Smooth muscle cell-specific transcription is regulated by nuclear localization of the myocardin-related transcription factors

Department of Pathology and Laboratory Medicine and the Carolina Cardiovascular Biology Center, University of North Carolina, Chapel Hill, North Carolina Submitted 10 August 2006 ; accepted in final form 14 September 2006 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 SM22 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 ( B1/B2) that traps endogenous MRTFs in the cytoplasm inhibited the SM -actin, SM22 , and SM myosin heavy chain promoters in SMC and attenuated the effects of sphingosine 1-phosphate and transforming growth factor (TGF)- 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 -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. serum response factor; RhoA; cytoskeleton; differentiation Address for reprint requests and other correspondence: C. Mack, Campus Box 7525, Univ. of North Carolina, Chapel Hill, NC 27599 (e-mail: cmack{at}med.unc.edu )