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

Abstract

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.

Ouabain downregulates Mcl-1 and sensitizes lung cancer cells to TRAIL-induced apoptosis

“…7C). The above results are consistent with the earlier findings in H292 (4,11,23,35), the effects of noncytotoxic concentrations of such compounds, which may be linked to biological functions, are unknown. Initiating from the discovery of endogenous ouabain and related compounds in human plasma and tissue, the roles of ouabain in the regulation of physiological functions as well as in pathological conditions have been intensively investigated.…”

“…Ouabainmediated cancer cell death has been reported in some cancers (4,11,23,35), and we have found that ouabain at concentrations of 5-10 nM causes a significant increase in apoptosis compared with nontreated control cells ( Fig. 2A).…”

“…Initiating from the discovery of endogenous ouabain and related compounds in human plasma and tissue, the roles of ouabain in the regulation of physiological functions as well as in pathological conditions have been intensively investigated. Indeed, evidence has indicated that the plasma ouabain concentration increases in certain pathological conditions including hypertension and cardiac and renal failure (4,16); however, the function of ouabain in immune-related reaction processes is still largely unknown. Our study suggests for the first time that endogenous ouabain might play a role in rendering an immune defense against cancers.…”

“…As ouabain has been found in human plasma (6, 9, 19 -20, 25-28) and identified as a human hormone (21,26), information regarding other activities of this compound is of great interest and will benefit both physiological and pathological knowledge. Although some studies have reported the apoptotic induction property of ouabain in cancer cells (4,11,23,35), the molecular basis of this induction and the precise role of this compound in TRAILmediated cancer cell death are not known. Therefore, in the present study, we demonstrate that administration of ouabain significantly accentuates TRAIL-induced apoptosis in lung cancer cells.…”

Caspase-2 promotes cytoskeleton protein degradation during apoptotic cell death

“…Major degradation systems, including UPS, have been suggested to be activated by inactivation of the Rho GTPase effector, Rock-2, that has central roles in the organization of the actin cytoskeleton, which has previously shown to be cleaved by caspase-2. 32 It is also likely that deubiquitination enzymes, which regulate the stability of cytoskeletal proteins, may act as downstream targets of caspase-2, which when cleaved lead to constitutive ubiquitination of its substrates. Another possibility is that proteins essential for cytoskeleton polymerization and/or stabilization are targeted and may be cleaved by caspase-2 during apoptosis, leading to destabilization and degradation of the cytoskeleton filaments.…”