HORMONE REGULATION OF BONE COLLAGEN SYNTHESIS

我们建议分析DNA序列和蛋白质结合因子， 负责α-1（I）胶原基因转录的升高 与成纤维细胞相比，这些序列和 负责维生素A抑制α-1（I）合成的因子 D在骨细胞中，而不是成纤维细胞。 我们会跟进初步调查 有证据表明在-3521和-2291之间存在骨特异性增强子， 和维生素D反应元件，其位于-2291和-1665上游之间， RNA转录起始位点。 我们将分析DNA序列， 缺失和寡核苷酸定向诱变，然后分析 稳定表达突变体的转录活性和激素调控 转染的骨肉瘤和成纤维细胞系，以及转基因 小鼠 将测试启动子的亚片段对启动子表达的影响。 弱异源启动子的转录。 体外蛋白质/DNA 将使用凝胶迁移率变化和足迹法分析相互作用， 来自激素处理和对照骨细胞的核提取物， 成纤维细胞 将使用DNA酶分析体内染色质结构 敏感性研究。 了解不同的监管影响， 骨和成纤维细胞中胶原蛋白的产生对于理解 遗传性获得性骨骼疾病。

We propose to analyze the DNA sequences and protein binding factors which are responsible for elevated transcription of the alpha-1(I) collagen gene in bone cells as compared to fibroblast, and also these sequences and factors responsible for the inhibition of alpha-1(I) synthesis by vitamin D in bone cells but not fibroblasts. We will follow-up on our preliminary evidence that there is a bone-specific enhancer between -3521 and -2291, and a vitamin D responsive element between -2291 and -1665 upstream from the RNA transcription start site. We will analyze DNA sequences using deletions and oligonucleotide directed mutagenesis followed by analysis of transcriptional activity and hormone regulation of the mutants in stably transfected osteosarcoma and fibroblast cell lines, and also in transgenic mice. Subfragments of the promoter will be tested for their effect on the transcription of a weak heterologous promoter. In vitro protein/DNA interactions will be analyzed by gel mobility shifts and footprinting using nuclear extracts from hormone treated and control bone cells and fibroblasts. In vivo chromatin structure will be analyzed using DNase sensitivity studies. Appreciating the different regulatory influences on collagen production in bone and fibroblast is essential for understanding genetic acquired disorders of bone.