Retinoids, natural and synthetic derivatives of vitamin A, are promising anti-cancer drugs that have been shown to reduce neointimal formation associated with atherosclerosis and balloon angioplasty, apparently by inhibiting smooth muscle cell (SMC) growth and migration. How this inhibition is achieved at the molecular level is poorly understood. Recently, a tumor suppressor gene called SSeCKS (Src Suppressed C Kinase Substrate) was cloned in a subtractive screen for retinoid-inducible genes m SMC, SSeCKS encodes for a scaffolding protein that compartmentalizes cAMP-dependent protein kinase A (PICA), whose signaling has been linked to the inhibition of both SMC growth and neointimal formation. SSeCKS appears to be the first tumor suppressor gene whose mRNA and protein are rapidly and highly induced following retinoid administration. The induction of SSeCKS in retinoid-treated SMC, as well as its virtual extinguished expression following mechanical injury to the vessel wall, suggests an intriguing molecular pathway of SMC growth and neointimal inhibition. Accordingly, the long-term goal of this application is to elucidate the transcriptional regulation of SSeCKS and begin unraveling its function in SMC and the vessel wall. The broad hypothesis to be tested is that one specific isoform of SSeCKS (beta-SSeCKS) is a direct target of retinoids that functions to inhibit SMC growth, migration, and injury-induced ncointimal formation. Three specific aims are proposed to begin testing this general thesis. In Specific Aim 1, studies are designed to clone and characterize beta-SSeCKS regulatory regions. A major goal of Aim 1 is to functionally appraise a consensus retinoic acid response element we discovered in the 5' regulatory region of beta-SScCKS. Specific Aim 2 uses several m vitro model systems of SMC biology and innovative methods of over-expression and gene silencing of beta-SSeCKS to begin examining the functional link between beta-SSeCKS expression, PKA-mediated signaling and SMC growth/migration inhibition. Specific Aim 3 will then examine SSeCKS expression and function in two mouse models of vascular occlusive disease. An important goal in Aim 3 is to knockout beta-SSeCKS function using a SMC-restricted promoter (SM22) in the Cre-lox system of gene inactivation to directly evaluate its importance in the vessel wall's Iresponses to injury and retinoid therapy. Collectively, the proposed plan will provide important new data that could I illuminate a molecular pathway of retinoid-mediated induction of a tumor suppressor and subsequent inhibition of SMC Igrowth and neointimal formation. Such information has enormous potential for understanding and treating human vascular Idisease and other pathological disorders, including cancer where retinoids have been in clinical use for years.
NIH ExPorter
NIH ExPorter