L-type voltage-gated Ca2+ channels modulate expression of smooth muscle differentiation marker genes via a rho kinase/myocardin/SRF-dependent mechanism.
Vascular smooth muscle cell (SMC) contraction is mediated in part by calcium influx through L-type voltage-gated Ca2+ channels (VGCC) and activation of the RhoA/Rho kinase (ROK) signaling cascade. We tested the hypothesis that Ca2+ influx through VGCCs regulates SMC differentiation marker expression and that these effects are dependent on RhoA/ROK signaling. Depolarization-induced activation of VGCCs resulted in a nifedipine-sensitive increase in endogenous smooth muscle myosin heavy chain (SMMHC) and SM alpha-actin expression and CArG-dependent promoter activity, as well as c-fos promoter activity. The ROK inhibitor, Y-27632, prevented depolarization-induced increase in SMMHC/SM alpha-actin but had no effect on c-fos expression. Conversely, the Ca2+/calmodulin-dependent kinase inhibitor, KN93, prevented depolarization-induced increases in c-fos expression with no effect on SMMHC/SM alpha-actin. Depolarization increased expression of myocardin, a coactivator of SRF that mediates CArG-dependent transcription of SMC marker gene promoters containing paired CArG cis regulatory elements (SMMHC/SM alpha-actin). Both nifedipine and Y-27632 prevented the depolarization-induced increase in myocardin expression. Moreover, short interfering RNA (siRNA) specific for myocardin attenuated depolarization-induced SMMHC/SM alpha-actin transcription. Chromatin immunoprecipitation (ChIP) assays revealed that depolarization increased SRF enrichment of the CArG regions in the SMMHC, SM alpha-actin, and c-fos promoters in intact chromatin. Whereas Y-27632 decreased basal and depolarization-induced SRF enrichment in the SMMHC/SM alpha-actin promoter regions, it had no effect of SRF enrichment of c-fos. Taken together, these results provide evidence for a novel mechanism whereby Ca2+ influx via VGCCs stimulates expression of SMC differentiation marker genes through mechanisms that are dependent on ROK, myocardin, and increased binding of SRF to CArG cis regulatory elements.
Wamhoff
B R
BR
Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, VA 22908-0736, USA.
Bowles
D K
DK
McDonald
O G
OG
Sinha
S
S
Somlyo
A P
AP
Somlyo
A V
AV
Owens
G K
GK
D001498:3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester / Q000494:pharmacology; D000199:Actins / Q000502:physiology; D000818:Animals; D001011:Aorta; D002121:Calcium Channel Blockers / Q000494:pharmacology; D020746:Calcium Channels, L-Type / Q000187:drug effects / Q000502:physiology*; D002454:Cell Differentiation / Q000502:physiology; D002478:Cells, Cultured / Q000166:cytology / Q000378:metabolism; D020869:Gene Expression Profiling; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects; D016762:Genes, fos; D047908:Intracellular Signaling Peptides and Proteins; D009131:Muscle, Smooth, Vascular / Q000166:cytology* / Q000378:metabolism; D032389:Myocytes, Smooth Muscle / Q000166:cytology* / Q000378:metabolism; D018995:Myosin Heavy Chains / Q000502:physiology; D009543:Nifedipine / Q000494:pharmacology; D009687:Nuclear Proteins / Q000502:physiology; D009940:Organoids / Q000166:cytology; D018408:Patch-Clamp Techniques; D011189:Potassium Chloride / Q000494:pharmacology; D017346:Protein Serine-Threonine Kinases / Q000502:physiology*; D021381:Protein Transport; D034741:RNA, Small Interfering / Q000494:pharmacology; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D027602:Serum Response Element / Q000235:genetics; D026362:Serum Response Factor / Q000502:physiology; D015534:Trans-Activators / Q000502:physiology; D014162:Transfection; D054460:rho-Associated Kinases; D020742:rhoA GTP-Binding Protein / Q000502:physiology; D000099251:Myocardin
D001498:3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; D000199:Actins; D002121:Calcium Channel Blockers; D020746:Calcium Channels, L-Type; D047908:Intracellular Signaling Peptides and Proteins; D018995:Myosin Heavy Chains; D009543:Nifedipine; D009687:Nuclear Proteins; D011189:Potassium Chloride; D017346:Protein Serine-Threonine Kinases; D034741:RNA, Small Interfering; D026362:Serum Response Factor; D015534:Trans-Activators; D054460:rho-Associated Kinases; D020742:rhoA GTP-Binding Protein; D000099251:Myocardin