeletal-site, respectively. In early adulthood, HOCs of both sexes appear to express somewhat higher AR mRNA levels and to possess more androgen binding sites than prepubertal and senescent HOC. Because sex hormone serum levels rise in puberty, we investigated the regulation of the AR mRNA expression by various steroids. We found that dexamethasone (dexa) and in some experiments also 17beta-estradiol (E2) and 1,25-dihydroxyvitamin D3 (D3) increased AR mRNA levels and androgen binding in HOC cultures. A pretreatment with dexa, E2, and D3 significantly increased the mitogenic response of HOCs to DHT. We conclude that (1) higher androgen serum levels in males together with a higher AR expression at certain skeletal sites may contribute to the development of sex-related differences in skeletal morphology, (2) glucocorticoids induce AR gene expression in HOC cultures, and (3) glucocorticoids, E2, and D3 enhance the mitogenic action of DHT.

Endothelial cell products may affect bone cell function, since trabecular and cortical bone are in close proximity to vascular endothelial cells. Incubation of cultured human osteoblastic cells with the endothelial cell polypeptide endothelin-1 (ET-1) resulted in a time- and dose-dependent stimulation of cell proliferation. Furthermore, markers of differentiated osteoblastic function, i.e., alkaline phosphatase and type-I collagen, were dose-dependently increased in response to ET-1. The effects of ET-1 on cell growth and function reached a maximum at higher ET-1 concentrations, and osteoblastic cells bound ET-1 specifically with a KD of 35 pM, corresponding to the biologic effects of ET-1 on bone cells. Under baseline conditions osteoblastic cells expressed 16,800 binding sites per cell. The effect of ET-1 was dependent on its binding to the endothelin-1 receptor A (ETRA), since an inhibitor of ET-1 binding blocked the biologic effects of ET-1. Northern blot analyses revealed that cultured human osteoblastic cells possess the transcript for the ETRA. Expression of ETRA mRNA was under control of 1,25-dihydroxyvitamin D3 [1,25 (OH)2D3]. Incubation of osteoblastic cells with 1,25(OH)2D3 increased ETRA mRNA levels, corresponding to an increased effect of ET-1 on osteoblastic proliferation and function. Thus, a concerted action of the endothelial cell polypeptide ET-1 and 1,25(OH)2D3 may mediate an osteoanabolic effect of the vascular and endocrine vitamin D system.

SummaryThe endothelial cell-derived peptide endothelin 1 (ET1) stimulates cell proliferation and differentiated functions of human osteoblastic cells (HOC), and HOC constitutively express the endothelin A receptor (ETR A ). Therefore, ET1 may play an important role in the regulation of bone cell metabolism. As glucocorticoids (GC) exert a profound influence on bone metabolism and increase the effects of ET1 on bone cell metabolism in vitro, the effects of GC on ETR A expression in HOC were investigated. Dexamethasone (DEX) increased ETR A mRNA levels in a dose-and time-dependent fashion. The effects of dexamethasone, prednisolone, and deflazacort on the increase of ETR A mRNA levels correlate positively with their binding affinity to the GC receptor. Scatchard analysis of ET1 binding data to HOC revealed that DEX increased the binding capacity for ET1 from 25,300 to 62,800 binding sites per osteoblastic cell, leading to an enhanced mitogenic effect of ET1 on HOC after preincubation with DEX. Transiently transfected primary HOC with a reporter gene construct, containing the 5 Ј -flanking region of the ETR A gene fused to luciferase gene, showed a promoter-dependent expression of the reporter gene and the induction of reporter gene expression by DEX treatment. Total RNA extracts of femoral head biopsies with osteonecrotic lesions from GC-treated patients showed threefold higher ETR A mRNA levels compared with extracts of bone biopsies from patients with traumatically induced osteonecrosis and coxarthrosis. Furthermore, GC treatment increased plasma ET1 levels by 50% compared with pretreatment values. These findings suggest that GC induced upregulation of ETR A , and ET1 plasma levels enhance ET1's anabolic action on bone cell metabolism. Increased ET1 concentrations may also impair bone perfusion by vasoconstriction in a metabolically activated skeletal region.

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Previously, we showed that androgens stimulate murine and human osteoblast-like cell proliferation and differentiation by mechanisms involving increased responses to mitogenic growth factors (GF) and increased GF production. To explain this dual action of androgens on primary osteoblastic cell populations we advanced the hypothesis that androgens exert differential effects on osteoblastic subpopulations. We subcloned a human osteosarcoma cell line (SaOS2) into subpopulations expressing high (HAS) and low (LAS) levels of alkaline phosphatase (ALP). The obtained subclones differed significantly in their ALP production and expressed a high and low ALP phenotype, respectively, for the entire experimental period. Dihydrotestosterone (DHT) increased specific ALP activity and type-I procollagen peptide secretion in both HAS and LAS. DHT pretreatment enhanced the mitogenic action of basic fibroblast growth factor (bFGF) and insulinlike growth factor 2 (IGF2) only in HAS. The enhanced mitogenic effect of IGF2 in HAS after DHT pretreatment was associated with increased IGF2-receptor mRNA levels. Therefore, we conclude that androgens exert their osteoanabolic action (1) by stimulating differentiated functions of osteoblastic cells with a high and a low ALP phenotype, and (2) via increased growth factor receptor expression and thereby enhancing mitogenic growth factor responses only in HAS.