2 MCAT ELEMENTS OF THE SM ALPHA-ACTIN PROMOTER FUNCTION DIFFERENTIALLY IN SM VS. NON-SM CELLS

Transcriptional activity of the smooth muscle (SM) alpha-actin gene is differentially regulated in SM vs. non-SM cells. Contained within the rat SM cu-actin promoter are two MCAT motifs, binding sites for trans cription enhancer factor 1 (TEF-1) transcriptional factors implicated in the regulation of many muscle-specific genes. Transfections of SM a lpha-actin promoter-CAT constructs containing wild-type or mutagenized MCAT elements were performed to evaluate their functional significanc e. Mutation of the MCAT elements resulted in increased transcriptional activity in SM cells, whereas these mutations either had no effect or decreased activity in L6 myotubes or endothelial cells. High-resoluti on gel shift assays resolved several complexes of different mobilities that were formed between MCAT oligonucleotides and nuclear extracts f rom the different cell types, although no single band was unique to SM . Western blot analysis of nuclear extracts with polyclonal antibodies to conserved domains of the TEF-1 gene family revealed multiple react ive bands, some that were similar and others that differed between SM and non-SM. Supershift assays with a polyclonal antibody to the TEF-re lated protein family demonstrated that TEF-1 or TEF-1-related proteins were contained in the shifted complexes. Results suggest that the MCA T elements may contribute to cell type-specific regulation of the SM a lpha-actin gene. However, it remains to be determined whether the diff erential transcriptional activity of MCAT elements in SM vs. non-SM is due to differences in expression of TEF-1 or TEF-1-related proteins o r to unique (cell type specific) combinatorial interactions of the MCA T elements with other cis-elements and trans-factors.