Oursler M J, Cortese C, Keeting P, Anderson M A, Bonde S K, Riggs B L, Spelsberg T C
Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, Minnesota 55905.
Endocrinology. 1991 Dec;129(6):3313-20. doi: 10.1210/endo-129-6-3313.
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.
尽管我们实验室已报告正常人类成骨细胞样（hOB）细胞含有雌激素受体，但我们未能发现17β-雌二醇（E2）对hOB细胞增殖或骨基质蛋白产生调节的主要作用。由于E2在体内的主要作用是减少骨吸收，且据报道转化生长因子-β（TGF-β）可减少破骨细胞介导的骨吸收，我们检验了这样一个假设，即E2对破骨细胞活性的影响至少部分是通过增强成骨细胞TGF-β的产生而间接介导的。因此，我们扩展了研究，以检查hOB细胞近乎同质群体中可能的TGF-β基因表达，包括E2对TGF-β释放的调节。使用CCL-64细胞的生长抑制来测量TGF-β蛋白的产生，并用抗TGF-β抗体的阻断作用进行验证。通过Northern印迹分析评估TGF-β1信使核糖核酸（mRNA）的稳态水平，并以18S核糖体RNA作为参照通过光密度测量进行定量。用E2刺激24小时内，TGF-β蛋白产生呈E2剂量依赖性增加。这些细胞的Northern印迹显示，处理6小时内TGF-β1的稳态mRNA水平呈剂量依赖性增加。甲状旁腺激素（PTH）也是TGF-β蛋白和信使水平的有效刺激剂，呈剂量依赖性。有趣的是，等摩尔浓度的E2和PTH（10^(-8)M）共同孵育可消除对TGF-β1mRNA和蛋白的刺激。在与E2共同孵育时降低PTH的相对浓度会增加TGF-β1mRNA和蛋白水平。这些数据支持E2调节成骨细胞中TGF-β产生这一事实。通过这种方式，TGF-β可能介导E2对破骨细胞活性的抑制作用。
