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.
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Abbreviations
- TG2:
-
tissue transglutaminase
- DLK:
-
dual leucine zipper-bearing kinase
- JNK:
-
c-Jun amino-terminal kinase
- PARP:
-
poly-ADP-ribose polymerase
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Acknowledgements
We thank Dr. Alain Lavigueur for critical reading of the article and Dr. Nicolas Gévry for his help with the design of the short hairpin sequences. This work was supported by a grant from the Canadian Institutes of Health Research (CIHR). KR was recipient of a studentship from the Fonds Québécois de la Recherche sur la Nature et les Technologies.
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Robitaille, K., Daviau, A., Lachance, G. et al. Calphostin C-induced apoptosis is mediated by a tissue transglutaminase-dependent mechanism involving the DLK/JNK signaling pathway. Cell Death Differ 15, 1522–1531 (2008). https://doi.org/10.1038/cdd.2008.77
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DOI: https://doi.org/10.1038/cdd.2008.77
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