title: Nuclear factor of activated T cells and serum response factor cooperatively regulate the activity of an alpha-actin intronic enhancer.
abstract: Expression of alpha-actin in smooth muscle cells (SMCs) is regulated, in part, by an intronic serum response factor (SRF)-binding CArG element. We have identified a conserved nuclear factor of activated T cells (NFAT) binding site that overlaps this CArG box and tested the hypothesis that this site plays a previously unrecognized role in regulating alpha-actin expression. A reporter construct prepared using a 56-bp region of the mouse alpha-actin first intron containing SRF, NFAT, and AP-1 sites (SNAP) acted as an enhancer element in the context of a minimal thymidine kinase promoter. Basal reporter activity following expression in SMCs was robust and sensitive to the calcineurin-NFAT pathway inhibitors cyclosporin A and FK506. Mutating either the NFAT or SRF binding site essentially abolished reporter activity, suggesting that both NFAT and SRF binding are required. Basal activity in non-smooth muscle HEK293 cells was SRF-dependent but NFAT-independent and approximately 8-fold lower than that in SMCs. Activation of NFAT in HEK293 cells induced an approximately 4-fold increase in activity that was dependent on the integrity of both NFAT and SRF binding sites. NFATc3.SRF complex formation, demonstrated by co-immunoprecipitation, was facilitated by the presence of SNAP oligonucleotide. Inhibition of the calcineurin-NFAT pathway decreased alpha-actin expression in cultured SMCs, suggesting that the molecular interaction of NFAT and SRF at SNAP may be physiologically relevant. These data provide the first evidence that NFAT and SRF may interact to cooperatively regulate SMC-specific gene expression and support a role for NFAT in the phenotypic maintenance of smooth muscle.
authors.affiliation: Department of Pharmacology, College of Medicine, University of Vermont, Burlington, Vermont 05405, USA.
authors.forename: Laura V
authors.initials: LV
authors.lastname: Gonzalez Bosc
authors.forename: Jeff J
authors.initials: JJ
authors.lastname: Layne
authors.forename: Mark T
authors.initials: MT
authors.lastname: Nelson
authors.forename: David C
authors.initials: DC
authors.lastname: Hill-Eubanks
mesh_terms: D000199:Actins / Q000235:genetics*; D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D019703:Calcineurin / Q000378:metabolism; D002460:Cell Line; D016572:Cyclosporine / Q000494:pharmacology; D018076:DNA, Complementary / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism / Q000502:physiology*; D004742:Enhancer Elements, Genetic; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005091:Exons; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation; D007166:Immunosuppressive Agents / Q000494:pharmacology; D007438:Introns; D008156:Luciferases / Q000378:metabolism; D051379:Mice; D008856:Microscopy, Fluorescence; D008957:Models, Genetic; D009154:Mutation; D032389:Myocytes, Smooth Muscle / Q000378:metabolism; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins / Q000378:metabolism / Q000502:physiology*; D010641:Phenotype; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D051381:Rats; D026362:Serum Response Factor / Q000378:metabolism / Q000502:physiology*; D013045:Species Specificity; D016559:Tacrolimus / Q000494:pharmacology; D014157:Transcription Factors / Q000378:metabolism / Q000502:physiology*; D014162:Transfection
chemical_list: D000199:Actins; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D007166:Immunosuppressive Agents; D050778:NFATC Transcription Factors; C494008:NFATC3 protein, human; C494009:Nfatc3 protein, mouse; D009687:Nuclear Proteins; D026362:Serum Response Factor; D014157:Transcription Factors; D049452:Green Fluorescent Proteins; D016572:Cyclosporine; D008156:Luciferases; D019703:Calcineurin; D016559:Tacrolimus