The MADS box transcription factors, MEF2 and SRF, activate contractile protein gene expression in differentiated myocytes and also activate the expression of genes required for cell cycle progression. The regulation of cardiac and skeletal muscle-specific genes by the MADS box factors is well documented, but little is known about the role of these proteins in vascular smooth muscle cells (VSMCs). These cells do not terminally differentiate and reversibly coordinate the expression of myocyte specific and cell cycle genes. Prior studies in the applicant's lab have found that MEF2A expression is induced by mitogens in VSMCs, and this regulation occurs at the level of translation. Translational regulation of MEF2 during development is also indicated by discordant spatial expression patterns of MEF2 mRNA, which is ubiquitous, and MEF2 protein, which tends to be specific to muscle and brain. To elucidate the molecular basis of the mitogen-induced up-regulation of MEF2A expression, a modular system will be devised to map the cis-regulatory elements within the MEF2A transcript that confer translational regulation. These constructs will also be tested in a skeletal muscle cell line to determine whether the same translational regulatory mechanisms function to up-regulate MEF2A expression upon terminal differentiation. Once identified, the RNA-binding proteins that interact with these elements will be characterized. The role of MADS box factors in the control of cell proliferation will be assessed in VSMCs and in skeletal muscle cells. The levels of different MEF2 isoforms and SRF will be manipulated by transfection and by the microinjection of recombinant proteins and antibodies. The consequences of these manipulations on cell cycle will be assessed. These assays will test for functional differences between the MADS box family members and will test for cell type-specific differences on proliferation. The cyclin- dependent kinase (cdk) inhibitors represent logical targets for MADS box and other growth-regulatory transcription factors because their expression can be regulated at the level of transcription. Prior studies in the applicant's lab have found that the cdk inhibitor p21 is markedly up-regulated upon myocyte terminal differentiation. Experiments will explore the mechanism of the p21 cdk inhibitor up-regulation during myogenesis, and over-expression and under-expression experiments will assess the functional consequences of this regulation on myocyte differentiation.
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