Ouabain Induces Caspase‐2 Dependent Rock II Cleavage In HeLa Cells

We have previously showed that ouabain induced Rho kinase II (Rock II) cleavage via caspase‐2 dependent manner and produced ~140 kDa fragment of the enzyme in HUVEC during apoptosis. In the current work we evaluated that whether this phenomenon is unique for HUVEC or appears in different cell types especially in cancer cell lines. Subconfluent HeLa cells were exposed to ouabain (10 μM, 18 h), Rock inhibitor Y‐27632 (10 μM, 30 min before ouabain), caspase‐2 inhibitor z‐VDVAD‐fmk (25 μM, 2h before ouabain), caspase‐3 inhibitor Z‐DEVD‐fmk (25 μM, 2h before ouabain) and pan caspase inhibitor z‐VAD‐fmk (25μM, 2h before ouabain). After the treatments, pictures were taken through a phase‐contrast microscope at random sites and cells with blebs were counted. Rock II cleavage was evaluated by Western Blot and an xCelligence real time cell analyzer DP were used for cell detachment assay. Ouabain treatment resulted in membrane blebbing and cell‐substratum detachment. Y‐27632 did not prevent ouabain‐induced cell detachment. Neither Y‐27632 nor caspase inhibitors inhibited the formation of blebs. Therefore our results suggest that in contrast to previous works, Rock may not play a profound role in the formation of membrane blebs. Very little basal Rock II cleavage was detected in HeLa cells. Ouabain treatment enhanced this basal cleavage of Rock II. Although this cleavage was not inhibited by Y‐27632, all the caspase inhibitors prevented the proteolytic clevage of Rock II. It has been shown that activation of caspase‐3 results in cleavage and activation of caspase‐2. Therefore our results indicate that ouabain induces caspase‐3, which induces caspase‐2 activation causing proteolytic cleavage of Rock II in HeLa cells. As a result, ouabain‐induced Rock II cleavage by caspase‐2 may be a common process in different cell types.