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

Jaeyeaon Cho,Pranela Rameshwar,Junichi Sadoshima

doi:10.1074/jbc.m109.019109

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

Highlights

Rebuilding the injured heart from its component parts
Expression of any of these marker genes (Fig. 5F). These results suggest that Glycogen synthase kinase (GSK)-3␤ induces cardiomyocyte differentiation of mesenchymal stem cells (MSCs) more potently than the Wnt agonists
BIO and SB216743 inhibited 5-Aza-induced up-regulation of Flk-1, Nkx2.5, cardiac troponin C (cTnC), and ␣-MHC mRNA (Fig. 6E) and sarcomeric ␣-actinin protein expression. These results suggest that GSK-3 plays an essential role in mediating 5-Aza-induced cardiomyocyte differentiation in

Summary

Transgenic Mice and MSC

The isoform-specific functions of GSK-3␣ and GSK-3␤ during cardiomyocyte differentiation are not well understood in mesenchymal stem cells (MSCs). Through initial screening of signaling molecules modulated by 5-Aza during cardiomyocyte differentiation in MSCs, we found that GSK-3 plays an important role in regulating this process. GSK-3␤ Conditional Overexpression Using Tet-On or Tet-Off roles in mediating cardiomyocyte differentiation and whether System—Adenovirus vector harboring tTA or rtTA was introregulation of ␤-catenin is involved in modulation of cardiom- duced into Tet-MSCs isolated from Tg-Tet-GSK-3␤ mice. MSCs were transfected with the TOP-FOP flash reporter genes and treated with or without 5-Aza. The luciferase activity was evaluated on day 3 and the experiments were conducted 3 times. Statistical analyses were performed using analysis of variance and NewmanKeuls multiple comparison test with a p Ͻ 0.05 considered significant

MSCs at the third passage expressed

Tubular structures were not observed in MSCs treated with

DISCUSSION