Tissue Transglutaminase Serves as an Inhibitor of Apoptosis by Cross-Linking Caspase 3 in Thapsigargin-Treated Cells

Abstract

Thapsigargin (THG) is an inhibitor of the endoplasmic reticulum Ca²⁺-ATPase that induces caspase 3 activation and apoptosis in HCT116 cells through a Bax-dependent pathway. In Bax-deficient HCT116 cells, however, THG specifically generates two additional species of caspase 3, termed p40 and p64, with molecular masses of approximately 40 and 64 kDa, respectively, through unknown mechanisms. Here, we report that the Ca²⁺-dependent protein cross-linking enzyme tissue transglutaminase (tTGase) is involved in THG-induced p40 and p64 formation by catalyzing caspase 3 cross-linking reactions, thereby inactivating caspase 3 and apoptosis in Bax-deficient cells. Overexpression of tTGase increases p40 and p64 in THG-treated cells, and purified tTGase catalyzes procaspase 3 cross-linking in vitro. Inhibition of tTGase activity by either the tTGase inhibitor monodansylcadaverine or short-hairpin RNA reduces the cross-linked species p40 and p64 and restores caspase 3 activation in response to THG treatment. Moreover, prolonged exposure to THG results in a decrease in protein levels of XIAP and cIAP-1, which is subsequently followed by an increase in tTGase protein expression and activity. Expression of cytosolic Smac sensitizes Bax-deficient cells to THG-induced apoptosis; however, this effect is diminished by coexpression of tTGase. Taken together, these results suggest a novel role for tTGase as a new type of caspase 3 inhibitor in THG-mediated apoptosis.

We thank Bert Vogelstein (Johns Hopkins University) for HCT116 Bax^+/− and Bax^−/− cells, Emad Alnemri (Kimmel Cancer Institute) for GFP-IETD-Smac plasmid, and Nicholas Woods for reading the manuscript. We acknowledge the services provided by the Moffitt Molecular Biology Core and Molecular Imaging Core.

This work was supported by NIH grant CA82197.