L-type voltage-gated Ca2+ channels modulate expression of smooth muscle differentiation marker genes via a rho kinase/myocardin/SRF-dependent mechanism.

Wamhoff BR, Bowles DK, McDonald OG, Sinha S, Somlyo AP, Somlyo AV, Owens GK
Circ Res. 2004 95 (4): 406-14

PMID: 15256479 · DOI:10.1161/01.RES.0000138582.36921.9e

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.

MeSH Terms (31)

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester Actins Animals Aorta Calcium Channel Blockers Calcium Channels, L-Type Cell Differentiation Cells, Cultured Gene Expression Profiling Gene Expression Regulation, Developmental Genes, fos Intracellular Signaling Peptides and Proteins Muscle, Smooth, Vascular Myocytes, Smooth Muscle Myosin Heavy Chains Nifedipine Nuclear Proteins Organoids Patch-Clamp Techniques Potassium Chloride Protein-Serine-Threonine Kinases Protein Transport Rats Reverse Transcriptase Polymerase Chain Reaction rho-Associated Kinases rhoA GTP-Binding Protein RNA, Small Interfering Serum Response Element Serum Response Factor Trans-Activators Transfection

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