Transcriptional regulation of SM22alpha by Wnt3a: convergence with TGFbeta(1)/Smad signaling at a novel regulatory element.
The role of canonical Wnt signaling in myofibroblast biology has not been fully investigated. The C3H10T1/2 mesenchymal cell line recapitulates myofibroblast differentiation in vitro and in vivo, including SM22alpha expression. Using this model, we find that Wnt3a upregulates SM22alpha in concert with TGFbeta(1). Wnt1, Wnt5a and BMP2 could not replace Wnt3a and TGFbeta(1) signals. Chromatin immunoprecipitation identified that Wnt3a enhances both genomic SM22alpha histone H3 acetylation and beta-catenin association, hallmarks of transcriptional activation. By analyzing a series of SM22alpha promoter-luciferase (LUC) reporter constructs, we mapped Wnt3a-regulated DNA transcriptional activation to nucleotides -213 to -192 relative to the transcription initiation site. In gel shift assays, DNA-protein complexes assembled on this element were disrupted with antibodies to beta-catenin, Smad2/3, and TCF7, confirming the participation of known Wnt3a and TGFbeta transcriptional mediators. Mutation of a CAGAG motif within this region abrogated recognition by these DNA binding proteins. Wnt3a treatment increased Smad2/3 binding to this element. Mutation of the cognate within the context of the native 0.44 kb SM22alpha promoter resulted in a 70% decrease in transcription, and reduced Wnt3a+TGFbeta(1) induction. A concatamer of SM22alpha [-213 to -192] conveyed Wnt3a+TGFbeta(1) activation to the unresponsive RSV promoter. Dominant negative TCF inhibited SM22alpha [-213 to -192] x 6 RSVLUC activation. Moreover, ICAT (inhibitor of beta-catenin and TCF) decreased while TCF7L2 and beta-catenin enhanced 0.44 kb SM22alpha promoter induction by Wnt3a+TGFbeta(1). RNAi "knockdown" of beta-catenin inhibited Wnt3a induction of SM22alpha. Thus, Wnt/beta-catenin signaling interacts with TGFbeta/Smad pathways to control SM22alpha gene transcription.
Shafer
Shawn L
SL
Washington University School of Medicine, Center for Cardiovascular Research, Internal Medicine/BMD, Campus Box 8301, 660 South Euclid Ave, St. Louis, MO 63110, USA.
Towler
Dwight A
DA