C/EBP Regulation of Osteoblast Function

C/EBP 对成骨细胞功能的调节

• 批准号: 6435131
• 负责人: JOHN R HARRISON
• 金额: $ 32.49万
• 依托单位: UNIVERSITY OF CONNECTICUT SCH OF MED/DNT
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-09-15 至 2006-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6435131
• 关键词: bone sialoprotein; cell differentiation; computed axial tomography; developmental genetics; gel mobility shift assay; genetically modified animals; immunocytochemistry; immunoprecipitation; laboratory mouse; northern blottings; osteoblasts; osteocalcin; osteogenesis; tissue /cell culture; transcription factor

DESCRIPTION (provided by applicant): C/EBP transcription factors are known to stimulate adipocyte differentiation, and mounting evidence suggests that osteoblasts and adipocytes share a common pluripotent progenitor in the bone marrow. We prepared transgenic mice with Col1a1 promoter-targeted expression of FLAG-tagged p20C/EBPbeta in an effort to enhance osteoblast differentiation at the expense of adipogenesis. Surprisingly, however, we found that all four lines of transgenic pOBCol3.6-FLp20C/EBPbeta mice exhibit osteopenia. Preliminary histological evidence suggests that low bone mass may be due to impaired osteoblast differentiation or function. We hypothesize that mice with targeted expression of p20C/EBPbeta have osteopenia resulting from decreased bone formation, and that p20C/EBPbeta inhibits osteoblast differentiation in vivo. In Specific Aim 1, we will characterize the bone phenotype of transgenic mice using microcomputed tomography, static and dynamic histomorphometry, and measurement of transgene expression by Northern blotting and immunohistochemistry. C/EBP gene knock-out mice will be screened for the presence of a similar phenotype. In Specific Aim 2, we will determine the effects of p20C/EBPbeta expression on osteoblast differentiation and function using ex vivo bone marrow stromal and primary calvarial cell culture models. Cell proliferation will be determined in primary calvarial cultures by cell number and by (3H)thymidine incorporation. Osteoblast differentiation will be assessed in both models by the formation of mineralized bone nodules and the expression of osteoblast markers such as Col1a1, bone sialoprotein and osteocalcin. As a first step toward understanding the mechanism of p20C/EBPbeta action, its dimerization partners in primary osteoblastic cells will be identified by immunoprecipitation and electrophoretic mobility shift analysis. The proposed studies should help to elucidate role of transcription factors, specifically members of the C/EBP family, in the control of osteoblast differentiation and bone mass.

描述（由申请人提供）：已知C/EBP转录因子刺激脂肪细胞分化，而越来越多的证据表明 成骨细胞和脂肪细胞在骨头中共有一个常见的多能祖细胞 骨髓。我们用Col1a1启动子靶向的表达制备了转基因小鼠 标记标签的P20C/EBPBETA，以增强成骨细胞的分化 脂肪形成的费用。令人惊讶的是，我们发现这四个 转基因POBCOL3.6-FLP20C/EBPBETA小鼠的线表现出骨质减少。 初步组织学证据表明，低骨量可能是由于 成骨细胞分化或功能受损。我们假设与 P20C/EBPBETA的靶向表达的骨质减少症降低。 骨形成，P20C/EBPBETA抑制成骨细胞的分化 体内。在特定目标1中，我们将使用微型层析成像，静态和动态来表征转基因小鼠的骨表型 组织形态测定法和通过北印迹测量转基因表达 和免疫组织化学。 C/EBP基因敲除小鼠将被筛选 存在类似的表型。在特定目标2中，我们将确定 P20C/EBPBETA表达对成骨细胞分化和功能的影响 使用离体骨髓基质和原代细胞培养模型。 细胞增殖将通过细胞在原发性钙钙培养物中确定 数量和（3H）胸苷掺入。成骨细胞的分化将是 通过形成矿化骨结节和 成骨细胞标记物的表达，例如Col1a1，骨唾液酸蛋白和 骨钙素。作为了解P20C/EBPBETA机制的第一步 作用，其在原始成骨细胞中的二聚化伙伴将是 通过免疫沉淀和电泳迁移率转移分析确定。 拟议的研究应有助于阐明转录因子的作用， C/EBP家族的特定成员，控制成骨细胞 分化和骨骼质量。