Regulation of the versican promoter by the β-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)- 3 beta, 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-3 beta 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 andsupershift 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- 3 beta 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.