SEX STEROIDS, GROWTH FACTORS AND BONE CELL FUNCTION

本申请的主要重点将是使用培养的正常人 成骨细胞样（hOB）细胞作为模型系统，用于了解 正常成骨细胞功能的调节，并提供有关 骨质疏松症的细胞功能异常。 研究制定一个明确的， 将继续使用无血清培养基进行最佳细胞生长，因为 培养基应该1）增强这些细胞的生产， 增长，劳动密集型和昂贵的，和2）允许更准确的 评估个体生长相关因子对这些 细胞 我们目前的数据表明雌激素受体的存在 在hOB细胞中存在雄激素的迹象， 孕酮受体 我们计划继续分析 hOB细胞中的功能性类固醇受体，并选择 骨肉瘤细胞系使用核结合试验，活性炭试验， 北方印迹（mRNA）和蛋白质印迹（蛋白质）分析。 我们有 获得了针对骨蛋白、生长和骨形成的各种mRNA的cDNA探针， 因子、原癌基因和其他类固醇调节基因，包括 类固醇受体 我们计划筛选这些cDNA探针， 显示出最佳的雌激素调节。 然后，利用这些cDNA， 将更详细地评估雌激素、雄激素和 孕激素对hOB细胞和选定骨肉瘤中mRNA水平的影响 细胞系 类固醇激动和拮抗作用的研究 类固醇和其他激素（PTH，1，25二羟维生素D3， 糖皮质激素）将在hOB细胞中进行。 类固醇对 骨蛋白的水平将在类固醇 这些重要基因表达的调节发生在 翻译/翻译后的水平。 最后，我们计划继续我们的 性类固醇激素对大鼠睾丸激素分泌影响的研究 转化生长因子（TGF-α、TGF-β）和胰岛素样生长 在mRNA，蛋白质， 和活动。 与Greg Mundy博士和同事合作， 位于圣安东尼奥的德克萨斯大学将研究 雌激素与我们的性类固醇和PTH的相互作用， 影响TGF-β的产生。 在同一次合作中， 雌激素处理的hOB细胞的条件培养基对破骨细胞的作用 将研究功能（骨吸收）。 希望广大 上述研究将有助于深入了解骨调节途径 并最终转化为这些异常[在退化中的途径] 骨质疏松症导致更好的治疗策略。

The major focus of this application will be to use cultured normal human osteoblast-like (hOB) cells as a model system for understanding the regulation of normal osteoblast function and to provide insights on abnormal cell function in osteoporosis. Studies to develop a defined, serum-free medium for optimal cell growth will be continued since this medium should 1) enhance the production of these cells which are slow growing, labor intensive and costly, and 2) permit more accurate assessment of the effects of individual growth related agents on these cells. Our present data demonstrate the presence of estrogen receptors in hOB cells with indications of the presence of androgen and progesterone receptors. We plan to continue our analyses of the functional sex steroid receptors in the hOB cells and selected osteosarcoma lines using a nuclear binding assay, a charcoal assay, Northern blot (mRNA), and Western blot (protein) analyses. We have obtained cDNA probes to a variety of mRNAs for bone proteins, growth factors, proto-oncogenes and other steroid regulated genes including steroid receptors. We plan to screen these cDNA probes to select those which display optimal estrogen regulation. Then, using these cDNAs, we will assess in greater detail the effects of estrogens, androgens and progestins on the mRNA levels in hOB cells and in selected osteosarcoma cell lines. Studies of steroid agonism and antagonism with other sex steroids and other hormones (PTH, 1,25 dihydroxyvitamin D3, and glucocorticoids) will be performed in hOB cells. The steroid action on the levels of bone proteins will be measured in the instance the steroid regulation of the expression of these important genes occurs at the level of translation/post-translation. Finally, we plan to continue our investigations on the effects of sex steroids on the production of the transforming growth factors (TGF-a, TGF-b) and insulin-like growth factors (IGF-I adn IGF-II) in hOB cells at the level of mRNA, protein, and activity. Collaborations with Dr. Greg Mundy and coworkers, University of Texans at San Antonio, will involve studies of the interactions of estrogens with our sex steroids and with PTH to influence TGF-B production. Also in the same collaboration, the effects of conditioned media from estrogen treated hOB cells on osteoclast cell function (bone resorption) will be investigated. It is hoped that the above investigations will yield insights into bone regulatory pathways and ultimately into abnormalities of these [pathways in involutional osteoporosis resulting in better strategies for its treatment.