Regulation of the versican promoter by the beta-catenin-T-cell factor complex in vascular smooth muscle cells.
The proteoglycan versican is pro-atherogenic and central to vascular injury and repair events. We identified the signaling pathways and promoter elements involved in regulation of versican expression in vascular smooth muscle cells. Phosphatidylinositol 3-kinase inhibitor, LY294002, significantly decreased versican-luciferase (Luc) promoter activity and endogenous mRNA levels. We further examined the roles of protein kinase B and glycogen synthase kinase (GSK)-3beta, downstream effectors of phosphatidylinositol 3-kinase, in the regulation of versican transcription. Co-transfection of dominant negative and constitutively active protein kinase B constructs with a versican-Luc construct decreased and increased promoter activity, respectively. Inhibition of GSK-3beta activity by LiCl augmented accumulation of beta-catenin and caused induction of versican-Luc activity as well as versican mRNA levels. Beta-catenin has no DNA binding domain, therefore it cannot directly induce transcription of the versican promoter. Software analysis of the versican promoter revealed two potential binding sites for T-cell factors (TCFs), proteins that confer transcriptional activation of beta-catenin. Electrophoretic mobility shift and supershift assays revealed specific binding of human TCF-4 and beta-catenin to oligonucleotides corresponding to a potential TCF binding site in the versican promoter. In addition to binding assays, we directly assessed the dependence of versican promoter activity on TCF binding sites. Site-directed mutagenesis of the TCF site located -492 bp relative to the transcription start site markedly diminished versican-Luc activity. Co-transfection of TCF-4 with versican-Luc did not increase promoter activity, but addition of beta-catenin and TCF-4 significantly stimulated basal versican promoter activity. Our findings suggest that versican transcription is predominantly mediated by the GSK-3beta pathway via the beta-catenin-TCF transcription factor complex in smooth muscle cells, wherein such regulation contributes to the normal or aberrant formation of provisional matrix in vascular injury and repair events.
Rahmani
Maziar
M
James Hogg iCAPTURE Centre for Cardiovascular and Pulmonary Research, St. Paul's Hospital, 1081 Burrard Street, Vancouver, British Columbia V6Z 1Y6, Canada.
Read
Jason T
JT
Carthy
Jon M
JM
McDonald
Paul C
PC
Wong
Brian W
BW
Esfandiarei
Mitra
M
Si
Xiaoning
X
Luo
Zongshu
Z
Luo
Honglin
H
Rennie
Paul S
PS
McManus
Bruce M
BM
D000818:Animals; D001011:Aorta / Q000166:cytology; D001665:Binding Sites; D045744:Cell Line, Tumor; D011508:Chondroitin Sulfate Proteoglycans / Q000096:biosynthesis* / Q000235:genetics*; D002867:Chromones / Q000494:pharmacology; D003598:Cytoskeletal Proteins / Q000378:metabolism*; D004247:DNA / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D017353:Gene Deletion; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism; D000071679:Glycogen Synthase Kinase 3 beta; D006801:Humans; D015151:Immunoblotting; D037181:Lectins, C-Type; D018021:Lithium Chloride / Q000494:pharmacology; D008156:Luciferases / Q000378:metabolism; D051821:Lymphoid Enhancer-Binding Factor 1; D008957:Models, Genetic; D009025:Morpholines / Q000494:pharmacology; D009131:Muscle, Smooth, Vascular / Q000166:cytology*; D032389:Myocytes, Smooth Muscle / Q000166:cytology*; D009841:Oligonucleotides / Q000737:chemistry; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D012313:RNA / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D015534:Trans-Activators / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic; D014162:Transfection; D053675:Versicans; D014945:Wound Healing; D051176:beta Catenin
C495270:CTNNB1 protein, human; D011508:Chondroitin Sulfate Proteoglycans; D002867:Chromones; C495268:Ctnnb1 protein, rat; D003598:Cytoskeletal Proteins; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D037181:Lectins, C-Type; D051821:Lymphoid Enhancer-Binding Factor 1; D009025:Morpholines; D009841:Oligonucleotides; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; D015534:Trans-Activators; D014157:Transcription Factors; C508322:VCAN protein, human; C508324:Vcan protein, rat; D051176:beta Catenin; D053675:Versicans; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D012313:RNA; D004247:DNA; D008156:Luciferases; C000605506:GSK3B protein, human; D000071679:Glycogen Synthase Kinase 3 beta; C000605508:Gsk3b protein, rat; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D038362:Glycogen Synthase Kinase 3; D018021:Lithium Chloride