Ouabain-induced apoptosis and Rho kinase: a novel caspase-2 cleavage site and fragment of Rock-2

Ouabain, a specific Na(+)/K(+)-ATPase inhibitor, has recently been identified as a mammalian hormone. Its elevated concentrations in human plasma have also been associated with pathogenesis of several diseases. Recent studies have shown that ouabain induces aponecrotic cell death in a cell-type- and dose-dependent manner. However, the exact mechanism of ouabain-induced cell death is not fully understood. The Rho GTPase effectors Rho kinases-1 and -2 (Rock-1 and Rock-2) which play central roles in the organization of the actin cytoskeleton, involve in several models of apoptosis. In this study, we investigated the possible involvement of Rocks in ouabain-induced human umbilical vein endothelial cell (HUVEC) apoptosis. Ouabain treatment resulted in loss of cell-cell and cell-substratum adhesion and apoptotic blebbing. Pretreatment of cells with Y-27632, a specific Rock inhibitor, resulted in the inhibition of cell-to-cell detachment and formation of membrane blebs. However, Y-27632 did not prevent ouabain-induced cell-substratum detachment. Instead, treatment with Y-27632 actually accelerated this process. Ouabain treatment induced cleavage of Rock-1 and Rock-2, which was prevented by caspase-3 and caspase-2 specific inhibitors z-DEVD-fmk and z-VDVAD-fmk, respectively. Ouabain-induced Rock-2 cleavage generated a fragment of approximately 140 kDa corresponding to the consensus sequence of caspase-2 on the carboxy terminus of Rock-2. Although it has been previously shown that Rock-2 was cleaved by caspase-2, we have identified here a novel cleavage site and fragment of Rock-2. Our data indicate that ouabain induces both Rock-1 and Rock-2 cleavage via caspase-dependent mechanisms and provide evidence that Rocks are involved in ouabain-induced cell-to-cell detachment and apoptosis.