RESUMEN
Bone morphogenetic proteins (BMPs) induce differentiation of osteoblast and chondroblast lineage cells from uncommitted mesenchymal precursors. Because estrogen has potent osteochondrogenic actions, we investigated its effect on BMP production in two estrogen-responsive, human immortalized cell lines (hFOB/ER3 and hFOB/ER9) that display the mature osteoblast phenotype. These cell lines were produced by stable transfection of the estrogen receptor (ER) gene into immortalized fetal osteoblasts at low ( approximately 800 ER/ nucleus) and at high ( approximately 3, 900 ER/nucleus) levels, respectively. As assessed by reverse transcriptase PCR, treatment with 17beta-estradiol (10(-)10 - 10(-)7 M) increased steady-state levels of BMP-6 mRNA dose dependently by twofold in the hFOB/ER3 cells and by over threefold in the hFOB/ER9 cells. Messenger RNA levels for transforming growth factors-beta1 and -beta2 and BMPs-1 through -5 and -7 levels were unchanged. The results were confirmed by sequence determination of the PCR product and by Northern blot analysis for total RNA. 17beta-estradiol increased BMP-6 protein production sixfold by Western analysis. Cotreatment with antiestrogens (ICI 182,780 or 4-hydroxytamoxifen) antagonized the effects of 17beta-estradiol. These data suggest that some of the skeletal effects of estrogen on bone and cartilage may be mediated by increased production of BMP-6 by osteoblasts.
Asunto(s)
Proteínas Morfogenéticas Óseas/biosíntesis , Estrógenos/farmacología , Osteoblastos/metabolismo , Northern Blotting , Western Blotting , Proteína Morfogenética Ósea 6 , Proteínas Morfogenéticas Óseas/genética , Línea Celular , Estradiol/análogos & derivados , Estradiol/farmacología , Fulvestrant , Humanos , Reacción en Cadena de la Polimerasa , ARN Mensajero/análisis , Tamoxifeno/análogos & derivados , Tamoxifeno/farmacología , Factor de Crecimiento Transformador beta/genéticaRESUMEN
Individually, transforming growth factor beta (TGF beta) and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) alter the growth and differentiation of normal and transformed osteoblast-like (OB) cells. Although recent evidence suggests interactions between TGF beta and 1,25(OH)2D3 may occur, little is known of the individual or combined effects of these hormones on the expression of the osteoblast phenotype at the cytochemical and biochemical levels in normal human OB (hOB) cells. Primary cultures of hOBs were treated with TGF beta (0.001-10 ng/ml) and 1,25(OH)2D3 (0.1 pM-100 nM) either alone or in combination. TGF beta and 1,25(OH)2D3 stimulated spindle-shaped cells to become stellate in appearance and increased the number of cytoplasmic processes. TGF beta increased 3H-thymidine incorporation and 1,25(OH)2D3 reduced this effect. Conversely, procollagen type-I synthesis and secretion were increased in a dose-dependent manner in the presence of TGF beta but were not significantly affected in the presence of 1,25(OH)2D3. TGF beta and 1,25(OH)2D3 each marginally increased alkaline phosphatase (ALP) activity, but the combination synergistically increased ALP activity in a dose- and time-dependent manner at the cytochemical and biochemical level (three to tenfold over vehicle controls; n = 12). In contrast, TGF beta reduced 1,25(OH)2D3-stimulated osteocalcin secretion. These data suggest that TGF beta stimulates hOB cells to actively produce collagen matrix and proliferate. The combination of TGF beta and 1,25(OH)2D3, however, produces a synergistic increase in ALP activity and maintenance of collagen synthesis. 1,25(OH)2D3 stimulation may induce cells to advance to an endstage where cell proliferation is reduced and osteocalcin expression is promoted. Interactions between TGF beta and 1,25(OH)2D3 may represent important steps in the regulation of osteoblast differentiation and matrix production.
Asunto(s)
Calcitriol/farmacología , Osteoblastos/citología , Osteoblastos/metabolismo , Factor de Crecimiento Transformador beta/farmacología , Fosfatasa Alcalina/metabolismo , División Celular/efectos de los fármacos , Células Cultivadas , ADN/biosíntesis , Relación Dosis-Respuesta a Droga , Humanos , Cinética , Osteoblastos/efectos de los fármacos , Osteocalcina/análisis , Osteocalcina/metabolismo , Procolágeno/análisis , Procolágeno/metabolismo , Radioinmunoensayo , Timidina/metabolismoRESUMEN
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.
Asunto(s)
Silicatos de Aluminio/farmacología , Osteoblastos/efectos de los fármacos , Factor de Crecimiento Transformador beta/biosíntesis , Fosfatasa Alcalina/metabolismo , Northern Blotting , Calcitriol/farmacología , Diferenciación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Células Cultivadas , Colágeno/metabolismo , ADN/biosíntesis , Relación Dosis-Respuesta a Droga , Humanos , Osteoblastos/citología , Osteoblastos/metabolismo , Osteocalcina/metabolismo , Análisis de Regresión , Factor de Crecimiento Transformador beta/farmacología , ZeolitasRESUMEN
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.
Asunto(s)
Estradiol/farmacología , Osteoblastos/metabolismo , Hormona Paratiroidea/farmacología , Factor de Crecimiento Transformador beta/biosíntesis , Northern Blotting , Células Cultivadas , Interacciones Farmacológicas , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Osteoblastos/efectos de los fármacos , ARN Mensajero/metabolismo , Factor de Crecimiento Transformador beta/genéticaRESUMEN
The NH2-terminal cleavage peptide of procalcitonin (N-proCT) recently was reported to be a bone cell mitogen (Burns DM et al., Proc Natl Acad Sci USA 86:9519-9523, 1989). We have investigated the effect of N-proCT on the proliferation of normal human cells that have the phenotype of mature osteoblasts (hOB cells). N-proCT treatment for 24, 48, or 96 h in concentrations from 1 nM to 1 microM did not significantly increase [3H]thymidine uptake (means ranged from -19% to 38% of control, no significant differences) in hOB cells (6-10 cell strains per experiment) plated at four different densities. However, the hOB cells responded significantly to treatment with transforming growth factor beta (3 ng/ml), bovine insulin (300 micrograms/ml), or 30% fetal calf serum, which were included in all experiments as positive controls. The [3H]thymidine uptake data were confirmed in a direct cell count experiment tested at 96 h. Thus our data do not support the hypothesis that N-proCT is a potent mitogen for normal human osteoblasts.