Transcriptional regulation of SM22α by Wnt3a: Convergence with TGFβ₁/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 SM22α expression. Using this model, we find that Wnt3a upregulates SM22α in concert with TGFβ₁. Wnt1, Wnt5a and BMP2 could not replace Wnt3a and TGFβ₁ signals. Chromatin immunoprecipitation identified that Wnt3a enhances both genomic SM22α histone H3 acetylation and β-catenin association, hallmarks of transcriptional activation. By analyzing a series of SM22α 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 β-catenin, Smad2/3, and TCF7, confirming the participation of known Wnt3a and TGFβ 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 SM22α promoter resulted in a 70% decrease in transcription, and reduced Wnt3a + TGFβ₁ induction. A concatamer of SM22α [- 213 to - 192] conveyed Wnt3a + TGFβ₁ activation to the unresponsive RSV promoter. Dominant negative TCF inhibited SM22α [- 213 to - 192]x6 RSVLUC activation. Moreover, ICAT (inhibitor of β-catenin and TCF) decreased while TCF7L2 and β-catenin enhanced 0.44 kb SM22α promoter induction by Wnt3a + TGFβ₁. RNAi "knockdown" of β-catenin inhibited Wnt3a induction of SM22α. Thus, Wnt/β-catenin signaling interacts with TGFβ/Smad pathways to control SM22α gene transcription.