title: Potential Role of Glycogen Synthase Kinase-3β in Regulation of Myocardin Activity in Human Vascular Smooth Muscle Cells.
abstract: Glycogen synthase kinase (GSK)-3β, a serine/threonine kinase with an inhibitory role in glycogen synthesis in hepatocytes and skeletal muscle, is also expressed in cardiac and smooth muscles. Inhibition of GSK-3β results in cardiac hypertrophy through reducing phosphorylation and increasing transcriptional activity of myocardin, a transcriptional co-activator for serum response factor. Myocardin plays critical roles in differentiation of smooth muscle cells (SMCs). This study, therefore, aimed to examine whether and how inhibition of GSK-3β regulates myocardin activity in human vascular SMCs. Treatment of SMCs with the GSK-3β inhibitors AR-A014418 and TWS 119 significantly reduced endogenous myocardin activity, as indicated by lower expression of myocardin target genes (and gene products), CNN1 (calponin), TAGLN1 (SM22), and ACTA2 (SM α-actin). In human SMCs overexpressing myocardin through the T-REx system, treatment with either GSK-3β inhibitor also inhibited the expression of CNN1, TAGLN1, and ACTA2. These effects of GSK-3β inhibitors were mimicked by transfection with GSK-3β siRNA. Notably, both AR-A014418 and TWS 119 decreased the serine/threonine phosphorylation of myocardin. The chromatin immunoprecipitation assay showed that AR-A014418 treatment reduced myocardin occupancy of the promoter of the myocardin target gene ACTA2. Overexpression of a dominant-negative GSK-3β mutant in myocardin-overexpressing SMCs reduced the expression of calponin, SM22, and SM α-actin. As expected, overexpression of constitutively active or wild-type GSK-3β in SMCs without myocardin overexpression increased expression of these proteins. In summary, our results indicate that inhibition of GSK-3β reduces myocardin transcriptional activity, suggesting a role for GSK-3β in myocardin transcriptional activity and smooth muscle differentiation.
authors.affiliation: Department of Biochemistry and Molecular Biology, Smooth Muscle Research Group, Libin Cardiovascular Institute of Alberta, Cumming School of Medicine, University of Calgary, Calgary, Alberta, Canada.
authors.forename: Yi-Xia
authors.initials: YX
authors.lastname: Zhou
authors.affiliation: Department of Biochemistry and Molecular Biology, Smooth Muscle Research Group, Libin Cardiovascular Institute of Alberta, Cumming School of Medicine, University of Calgary, Calgary, Alberta, Canada.
authors.forename: Zhan
authors.initials: Z
authors.lastname: Shi
authors.affiliation: Department of Biochemistry and Molecular Biology, Smooth Muscle Research Group, Libin Cardiovascular Institute of Alberta, Cumming School of Medicine, University of Calgary, Calgary, Alberta, Canada.
authors.forename: Pavneet
authors.initials: P
authors.lastname: Singh
authors.affiliation: Department of Biochemistry and Molecular Biology, Smooth Muscle Research Group, Libin Cardiovascular Institute of Alberta, Cumming School of Medicine, University of Calgary, Calgary, Alberta, Canada.
authors.forename: Hao
authors.initials: H
authors.lastname: Yin
authors.affiliation: Guiyang Medical University, Guizhou, China.
authors.forename: Yan-Ni
authors.initials: YN
authors.lastname: Yu
authors.affiliation: Guiyang Medical University, Guizhou, China.
authors.forename: Long
authors.initials: L
authors.lastname: Li
authors.affiliation: Department of Biochemistry and Molecular Biology, Smooth Muscle Research Group, Libin Cardiovascular Institute of Alberta, Cumming School of Medicine, University of Calgary, Calgary, Alberta, Canada.
authors.forename: Michael P
authors.initials: MP
authors.lastname: Walsh
authors.affiliation: Department of Physiology and Pharmacology, Smooth Muscle Research Group, Libin Cardiovascular Institute of Alberta, Cumming School of Medicine, University of Calgary, Calgary, Alberta, Canada.
authors.forename: Yu
authors.initials: Y
authors.lastname: Gui
authors.affiliation: Department of Biochemistry and Molecular Biology, Smooth Muscle Research Group, Libin Cardiovascular Institute of Alberta, Cumming School of Medicine, University of Calgary, Calgary, Alberta, Canada.
authors.forename: Xi-Long
authors.initials: XL
authors.lastname: Zheng
mesh_terms: D000199:Actins / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects / Q000235:genetics / Q000502:physiology; D002460:Cell Line; D005786:Gene Expression Regulation; D055786:Gene Knockout Techniques; D038362:Glycogen Synthase Kinase 3 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D000071679:Glycogen Synthase Kinase 3 beta; D006801:Humans; D008565:Membrane Proteins; D008840:Microfilament Proteins / Q000378:metabolism; D009124:Muscle Proteins / Q000378:metabolism; D032389:Myocytes, Smooth Muscle / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D009687:Nuclear Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D010750:Phosphoproteins; D011401:Promoter Regions, Genetic; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011743:Pyrimidines / Q000494:pharmacology; D011758:Pyrroles / Q000494:pharmacology; D034741:RNA, Small Interfering / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012694:Serine / Q000737:chemistry; D013844:Thiazoles / Q000494:pharmacology; D013912:Threonine / Q000737:chemistry; D015534:Trans-Activators / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D014508:Urea / Q000031:analogs & derivatives / Q000494:pharmacology; D000096985:Calponins; D000099251:Myocardin
chemical_list: D000199:Actins; D002135:Calcium-Binding Proteins; D038362:Glycogen Synthase Kinase 3; D000071679:Glycogen Synthase Kinase 3 beta; D008565:Membrane Proteins; D008840:Microfilament Proteins; D009124:Muscle Proteins; D009687:Nuclear Proteins; D010750:Phosphoproteins; D047428:Protein Kinase Inhibitors; D011743:Pyrimidines; D011758:Pyrroles; D034741:RNA, Small Interfering; D011994:Recombinant Proteins; D012694:Serine; D013844:Thiazoles; D013912:Threonine; D015534:Trans-Activators; D014508:Urea; D000096985:Calponins; D000099251:Myocardin; C541116:ACTA2 protein, human; C547688:TWS 119; C083557:p112 protein, rat; C051615:transgelin; C479831:N-(4-methoxybenzyl)-N'-(5-nitro-1,3-thiazol-2-yl)urea; C000605506:GSK3B protein, human; C000605508:Gsk3b protein, rat