Abstract 1100: GSK-3β Induces Cardiomyocyte Markers In Bone Marrow-derived Mesenchymal Stem Cells In Vitro
Although MSCs have cardiomyogenic properties, the underlying signaling mechanisms are not well understood. The Wnt pathway regulates cardiomyocyte differentiation in MSCs, but its effects vary substantially depending upon experimental conditions. The goal of this study was to clarify the role of GSK-3β, a major inhibitory component of the Wnt pathway, in regulating cardiomyocyte differentiation in MSCs. Human or mouse bone marrow-derived MSCs were treated with 5 μM of 5- azacytidine (5-Aza), which induced mRNA expression of cardiomyocyte markers, including Nkx2.5, troponin I (TnI), GATA4, atrial natriuretic factor (ANF), cardiac troponin C (cTnC), and Irx4, within 5 days. Protein expression of GSK-3β was increased by 5-Aza in a time-dependent manner, reaching a peak (~4 fold) on Day 3, and was accompanied by decreases in β-catenin (~50 %), suggesting that the activity of GSK-3β was increased, whereas the Wnt pathway was suppressed by 5-Aza. To test the effect of GSK-3β upon cardiomyocyte differentiation, MSCs were transduced with adenovirus (Ad) harboring GSK-3β, which induced 10 fold expression of GSK-3β and 90 % downregulation of β-catenin on Days 3–12. Ad-GSK-3β induced expression of Nkx2.5 and ANF mRNA, whereas Ad-LacZ did not. In order to stimulate GSK-3β by alternative methods, MSCs were isolated from conditional GSK-3β mice. Isolated MSCs were transduced with Ad-tTA or Ad-rtTA, to achieve GSK-3β expression regulated by the tetracycline (tet)-OFF and -ON systems, respectively. GSK-3β expression increased by the tet-OFF (4.7 fold) or tet-ON system (3 fold) induced mRNA expression of Nkx2.5, α-myosin heavy chain, and GATA4, as well as protein expression of sarcomeric α-actinin and TnI in MSCs. LiCl (10 mM), an inhibitor of GSK-3β, significantly attenuated 5-Aza-induced upregulation of ANF (−75%) and GATA4 (−71%), and abolished 5-Aza-induced upregulation of TnI, cTnC, Nkx2.5 and Irx4. In summary, 5-Aza-induced increases in cardiomyocyte markers in MSCs are critically mediated by activation of GSK-3β. Ex vivo induction of GSK-3β may be prime for differentiation of adult MSCs into cardiomyocytes before they are used for myocardial regeneration.