Distinct Roles of Glycogen Synthase Kinase (GSK)-3α and GSK-3β in Mediating Cardiomyocyte Differentiation in Murine Bone Marrow-derived Mesenchymal Stem Cells

The signaling mechanisms facilitating cardiomyocyte (CM) differentiation from bone marrow (BM)-derived mesenchymal stem cells (MSCs) are not well understood. 5-Azacytidine (5-Aza), a DNA demethylating agent, induces expression of cardiac-specific genes, such as Nkx2.5 and alpha-MHC, in mouse BM-derived MSCs. 5-Aza treatment caused significant up-regulation of glycogen synthase kinase (GSK)-3 beta and down-regulation of beta-catenin, whereas it stimulated GSK-3 alpha expression only modestly. The promoter region of GSK-3 beta was heavily methylated in control MSCs, but was demethylated by 5-Aza. Although overexpression of GSK-3 beta potently induced CM differentiation, that of GSK-3 alpha induced markers of neuronal and chondrocyte differentiation. GSK-3 inhibitors, including LiCl, SB 216743, and BIO, abolished 5-Aza-induced up-regulation of CM-specific genes, suggesting that GSK-3 is necessary and sufficient for CM differentiation in MSCs. Although specific knockdown of endogenous GSK-3 alpha abolished 5-Aza-induced expression of cardiac specific genes, surprisingly, that of GSK-3 alpha facilitated CM differentiation in MSCs. Although GSK-3 alpha is found in both the cytosol and nucleus in MSCs, GSK-3 alpha is localized primarily in the nucleus. Nuclear-specific overexpression of GSK-3 beta failed to stimulate CM differentiation. Down-regulation of beta-catenin mediates GSK-3 beta-induced CM differentiation in MSCs, whereas up-regulation of c-Jun plays an important role in mediating CM differentiation induced by GSK-3 alpha knockdown. These results suggest that GSK-3 alpha and GSK-3 beta have distinct roles in regulating CM differentiation in BM-derived MSCs. GSK-3 beta in the cytosol induces CM differentiation of MSCs through down-regulation of beta-catenin. In contrast, GSK-3 alpha in the nucleus inhibits CM differentiation through down-regulation of c-Jun.