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

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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.

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Acknowledgment

This work was supported by Gazi University Scientific Research Project Unit in the 02/2008-04 project.

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Authors and Affiliations

  1. Faculty of Pharmacy, Department of Pharmacology, Gazi University, Etiler, 06330, Ankara, Turkey

    Mustafa Ark & Aysun Özdemir

  2. Department of Perinatology, Zekai Tahir Burak Maternity Hospital, Ankara, Turkey

    Belgin Polat

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  1. Mustafa Ark
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Correspondence to Mustafa Ark.

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10495_2010_529_MOESM1_ESM.jpg

Supplementary Fig. 1 Schematic presentation of possible caspase-2 consensus sequence in Rock-2 C-terminus. Granzyme B consensus sequence and identification of a possible Rock-2 cleavage site in the Rock-2 C-terminus. Rock-2 is cleaved by granzyme B at the IGLD 1131 site, producing a ~130 kDa fragment. Possible caspase-2 cleavage sequence VDVAD is situated at position 1211–1215 (VTQTD). Cleavage by caspase-2 produces a ~140 kDa form of Rock-2. (JPEG 1228 kb)

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Ark, M., Özdemir, A. & Polat, B. Ouabain-induced apoptosis and Rho kinase: a novel caspase-2 cleavage site and fragment of Rock-2. Apoptosis 15, 1494–1506 (2010). https://doi.org/10.1007/s10495-010-0529-1

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