Elucidating the regulation of vascular smooth muscle alpha-actin gene expression in fibroblasts.

摘要

Myofibroblasts are contractile cells essential to the wound healing process that contain stress fibers composed of vascular smooth muscle alpha-actin (VSMA) and are thought to contribute to the mechanical force necessary for wound contraction. In a normal wound, myofibroblasts transiently and selectively undergo apoptosis concurrent with wound closure. However chronic activation and accumulation of VSMA-positive myofibroblasts have been associated with a number of pathological conditions associated with tissue remodeling including hypertrophic scarring and fibrosis. Therefore understanding the molecular mechanisms leading to the expression of VSMA in fibroblasts will be critical to understanding both normal and aberrant fibroproliferative processes.;Many studies have shown that cytokines, such as transforming growth factor beta1 (TGFbeta1), released in response to injury may be particularly important in the initiation of VSMA gene activation and myofibroblast differentiation. Previous studies from our laboratory have indicated that the VSMA gene is activated by TGFbeta1 and is regulated by both positive and negative cis-acting elements in both fibroblasts as well as myogenic cells. The aims of this dissertation were to determine the molecular mechanism controlling VSMA expression by determining the proteins involved in the activation and the repression of the VSMA gene and functional consequences of these transcriptional regulatory proteins working alone or in combination. We will test the hypothesis that VSMA gene regulation is the result of changes in protein-protein interactions and/or changes in interactions at the enhancer region of the VSMA promoter by using biochemical assays that focus on these interactions. Electrophoretic mobility shift assays (EMSAs) and DNA binding assays (DBAs) will determine changes in protein-DNA interactions while immunoprecipitations (IPs) and transient transfection assays will determine whether protein-protein interactions between putative activators and repressors are responsible for VSMA gene output. We will also elucidate the intracellular signaling pathways involved in VSMA gene activation using specific kinase and phosphatase inhibitors.;Our results indicate that VSMA gene activation is a phosphorylation dependent process involving specific kinases whose main mode of transient activation involves a mechanism of de-repression. This concept of de-repression may shine new light in an overly transactivator recruitment-dominated world. Overall, our data suggests that fibroblast to myofibroblast conversion involves promoter specific protein interactions as well as specific protein-protein interactions which may prove to be useful targets for antifibrotic therapeutic strategies.