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.
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