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.
Ark
Mustafa
M
Faculty of Pharmacy, Department of Pharmacology, Gazi University, Etiler, 06330 Ankara, Turkey. mark@gazi.edu.tr
Ozdemir
Aysun
A
Polat
Belgin
B
D000199:Actins / Q000378:metabolism; D000577:Amides / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology; D053143:Caspase 2 / Q000378:metabolism*; D053148:Caspase 3 / Q000378:metabolism*; D002478:Cells, Cultured; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D042783:Endothelial Cells / Q000187:drug effects* / Q000201:enzymology* / Q000648:ultrastructure; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005260:Female; D005333:Fetus; D006801:Humans; D008958:Models, Molecular; D010042:Ouabain / Q000378:metabolism / Q000494:pharmacology*; D010446:Peptide Fragments / Q000032:analysis* / Q000737:chemistry; D017434:Protein Structure, Tertiary; D011725:Pyridines / Q000494:pharmacology*; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors*; D014470:Umbilical Cord / Q000201:enzymology; D054460:rho-Associated Kinases / Q000378:metabolism*
D000199:Actins; D000577:Amides; D004791:Enzyme Inhibitors; D010446:Peptide Fragments; D011725:Pyridines; C108830:Y 27632; D010042:Ouabain; C515851:ROCK1 protein, human; C515852:ROCK2 protein, human; D054460:rho-Associated Kinases; C490360:CASP2 protein, human; C505407:CASP3 protein, human; D053143:Caspase 2; D053148:Caspase 3; D003546:Cysteine Endopeptidases; D000254:Sodium-Potassium-Exchanging ATPase