Vascular smooth muscle cells (VSMC) display distinct patterns of proliferation and differentiation during development and in association with vascular diseases. During development, VSMC differentiate from a proliferative phenotype to a non-proliferating contractile phenotype. One of the best markers for this process is the smooth muscle form of alpha-actin (SM-alpha-actin). SM-alpha-actin expression is low in proliferative cells and increases dramatically in contractile cells. In atherosclerotic plaques, VSMC undergo de-differentiation characterized by suppression of SM-alpha actin expression, which is associated with proliferation and migration into the intima. This growth response can be contrasted with hypertrophy of VSMC observed in many models of essential hypertension. Changes in expression of muscle-specific genes such as SM- alpha-actin associated with distinct patterns of growth occur in cultures of VSMC in response to specific agonists. Vasoconstrictors such as arginine vasopressin (AVP) increase expression of SM-alpha actin and induce hypertrophy, while growth factors such as PDGF suppress SM-alpha- actin expression and increase cell matrix proteins. Growth on Matrigel promotes features of the contractile phenotype and induces SM-alpha expression compared to cells grown on plastic. These effects are mediated through transcriptional regulation of the SM-alpha-actin promoter. The goal of this proposal is to use expression of SM-alpha- actin to define the molecular events mediating regulation of muscle- specific gene expression by external factors and extracellular matrix. Based on Preliminary Studies, we hypothesize that induction in response to AVP is mediated by members of the Gq family of G-proteins and involves the c-Jun amino terminal protein kinases (JNK), while suppression of SM-alpha expression is mediated by Ras and involves induction of cytosolic phospholipase A2. These pathways will be explored using a combination of molecular and pharmacologic approaches.
In Specific Aim 1 events mediating JNK activation will be identified, and potential downstream targets examined. Mechanisms mediating induction of SM-alpha actin induction by Matrigel will be identified.
In Specific Aim 2 mechanisms leading to suppression of SM-alpha actin will be investigated. Effectors of Ras will be identified and the role of eicosanoid production examined.
Specific Aim 3 will identify specific transcription factors acting directly at the SM-alpha-actin promoter. Transfection with dominant negative transcription factors encoding DNA binding domains will be employed. By obtained a detailed picture of the pathways regulating SM-alpha-actin expression, we hope to develop a better understanding of the events mediating VSMC differentiation, as well as the events regulating distinct growth patterns associated with disease.
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