GLUCOCORTICOID REGULATION OF PROCOLLAGEN GENE EXPRESSION

皮肤萎缩是局部用药的主要明显副作用。 皮质类固醇治疗和减少胶原沉积的结果。 皮肤中胶原沉积的减少是由于皮肤中 已在体内和体内观察到的前胶原合成 成纤维细胞培养。这个实验室证明了 糖皮质激素对前胶原合成的抑制作用 受体介导的糖皮质激素诱导的前胶原下调 基因表达。这项提案的一个主要目标是确定和 DNA糖皮质激素调节元件的功能特征 小鼠前α2(1)基因的5‘侧翼区和第一内含子 并将这些序列与糖皮质激素反应元件进行比较。5英尺 侧翼或内含子DNA缺失将与猫连接 (氯霉素乙酰转移酶)基因。这些DNA结构将是 转染小鼠胚胎成纤维细胞以确定其生物学特性 糖皮质激素介导降压所需序列的活性 基因表达的调控。我们将合成糖皮质激素受体 并测定其与5‘-DNA片段的结合。 侧翼区和第一内含子。糖皮质激素对血管紧张素转换酶的影响 糖皮质激素受体mRNA的稳定水平也将是 下定决心。糖皮质激素受体可能不直接与DNA结合，并可能 涉及协同调节蛋白(S)和可能的已知转录 通过一种涉及调控基因的间接机制， 其产物可能调节胶原基因的表达。因此，我们 将确定糖皮质激素对DNA结合蛋白的影响 通过凝胶滞留和阶梯试验。此外，序列(S) DNA结合元件的数量将通过足迹来确定。它有 研究表明，维生素A可以阻断糖皮质激素的某些作用。 胶原蛋白代谢。这项提案的一个目标是确定 全反式维甲酸逆转分子抑制作用的研究 糖皮质激素对前胶原基因表达的影响

Dermal atrophy is a major conspicuous side effect of topical corticosteroid therapy and results from decreased collagen deposition. The decreased deposition of collagen in skin results from a decrease of procollagen synthesis which has been observed both in vivo and in fibroblast cell cultures. This laboratory has demonstrated that glucocorticoid-induced inhibition of procollagen synthesis occurs by a receptor-mediated glucocorticoid-induced down regulation of procollagen gene expression. A major objective of this proposal is to identify and functionally characterize DNA glucocorticoid regulatory elements in the 5'-flanking region and the first intron of the mouse pro-alpha2(1) gene and compare these sequences to glucocorticoid response elements. The 5' flanking or intronic DNA deletions will be ligated to the CAT (chloramphenicol acetyltransferase) gene. These DNA constructs will be transfected into embryonic mouse fibroblasts to determine the biological activity of sequences necessary for glucocorticoid-mediated down regulation of gene expression. We will synthesize glucocorticoid receptor in vitro from cDNA and determine its binding to DNA fragments of the 5'- flanking region and the first intron. The effect of glucocorticoid on steady state levels of the glucocorticoid receptor mRNA will also be determined. Glucocorticoid receptor may not bind directly to DNA and may involve cooperating regulatory protein(s) and possible known transcription factors through an indirect mechanism involving a regulatory gene, the product of which may regulate collagen gene expression. Accordingly, we will determine the effects of glucocorticoids on DNA binding proteins through gel retardation and stairway assays. In addition, the sequence(s) of the DNA binding elements will be determined by footprinting. It has been shown that Vitamin A blocks some of the glucocorticoid effects on collagen metabolism. An objective of this proposal will be to determine the ability of all-trans retinoic acid to reverse the molecular inhibitory effects of glucocorticoids on procollagen gene expression.