Tissue transglutaminase prevents thapsigargin-induced cell death through cross-linking of caspase-3 in Bax-deficient HCT116 cells Free

720

We have reported previously that thapsigargin (THG), an inhibitor of the endoplasmic reticulum Ca²⁺-ATPase, induces caspase-3 activation and apoptosis in HCT116 cells through a DR5-initiated and Bax-dependent pathway. In Bax-deficient HCT116 cells, THG treatment generates high-molecular-weight inactive species of caspase-3 that are highly resistant to SDS and 2-mercaptoethanol through an unknown mechanism. Here, we show that the Ca²⁺-dependent protein cross-linking enzyme tissue transglutaminase (tTGase) catalyzes caspase-3 cross-linking reactions, thereby inactivating caspase-3 and blocking apoptosis in HCT116 cells in response to THG. A prolonged exposure of Bax-deficient cells to THG caused cell rounding and reduced clonogenic cells but did not induce caspase-3 activation and cell membrane permeabilization. Concurrently, two additional species of caspase-3, termed p40 and p64, with molecular mass approximately 40 and 64 kDa, respectively, were observed. However, this formation of p40 and p64 species was inhibited by the tTGase inhibitor monodansylcadaverine. Overexpression of tTGase increased p40 and p64 in THG-treated Bax-deficient cells and purified tTGase catalyzed caspase-3 cross-linking in vitro. In contrast, knockdown of tTGase by shRNA in Bax-deficient cells reduced the cross-linked species p40 and p64 and restored caspase-3 activation in response to THG treatment. Moreover, the protein levels of XIAP and cIAP-1were decreased after a prolonged exposure to THG, but the protein levels and enzymatic activity of tTGase were subsequently upregulated. Expression of cytosolic Smac sensitized Bax-deficient cells to THG-induced apoptosis, while this effect was diminished by co-expression of tTGase. Taken together, these results suggest that tTGase serves as an inhibitor of apoptosis by cross-linking caspase-3 in THG-treated cells.