Distinct roles of glycogen synthase kinase (GSK)-3α and GSK-3β in mediating cardiomyocyte differentiation in murine bone marrow-derived mesenchymal stem cells

Abstract

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 α-MHC, in mouse BM-derived MSCs. 5-Aza treatment caused significant up-regulation of glycogen synthase kinase (GSK)-3β and down-regulation of β-catenin, whereas it stimulated GSK-3α expression only modestly. The promoter region of GSK-3β was heavily methylated in control MSCs, but was demethylated by 5-Aza. Although overexpression of GSK-3β potently induced CM differentiation, that of GSK-3α 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-3β abolished 5-Aza-induced expression of cardiac specific genes, surprisingly, that of GSK-3α facilitated CM differentiation in MSCs. Although GSK-3β is found in both the cytosol and nucleus in MSCs, GSK-3α is localized primarily in the nucleus. Nuclear-specific overexpression of GSK-3β failed to stimulate CM differentiation. Down-regulation of β-catenin mediates GSK-3β-induced CM differentiation in MSCs, whereas up-regulation of c-Jun plays an important role in mediating CM differentiation induced by GSK-3α knockdown. These results suggest that GSK-3α and GSK-3β have distinct roles in regulating CM differentiation in BM-derived MSCs. GSK-3β in the cytosol induces CM differentiation of MSCs through down-regulation of β-catenin. In contrast, GSK-3α in the nucleus inhibits CM differentiation through down-regulation of c-Jun. © 2009 by The American Society for Biochemistry and Molecular Biology, Inc.