The long-term objective of our laboratory is to understand changes in gene expression and regulation that occur during insulin resistance and diabetes which permit excessive SMC activation. We recently reported that overexpression of the cAMP Response Element Binding Protein (CREB) transcription factor decreases SMC migration and proliferation. One of the genes downregulated by CREB expression likely to mediate a decrease SMC proliferation is the platelet-derived growth factor receptor-alpha (PDGFRa) gene. Animal studies demonstrate the PDFGRa expression is increased in the vessel wall in diabetes and insulin resistance (models where CREB content is decreased). The goals of the current proposal are to clarify molecular details of how changes in CREB and C/EBP delta in diabetes and insulin resistance affect gene expression using the PDGFRa as a model target gene. HYPOTHESIS: Loss of vascular CREB function and augmented C/EBPdelta function in diabetes promotes smooth muscle cell proliferation, in part, by increasing expression of PDGFRa. Manipulation of vascular CREB function will impact proliferative capacity and development of atherosclerosis. SPECIFIC AIMS: 1. To determine the impact of targeted vascular overexpression of active and inactive CREB on gene expression, mitogenic capacity and atherosclerosis, a. To examine SMC proliferation and PDFGRa expression in transgenic mice with vascular specific expression of CREB and dominant negative CREB (A-CREB). b To test the hypothesis that loss of CREB contributes to atherosclerotic burden using inducible expression of active and inactive CREB on an ApoE null background. 2. To employ the promoter of the PDGFRa gene as a model to define the regions/cis-acting elements in that are responsive to regulation through CREB and C/EBPdelta. a. Mutagenesis and Footprinting studies of the PDGFRa promoter, b. Confirm CREB and C/EBPdelta DNA binding using EMSA and Affinity purification, c. Identify in vivo occupancy using ChIP analysis of relevant CREB and C/EBP binding elements, d. Determine how PDGFRa expression is affected by changing concentrations and activation states of CREB and C/EBPdelta. 3. To determine whether PPARa mediated suppression of PDGFRa gene expression works through CREB and C/EBPs.

R01DK064741

2004-04-15

2010-03-31