CONTROL OF MONOCYTE CHEMOTACTIC FACTOR EXPRESSION

这个项目的长期目标是了解细胞内 细胞因子和激素调节转录的机制 否则就是静止基因。 用血小板刺激成纤维细胞- 衍生生长因子（PDGF）导致转录的增加， JE基因。 JE编码的蛋白质是单核细胞 趋化蛋白-I（MCP-1）是趋化蛋白超家族的成员， 相关的诱导性细胞因子。 JE/MCP-1已被确定为 平滑肌细胞单核细胞特异性趋化因子 肿瘤细胞系，并可能在发病机制中发挥重要作用， 动脉粥样硬化，伤口愈合和炎症过程。 强效 抗炎糖皮质激素抑制JE的PDGF诱导 具有与其药理学一致的效力等级顺序的基因 作为一种抗炎剂。 DNA的缺失和突变 将制备JE基因侧翼或内部的序列以鉴定关键的 负责PDGF转录诱导的调控序列 以及糖皮质激素的抑制作用。 转录因子结合 将通过足迹法和凝胶电泳来表征这些序列 阻滞测定。 这些因素的大规模纯化将是 他们的基因将被克隆。 实验旨在 区分糖皮质激素介导的四种模型机制 提出了转录抑制。 为了进一步研究 JE转录活性的正调控和负调控， 建议的具体目标如下： 具体目的1：构建和分析JE启动子缺失突变体 为了鉴定转录增强子区域（PDGF应答元件）， 负责PDGF诱导的基因表达。 具体目标2：定位和鉴定负责 糖皮质激素介导的转录抑制。 具体目标3：鉴定和表征转录因子， 识别并结合PDGF应答元件和糖皮质激素介导的 转录抑制区。 具体目标4：将亲和力、数量和可能的相关性 转录因子DNA结合水平的位置效应 PDGF诱导JE表达和糖皮质激素介导的抑制。 具体目标5：纯化和克隆转录因子， 参与PDGF对JE基因的正向调节， 糖皮质激素的负调节。

The long term goal of this project is to understand the intracellular mechanism by which cytokines and hormones regulate the transcription of otherwise quiescent genes. Stimulation of fibroblasts with Platelet- derived growth factor (PDGF) results in the increased transcription of the JE gene. JE encodes a protein in which is the murine homolog of Monocyte Chemotactic Protein-I (MCP-1) and is a member of a growing superfamily of related inducible cytokines. JE/MCP-1 has been identified as the major monocyte specific chemotactic factor from smooth muscle cells and various tumor cell lines, and may play an important role in the pathogenesis of atherosclerosis, in wound healing and in the inflammatory process. Potent anti-inflammatory glucocorticoids inhibits the PDGF induction of the JE gene with the same rank order of potency consistent with its pharmacology as an anti-inflammatory agent. Deletions and mutations of the DNA sequences flanking or within the JE gene will be made to identify key regulatory sequences responsible for the transcriptional induction by PDGF and the repression by glucocorticoids. Transcriptional factors which bind to these sequences will be characterized by footprinting and gel retardation assays. Large scale purification of these factors will be attempted and their genes will be cloned. Experiments designed to distinguish between four model mechanisms of glucocorticoid mediated transcriptional repression are proposed. To further the study of the positive and negative regulation of JE transcriptional activity, the following specific aims are proposed: Specific Aim 1: Construction and analysis of JE promotor deletion mutants to identify transcriptional enhancer regions (PDGF response elements) which are responsible for PDGF induced gene expression. Specific Aim 2: Locate and identify DNA repressor regions responsible for the glucocorticoid mediated transcriptional repression. Specific Aim 3: To identify and characterize transcription factors which recognize and bind to PDGF response elements and to glucocorticoid mediated transcriptional repressor regions. Specific Aim 4: To correlate the affinity, number and possible relative positional effects of transcription factor DNA binding with the level of PDGF induced JE expression and glucocorticoid mediated repression. Specific Aim 5: To purify and clone transcription factors which are involved in the positive regulation of the JE gene by PDGF and in the negative regulation by glucocorticoids.