|About the Book|
Multipotent mesenchymal progenitor cells residing in the fibrous outer matrix of the aorta serve as a reservoir for new vascular smooth muscle cells (VSMC), but can also contribute to disease. Here I present previously unreported evidence that theMoreMultipotent mesenchymal progenitor cells residing in the fibrous outer matrix of the aorta serve as a reservoir for new vascular smooth muscle cells (VSMC), but can also contribute to disease. Here I present previously unreported evidence that the canonical Wnt ligand, Wnt3a, can induce a vascular smooth muscle cell phenotype in multipotent mesenchymal progenitors cells, and may play an important role in vascular regeneration and pathology.-I initially noticed that mesenchymal progenitor cells significantly increase their expression of vascular smooth muscle cell (VSMC)-specific genes following in vitro treatment with recombinant Wnt3a. When I compared the expression levels with those of a known inducer of VSMC gene expression, TGFbeta 1, they were equivalent. Importantly, I discovered that when administered in combination, Wnt3a and TGFbeta1 together resulted in an additive increase in VSMC-specific transcript and protein accumulation for two of our marker genes, SM22alpha and Acta2.-Focusing on the most responsive of the genes, SM22alpha, I dissected its promoter in search of a Wnt3a/TGFbeta1 regulatory sequence. By constructing a deletion library, I was able to map the element to -213 to -192 of the proximal SM22alpha promoter.-In immuno-competition and supershift experiments, I show that the Wnt3a transcription factors, beta-catenin and TCF7, and the TGFbeta1 transcription factor, Smad2, form a complex on this promoter sequence. Cold competition and promoter mutation experiments identified a CAGAG sequence element at -203 to -199 that is required for mediating protein/DNA complex formation and robust reporter expression, respectively. A six-copy concatamer of the region was constructed and used as a reporter in co-transfection experiments with components of the Wnt and TGFbeta signaling pathways in mesenchymal progenitors. The results demonstrated that the two signaling pathways overlap and influence each other, an interpretation confirmed by anti-beta-catenin siRNA experiments.-Finally, treatment of mesenchymal progenitor cells with Wnt3a results in substantially increased acetylation of the SM22alpha promoter in vivo, and an increased association with RNA polymerase II, as shown by chromatin immunoprecipitation. The upcoming characterization of a transgenic reporter mouse carrying the SM22alpha concatamer will yield important information about when and where this motif is accessed in the vasculature.