Alterations in the differentiated state of the smooth muscle cell (SMC) are believed to play a key role in the development and/or progression of atherosclerotic disease. The long-term goal of this proposal is to identify molecular mechanisms that control differentiation/maturation of SMC through elucidation of mechanisms that control transcription of SM alpha-actin, a contractile protein that is selective for SMC and which is required for its major differentiated function, contraction.
Aim 1 of this proposal is to identify cellular and molecular mechanisms that regulate cell-type specific expression of the SM alpha-actin gene both in cultured SMC and in vivo in transgenic animals. Studies will include: a) further characterization of DNA sequences required for SMC specific transcriptional regulation via two highly conserved CC(AT)6GG (CarG) elements that the applicant has previously demonstrated are required for high transcriptional activity in SMC; b) identification of trans factors that interact with and regulate the activity of these CarG elements; c) determination of the minimal promoter sequences required for expression of the SM alpha-actin gene in transgenic animals; d) identification of cis elements responsible for the positive transcriptional activity of the first intron observed in our preliminary studies; e) identification of cis elements upstream of the core 125 bp SM alpha-actin promoter that are required for cell-type-specific expression of this gene; and f) determination if the positive activity of the E-boxes of the SM alpha-actin promoter are mediated by a novel SMC specific HLH transcription factor.
Aim 2 will be to determine the in vivo function of selected cis elements and trans factors identified in Aim 1. Studies will include testing the activity of selected cis elements in transgenics by site-directed mutagenesis, and determining the temporal pattern of expression of SM alpha-actin transcription factors relative to alterations in expression of SM alpha-actin and other SMC differentiation marker proteins during vasculogenesis, and following vascular injury.
Aim 3 will be to determine whether there is evidence for coordinate regulation of transcription of SM alpha-actin and genes encoding other SMC differentiation marker proteins, and to determine the underlying mechanisms. Studies represent an extension of ongoing work, and will contribute to understanding of mechanisms that contribute to alterations in SMC differentiation control processes in vascular disease. In addition, studies are likely to identify DNA regulatory sequences that confer SMC specific expression which could be used for construction of vectors for SMC specific gene knockouts and/or targeting gene therapies to the vasculature.
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