Bodine P V, Riggs B L, Spelsberg T C
Department of Biochemistry and Molecular Biology, Mayo Clinic and Foundation, Rochester, MN 55905.
J Steroid Biochem Mol Biol. 1995 Feb;52(2):149-58. doi: 10.1016/0960-0760(94)00165-i.
Although the role of estrogens in bone formation is becoming clarified, the function of androgens in this process remains to be defined. Consequently, we have explored the mechanism of action for both gonadal and adrenal androgens in normal human osteoblastic (hOB) cells, which are responsible for the synthesis and mineralization of bone. Changes in the steady-state mRNA levels for two nuclear proto-oncogenes (c-fos and c-jun) and one cytokine (TGF-beta 1) were quantified in response to short (30 min) and long (24-48 h) treatments of these cells with physiologic concentrations of steroids. In addition, the levels of TGF-beta protein in the hOB cells conditioned-media were measured using a bioassay. The results indicated that neither 10 nM dihydrotestosterone, 10-20 nM testosterone, nor 10-100 nM androstenedione had a significant effect on the steady-state levels of c-fos, c-jun, or TGF-beta 1 mRNAs. Interestingly, 10-1000 nM dehydroepiandrosterone (DHEA) and 1-10 microM DHEA-sulfate rapidly reduced the steady-state level of c-fos mRNA by 60-80% in a dose-dependent manner within 30 min. In contrast, neither of these adrenal steroids had a significant effect on the message levels for c-jun or TGF-beta 1. Surprisingly, although TGF-beta 1 mRNA levels remained unchanged, the total amount of TGF-beta activity in the hOB cell conditioned-media increased 2-5-fold in response to 24-48 h treatments of the cells with gonadal or adrenal androgens. This increase in TGF-beta activity by DHEA-sulfate was both time- and dose-dependent, and was not blocked by cotreatment with the specific aromatase inhibitor 4-hydroxyandrostenedione (1 microM). Immunoprecipitations of the hOB cell conditioned-media with isoform-specific TGF-beta neutralizing-antibodies indicated that TGF-beta 2 was predominantly produced by the cells in response to DHEA-sulfate treatment. These results demonstrate that differences exist between the actions of estrogens and androgens on normal human osteoblasts with regard to the regulation of c-fos expression and TGF-beta production. Moreover, these data suggest that DHEA and DHEA-sulfate may play a distinct role in the regulation of human osteoblast function via the rapid repression of c-fos message levels and the slower increase in TGF-beta 2 protein levels.
尽管雌激素在骨形成中的作用正逐渐明晰，但雄激素在此过程中的功能仍有待确定。因此，我们探究了性腺和肾上腺雄激素在正常人成骨细胞（hOB）中的作用机制，这些细胞负责骨的合成与矿化。在这些细胞用生理浓度的类固醇进行短期（30分钟）和长期（24 - 48小时）处理后，对两个核原癌基因（c - fos和c - jun）和一种细胞因子（TGF - β1）的稳态mRNA水平变化进行了定量分析。此外，使用生物测定法测量了hOB细胞条件培养基中TGF - β蛋白的水平。结果表明，10 nM双氢睾酮、10 - 20 nM睾酮或10 - 100 nM雄烯二酮对c - fos、c - jun或TGF - β1 mRNA的稳态水平均无显著影响。有趣的是，10 - 1000 nM脱氢表雄酮（DHEA）和1 - 10 μM硫酸脱氢表雄酮在30分钟内以剂量依赖方式迅速使c - fos mRNA的稳态水平降低60 - 80%。相比之下，这两种肾上腺类固醇对c - jun或TGF - β1的信使水平均无显著影响。令人惊讶的是，尽管TGF - β1 mRNA水平保持不变，但在性腺或肾上腺雄激素对细胞进行24 - 48小时处理后，hOB细胞条件培养基中TGF - β活性的总量增加了2 - 5倍。硫酸脱氢表雄酮引起的TGF - β活性增加具有时间和剂量依赖性，并且与特异性芳香化酶抑制剂4 - 羟基雄烯二酮（1 μM）共同处理时未被阻断。用亚型特异性TGF - β中和抗体对hOB细胞条件培养基进行免疫沉淀表明，TGF - β2主要是细胞在硫酸脱氢表雄酮处理后产生的。这些结果表明，在正常人类成骨细胞中，雌激素和雄激素在调节c - fos表达和TGF - β产生方面的作用存在差异。此外，这些数据表明，DHEA和硫酸脱氢表雄酮可能通过快速抑制c - fos信使水平和较慢增加TGF - β2蛋白水平，在人类成骨细胞功能调节中发挥独特作用。
