ID3 REGULATION OF SMOOTH MUSCLE CELL PROLIFERATION

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DESCRIPTION (Adapted from Investigator's Abstract): Smooth muscle cell (SMC) proliferation plays a key role in a number of vascular proliferative disorders, including atherosclerosis and restenosis after balloon angioplasty. The long-term goal of this project is to identify the molecular mechanisms that regulate the growth state of SMC in response to vascular injury. The PI has demonstrated that Id3 (inhibitor of DNA binding), a helix-loop-helix (HLH) factor expressed in the vessel wall, enhances growth of SMC in culture potentially through inhibition of expression of the cell cycle regulator p21. He has recently cloned a novel Id3 isoform (Id3a) from a rat aortic SMC library and demonstrated unique vascular expression of this isoform relative to ID3. The Id3a mRNA retains a "coding intron," generating an ID3a protein with a unique carboxyl terminus. He has further shown that the C-terminus of ID3 is essential for its ability to block p21 gene expression. Thus,he hypothesizes that vascular injury regulates Id2 isoform expression resulting in functionally unique proteins that work in concert to regulate the SMC proliferative response. Aim 1 is to identify specific mechanisms by which Id3 enhances SMC proliferation and to determine the effect of Id3a on SMC growth. Studies will include: a) co-transfection studies to determine if Id3 and Id3a have effects on p21 transcription in SMC. Determine if Pan-1 can transactivate p21 in a SMC context and if Id3 and Id3a have effects on Pan-1 mediated p21 transactivation; b) Western analysis to determine if Id3a protein expression in SMC is regulated by serum or other mitogens; c) Id3a over-expression experiments to determine effects of Id3a on SMC proliferation and specifically on G1-S progression; d) cotransfection studies in p21 null cells to determine if there are Id3 and Id3a effects on proliferation that are p21 independent; e) cotransfection experiments to determine if Id3a is a dominant negative regulator of Id3; and f) domain swapping and mutagenesis studies to determine the amino acids in the C-terminus of Id3 that are essential for its dominant negative function in blocking gene transactivation in SMC and enhancing SMC proliferation. Aim 2 is to identify the mechanisms responsible for the differences in the basic functional properties between Id3 and Id3a. Studies will include: a) in vitro binding assays to determine if differences in dimerization interactions between Id3 and Id3a are responsible for their differential effects on transactivation; b) cotransfection studies using the mammalian two-hybrid system to determine if additional cellular factors are involved in regulating dimerization partner selectivity; c) electrophoretic mobility shift assays to determine if the above Id3/E-protein dimerizations are sufficient for inhibiting E-protein binding to an Ebox containing oligonucleotide; and d) domain swapping and mutagenesis studies to determine the amino acids in the C-terminus of Id3 that are essential for its altered ability to block E-protein binding to E-box containing oligonucleotides.