REGULATION OF BONE CELL GENES BY 1,25-(OH)2 VITAMIN D

对成骨细胞活性的分析是理解 在正常生理和疾病中的骨骼稳态，因为 成骨细胞负责合成新骨， 成骨细胞吸收骨。 成骨细胞的核心作用与 各种各样的荷尔蒙和旁分泌因素影响着 成骨细胞活性 1.25-二羟维生素D3 [1，25（OH）2D 3]，一种主要的 钙调节激素，直接作用于成骨细胞， 基因程序的表达。 1，25（OH）_2D_3的结构分析 调节成骨细胞基因表达将有助于阐明成骨细胞 不同的角色被调节，并将作为一个有用的模型如何类固醇 激素受体与其他调节蛋白相互作用， 细胞的表型。 21，25（OH）_2D_3对大鼠骨钙素基因的影响 将进行详细分析。 该基因实际上只在 成骨细胞;骨钙素基因转录由1，25（OH）2D 3刺激， 培养的成骨细胞样细胞。 将使用克隆基因的转染 定义必要且足够的DNA序列， 到1，25（OH）2D 3到骨钙素基因。 DNA结合研究，使用细胞 提取物，纯化的受体，和遗传产生的受体，将是 进行定义结合1，25（OH）2D 3受体的DNA序列， 结合骨钙素基因表达所需的其他因子， 定义相关结合蛋白之间的相互作用。 的 其他激素和细胞信使（甲状腺素， 维甲酸，雌激素，糖皮质激素，蛋白激活因子 激酶C）影响骨钙素基因转录， 评估。 可能的累加、协同和拮抗作用将 在特异性DNA结合和激活的水平上进行分析， 转录。 转录应答因子之间的相互作用 并且将类似地分析骨特异性转录因子。 的 骨钙素基因在完整染色质中的结构将被评估， 定义对DNA酶I超敏感的调节区。 这些 这些研究将提供克隆基因研究和基因组研究之间的联系。 原地。 因此，该项目的目标是建立一个基础， 了解1，25（OH）2D 3和其他激素如何调节成骨细胞 基因计划

An analysis of the activity of osteoblasts is central to an understanding of skeletal homeostasis in normal physiology and in disease, since osteoblasts are responsible for synthesizing new bone and for stimulating osteoblasts to resorb bone. The central role of osteoblasts correlates with the wide variety of hormonal and paracrine factors that influence osteoblast activity. 1.25-dihydroxyvitamin D3 [1,25(OH)2D3], a major calcium-regulating hormone, acts directly on osteoblasts to alter expression of the genetic program. An analysis of how 1,25(OH)2D3 modulates osteoblast gene expression will help clarify how the osteoblast's varying roles are regulated and will serve as a useful model of how steroid hormone receptors interact with other regulatory proteins to determine a cell's phenotype. The effects of 2\1,25(OH)2D3 on the rat osteocalcin gene will be analyzed in detail. This gene is expressed virtually only In osteoblasts; osteocalcin gene transcription is stimulated by 1,25(OH)2D3 in cultured osteoblast-like cells. Transfection of cloned genes will be used to define DNA sequences necessary and sufficient to confer responsiveness to 1,25(OH)2D3 to the osteocalcin gene. DNA binding studies, using cell extracts, purified receptors, and genetically produced receptors, will be performed to define DNA sequences that bind the 1,25(OH)2D3 receptor, those that bind other factors required for osteocalcin gene expression, and to define the interactions between the relevant binding proteins. The possibility that other hormones and cellular messengers (thyroxine, retinoic acid, estrogen, gluccorticoids, factors activated by protein kinase C) affect osteocalcin gene transcription will be systematically evaluated. Possible additive, synergistic, and antagonistic effects will be analyzed at the levels of specific DNA binding and activation of transcription. The interaction of hormone-responsive transcription factors and bone-specific transcription factors will similarly be analyzed. The structure for the osteocalcin gene in intact chromatin will be evaluated to define regulatory regions that are hypersensitive to DNAse I. These studies will provide a link between studied of cloned genes and of genes in situ. This project's goal is thus to build a base for a comprehensive understanding of how 1,25(OH)2D3 and other hormones modulate the osteoblast genetic program.