Calphostin C-induced apoptosis is mediated by a tissue transglutaminase-dependent mechanism involving the DLK/JNK signaling pathway

Abstract

A role for tissue transglutaminase (TG2) and its substrate dual leucine zipper-bearing kinase (DLK), an upstream component of the c-Jun N-terminal kinase (JNK) signaling pathway, has been previously suggested in the apoptotic response induced by calphostin C. In the current study, we directly tested this hypothesis by examining via pharmacological and RNA-interference approaches whether inhibition of expression or activity of TG2, DLK and JNK in mouse NIH 3T3 fibroblasts and human MDA-MB-231 breast cancer epithelial cells affects calphostin C-induced apoptosis. Our experiments with the selective JNK inhibitor SP600125 reveal that calphostin C is capable of causing JNK activation and JNK-dependent apoptosis in both cell lines. Small interfering RNA-mediated depletion of TG2 alone strongly reduces calphostin C action on JNK activity and apoptosis. Consistent with an active role for DLK in this cascade of event, cells deficient in DLK demonstrate a substantial delay of JNK activation and poly-ADP-ribose polymerase (PARP) cleavage in response to calphostin C, whereas overexpression of a recombinant DLK resistant to silencing, but sensitive to TG2-mediated oligomerization, reverses this effect. Importantly, combined depletion of TG2 and DLK further alters calphostin C effects on JNK activity, Bax translocation, caspase-3 activation, PARP cleavage and cell viability, demonstrating an obligatory role for TG2 and DLK in calphostin C-induced apoptosis.

Transglutaminase-2 in cell adhesion

“…This mechanism of TG-2 on JNK has been reported in the context of cell death signaling. 37,38 Consistent with this observation, silencing of TG-2 has been associated with reduced pJNK in malignancies. 37,39,40 Moreover, it has been reported that JNK can localize to paxillin-containing focal adhesions in corneal epithelial cells.…”

Transglutaminase 2 Promotes Both Caspase-dependent and Caspase-independent Apoptotic Cell Death via the Calpain/Bax Protein Signaling Pathway

“…TG2 overexpression and activation have been linked to induction of apoptosis (3,11,12,33). Recently, we reported a possible role of TG2 in the PDT-induced apoptotic death of cancer cells (20).…”

“…TG2 promotes apoptosis of cardiomyocytes by reducing phospholipase C␦1 and active PKC levels during oxidative stress (10). TG2 also mediates calphostin C-induced apoptosis via a mechanism involving activation of the c-Jun N-terminal kinase and translocation of Bax to the mitochondria in NIH3T3 fibroblasts and breast cancer cells (11). These events ultimately stimulate a caspase-dependent pathway.…”

Transglutaminase 2 and Its Role in Pulmonary Fibrosis

“…To examine the role of TG2 in pulmonary fibroblast function, TG2 was knocked down using an SH-RNA lentivirus vector containing GFP, and cells were sorted for GFP expression (42). This strategy resulted in very efficient 85% knockdown of TG2 protein levels ( Figure 7A and data not shown).…”

“…Sh-RNA lentiviral vectors targeting TG2 and a scrambled Sh-RNA were used as described (42), and cells were sorted for green fluorescent protein (GFP) expression using a FACSAria machine (BD Biosciences). For collagen gel contraction assays, transduced cells were seeded in gels of rat-tail collagen, (Roche, Basel, Switzerland), floated in media, and weighed after 48 hours (43).…”