title: Smooth muscle cell-specific transcription is regulated by nuclear localization of the myocardin-related transcription factors.
abstract: On the basis of our previous studies on RhoA signaling in smooth muscle cells (SMC), we hypothesized that RhoA-mediated nuclear translocalization of the myocardin-related transcription factors (MRTFs) was important for regulating SMC phenotype. MRTF-A protein and MRTF-B message were detected in aortic SMC and in many adult mouse organs that contain a large SMC component. Both MRTFs upregulated SMC-specific promoter activity as well as endogenous SM22alpha expression in multipotential 10T1/2 cells, although to a lesser extent than myocardin. We used enhanced green fluorescent protein (EGFP) fusion proteins to demonstrate that the myocardin factors have dramatically different localization patterns and that the stimulation of SMC-specific transcription by certain RhoA-dependent agonists was likely mediated by increased nuclear translocation of the MRTFs. Importantly, a dominant-negative form of MRTF-A (DeltaB1/B2) that traps endogenous MRTFs in the cytoplasm inhibited the SM alpha-actin, SM22alpha, and SM myosin heavy chain promoters in SMC and attenuated the effects of sphingosine 1-phosphate and transforming growth factor (TGF)-beta on SMC-specific transcription. Our data confirmed the importance of the NH(2)-terminal RPEL domains for regulating MRTF localization, but our analysis of MRTF-A/myocardin chimeras and myocardin RPEL2 mutations indicated that the myocardin B1/B2 region can override this signal. Gel shift assays demonstrated that myocardin factor activity correlated well with ternary complex formation at the SM alpha-actin CArGs and that MRTF-serum response factor interactions were partially dependent on CArG sequence. Taken together, our results indicate that the MRTFs regulate SMC-specific gene expression in at least some SMC subtypes and that regulation of MRTF nuclear localization may be important for the effects of selected agonists on SMC phenotype.
authors.affiliation: Department of Pathology and Laboratory Medicine and the Carolina Cardiovascular Biology Center, University of North Carolina, Chapel Hill, North Carolina 27599, USA.
authors.forename: Jeremiah S
authors.initials: JS
authors.lastname: Hinson
authors.forename: Matthew D
authors.initials: MD
authors.lastname: Medlin
authors.forename: Kashelle
authors.initials: K
authors.lastname: Lockman
authors.forename: Joan M
authors.initials: JM
authors.lastname: Taylor
authors.forename: Christopher P
authors.initials: CP
authors.lastname: Mack
mesh_terms: D021581:Active Transport, Cell Nucleus / Q000187:drug effects; D000818:Animals; D001013:Aorta, Thoracic / Q000378:metabolism; D002454:Cell Differentiation; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism*; D002478:Cells, Cultured; D008246:Lysophospholipids / Q000494:pharmacology; D008840:Microfilament Proteins / Q000235:genetics / Q000378:metabolism; D009124:Muscle Proteins / Q000235:genetics / Q000378:metabolism; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000378:metabolism*; D009154:Mutation; D032389:Myocytes, Smooth Muscle / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D010641:Phenotype; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology; D011401:Promoter Regions, Genetic / Q000187:drug effects; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D026362:Serum Response Factor / Q000378:metabolism; D013110:Sphingosine / Q000031:analogs & derivatives / Q000494:pharmacology; D013997:Time Factors; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic / Q000187:drug effects; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000494:pharmacology; D020742:rhoA GTP-Binding Protein / Q000378:metabolism; D000099251:Myocardin
chemical_list: D008246:Lysophospholipids; D008840:Microfilament Proteins; D009124:Muscle Proteins; D009687:Nuclear Proteins; D010982:Platelet-Derived Growth Factor; D012333:RNA, Messenger; D026362:Serum Response Factor; D013110:Sphingosine; D015534:Trans-Activators; D016212:Transforming Growth Factor beta; D020742:rhoA GTP-Binding Protein; D000099251:Myocardin; C051615:transgelin; C060506:sphingosine 1-phosphate