The effects of triiodothyronine on human osteoblast-like cells metabolism and interactions with growth hormone.

The expression of thyroid hormones receptors in osteoblasts and osteoclasts has involved these cells as direct targets for triiodothyronine (T3), but thyroid hormones may also interact with other hormones or local growth factors to exert their actions on bone cells. Among these, growth hormone (GH) is recognised as participating in the acquisition and maintenance of bone mass and exerting stimulatory effects on human osteoblastic cells. The aim of this study was to investigate T3 effects on primary human adult osteoblast-like cells (HOB) as well as to test for possible interactions between T3 and GH on bone cell metabolism. Primary human bone cell cultures were obtained by outgrowth from trabecular bone fragments from the hip and knee. Dose-response studies demonstrated enhanced [3H]-thymidine incorporation for T3 at 10(-9), 10(-8), 10(-7) and 20(-7) M, with a maximal response of 162.81 +/- 12.97 % with T3 10(-8) M, compared to vehicle (p < 0.001). Time-course studies showed an increased osteoblast-like cell proliferation after 24 h, followed by a decrease of cell proliferation by 48 h and 72 h of culture, respectively, when compared to control cells, with a maximal response after 72 h (T3 10(-10) M: 45.21 +/- 6.97 %, p < 0.01). In addition, T3 markedly increased specific alkaline phosphatase (AP) activity in HOB (10(-10) M: 169.86 +/- 12.14 % vs. control, p < 0.001), but no significant influence on type I procollagen propeptide (PICP) production was observed. At 10(-9) - 10(-7) g/ml, GH significantly enhanced HOB proliferation (p < 0.001) however, GH effects were not dose-dependent. Triiodothyronine, at a high concentration (10(-7) M), stimulated GH-receptor (GHR) mRNA levels by 165.20 +/- 16.54 % after 24 h (p < 0.05). Correspondingly, a synergistic effect of T 3 with the same concentration and GH on cell proliferation in human adult osteoblast-like cells was found.

Effects of triiodothyronine on the insulin-like growth factor system in primary human osteoblastic cells in vitro.

Thyroid hormone plays a major role in the regulation of bone metabolism but the mechanism by which this is accomplished is not clear. Interactions of thyroid hormone with the growth hormone/insulin-like growth factors (IGFs) axis suggest an alternate pathway of action for triiodothyronine (T(3)) on bone formation, besides direct effects. The present study investigates the influence of T(3) on IGF-1, IGF-2, IGF-1 receptor (IGF-1R), and IGF binding protein (IGFBP) transcripts, and on IGF-1 action in human osteoblastic cells (hOB) under serum-free culture conditions. No influence of T(3) on IGF-1, IGF-2, IGFBP-3, or IGFBP-4 mRNA levels in hOB was observed. However, T(3) at concentrations of 10(-8) mol/L and 10(-7) mol/L increased IGF-1R mRNA levels in a dose-dependent manner (p < 0.01) and enhanced IGFBP-5 mRNA levels at a concentration of 10(-7) mol/L (p < 0.05), as assessed by reverse transcriptase-polymerase chain reaction. Correspondingly, Scatchard analysis of [(125)I]-IGF-1 binding revealed that T(3) at 10(-7) mol/L increased the number of IGF-1 binding sites in hOB, with small changes in receptor affinity. In addition, a synergistic effect of T(3) and IGF-1 on hOB proliferation was found (p < 0.05). We conclude that IGF-1R and IGFBP-5 are thyroid hormone target genes in human osteoblasts, whereas IGF-1 mRNA expression itself appears not to be regulated by T(3) in hOB. However, T(3) stimulates IGF-1R mRNA expression as well as IGF-1 binding and IGF-1 induced cell proliferation in osteoblasts, thus suggesting thyroid hormone may potentiate the effect of IGF-1 at the receptor level. This may contribute to the positive effects of thyroid hormone on bone formation, which, in addition, may be modulated by increased IGFBP-5 expression.

Glucocorticoids regulate the expression of the human osteoblastic endothelin A receptor gene.

The 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 (ETRA). 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 ETRA expression in HOC were investigated. Dexamethasone (DEX) increased ETRA mRNA levels in a dose- and time-dependent fashion. The effects of dexamethasone, prednisolone, and deflazacort on the increase of ETRA 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'-flanking region of the ETRA 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 ETRA 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 ETRA, 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.

Endothelin-1 is a potent regulator of human bone cell metabolism in vitro.

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.

Gonadal and adrenal androgens are potent regulators of human bone cell metabolism in vitro.

Androgens stimulate bone formation and play an important role in the maintenance of bone mass. Clinical observations suggest that both gonadal and adrenal androgens contribute to the positive impact of androgenic steroids on bone metabolism. We investigated the mechanism of action of the adrenal androgen dehydroepiandrosterone (DHEA) and its sulfated compound dehydroepiandrosterone sulfate (DHEAS) on human osteoblastic cells (HOCs) in vitro. The DHEA- and DHEAS-induced effects were analyzed in parallel with the actions elicited by the gonadal androgen dihydrotestosterone (DHT). There was no qualitative difference between the effects of gonadal and adrenal androgens on HOC metabolism in vitro. Both were stimulatory as regards cell proliferation and differentiated functions, but the gonadal androgen DHT was significantly more potent than DHEA. The actions of DHT and DHEA on HOC proliferation and alkaline phosphatase (ALP) production could be prevented by the androgen receptor antagonist hydroxyflutamide and inhibitory transforming growth factor beta antibodies (TGF-beta ab), respectively, but were not affected by the presence of the 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) and 5-alpha-reductase (5-AR) inhibitor 17 beta-N,N-diethylcarbamoyl-4-methyl- 4aza-5 alpha-androstan-3-one (4-MA). This indicates that DHT and DHEA (1) exert their mitogenic effects by androgen receptor-mediated mechanisms, (2) stimulate ALP production by increased TGF-beta expression, (3) that the action of DHT is not affected by the presence of 4-MA, and that (4) DHEA does not need to be metabolized by 3 beta HSD or 5-AR first to exert its effects on HOCs in vitro.

Effects of androgens on subpopulations of the human osteosarcoma cell line SaOS2.

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.

Interactions of growth factors present in bone matrix with bone cells: effects on DNA synthesis and alkaline phosphatase.

It has been shown that bone cells produce and secrete several growth factors (GFs) which are also found in the bone matrix. To investigate the role of these growth factors in bone cell metabolism, we compared the effects of different factors separately and in combination with respect to osteoblastic cell proliferation and differentiation. While basic fibroblast GF (FGF), transforming GF beta-1 (TGF beta), and platelet-derived GF (PDGF) enhance DNA synthesis, they had the opposite effect on alkaline phosphatase (ALP) activity in cell extracts: FGF, TGF beta, and PDGF inhibited cell ALP but strongly stimulated DNA synthesis. The IGFs had little effect on cell ALP but increased the release of ALP into the conditioned medium. In mitogenic tests of combinations of GFs, most had at least additive effects at low concentrations, and FGF, TGF beta, and IGF2 produced synergistic effects. Evidence is presented for (1) the modulation of the effects of one GF by the action of other GF, (2) synergistic interactions between FGF, TGF beta, and IGF2, and (3) a possible role for the observed interactions among GF for the mitogenic effect of human bone extract.

Androgens directly stimulate proliferation of bone cells in vitro.

This report describes the first observation of a direct mitogenic effect of androgens on isolated osteoblastic cells in serum-free culture. [3H]thymidine incorporation into DNA and cell counts were used as measures of cell proliferation. The percentage of cells that stained for alkaline phosphatase was used as a measure of differentiation. Dihydrotestosterone (DHT) enhanced mouse osteoblastic cell proliferation in a dose dependent manner over a wide range of doses (10(-8) to 10(-11) molar), and was maximally active at 10(-9) M. DHT also stimulated proliferation in human osteoblast cell cultures and in cultures of the human osteosarcoma cell line, TE89. Testosterone, fluoxymesterone (a synthetic androgenic steroid) and methenolone (an anabolic steroid) were also mitogenic in the mouse bone cell system. The mitogenic effect of DHT on bone cells was inhibited by antiandrogens (hydroxyflutamide and cyproterone acetate) which compete for binding to the androgen receptor. In addition to effects on cell proliferation, DHT increased the percentage of alkaline phosphatase (ALP) positive cells in all three bone cell systems tested, and this effect was inhibited by antiandrogens. We conclude that androgens can stimulate human and murine osteoblastic cell proliferation in vitro, and induce expression of the osteoblast-line differentiation marker ALP, presumably by an androgen receptor mediated mechanism.