HORMONE REGULATION OF BONE COLLAGEN SYNTHESIS

我们建议分析DNA序列和蛋白质结合因子 是导致α-1(I)胶原基因转录升高的原因 与成纤维细胞相比，这些序列和 维生素抑制α-1(I)合成的因素 D在骨细胞中表达，但不在成纤维细胞中表达。我们将跟进我们的初选 有证据表明在-3521和-2291之间存在骨骼特异性增强剂， 和-2291至-1665之间的维生素D反应元件 RNA转录起始点。我们将使用以下工具分析DNA序列 缺失和寡核苷酸定向突变后的分析 突变株SSTRISTY的转录活性和激素调节 转基因骨肉瘤细胞系和成纤维细胞系 老鼠。启动子的亚片段将被测试它们对 弱异源启动子的转录。体外蛋白质/DNA 将通过凝胶迁移率变化和足迹来分析相互作用 激素处理和对照骨细胞的核提取物和 成纤维细胞。体内染色质结构将使用DNA酶进行分析 敏感性研究。了解不同的监管对 骨和成纤维细胞中胶原的产生是理解 遗传性获得性骨病。

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.