Inhibition of Ref-1 Stimulates the Production of Reactive Oxygen Species and Induces Differentiation in Adult Cardiac Stem Cells

Redox effector protein-1 (Ref-1) plays an essential role in DNA repair and redox regulation of several transcription factors. In the present study, we examined the role of Ref-1 in maintaining the redox status and survivability of adult cardiac stem cells challenged with a subtoxic level of H(2)O(2) under inhibition of Ref-1 by RNA interference. Treatment of cardiac stem cells with a low concentration of H(2)O(2) induced Ref-1-mediated survival signaling through phosphorylation of Akt. However, Ref-1 inhibition followed by H(2)O(2) treatment extensively induced the level of intracellular reactive oxygen species (ROS) through activation of the components of NADPH oxidase, like p22(phox), p47(phox), and Nox4. Cardiac differentiation markers (Nkx2.5, MEF2C, and GATA4), and cell death by apoptosis were significantly elevated in Ref-1 siRNA followed by H(2)O(2)-treated stem cells. Further, inhibition of Ref-1 increased the level of p53 but decreased the phosphorylation of Akt, a molecule involved in survival signaling. Treatment with ROS scavenger N-acetyl-L-cysteine attenuated Ref-1 siRNA-mediated activation of NADPH oxidase and cardiac differentiation. Taken together, these results indicate that Ref1 plays an important role in maintaining the redox status of cardiac stem cells and protects them from oxidative injury-mediated cell death and differentiation. Antioxid. Redox Signal. 11, 589-599.