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

Circ Res. 2004 Aug 20;95(4):406-14. doi: 10.1161/01.RES.0000138582.36921.9e. Epub 2004 Jul 15.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

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

Substances

  • Actins
  • Calcium Channel Blockers
  • Calcium Channels, L-Type
  • Intracellular Signaling Peptides and Proteins
  • Nuclear Proteins
  • RNA, Small Interfering
  • Serum Response Factor
  • Trans-Activators
  • myocardin
  • Potassium Chloride
  • 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester
  • Protein Serine-Threonine Kinases
  • rho-Associated Kinases
  • Myosin Heavy Chains
  • rhoA GTP-Binding Protein
  • Nifedipine