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PKCdelta is required for mitochondrial-dependent apoptosis in salivary epithelial cells
A A Matassa1, L Carpenter, T J Biden
1Departments of Basic Science and Oral Research, School of Dentistry and Cell and Structural Biology, School of Medicine, University of Colorado Health Sciences Center, Denver, Colorado 80262, USA.
Abstract:
We report here that the novel protein kinase C isoform, PKCdelta, is required at or prior to the level of the mitochondria for apoptosis induced by a diverse group of cell toxins. We have used adenoviral expression of a kinase-dead (KD) mutant of PKCdelta to explore the requirement for PKCdelta in the mitochondrial-dependent apoptotic pathway. Expression of PKCdeltaKD, but not PKCalphaKD, in salivary epithelial cells resulted in a dose-dependent inhibition of apoptosis induced by etoposide, UV-irradiation, brefeldin A, and paclitaxel. DNA fragmentation was blocked up to 71% in parotid C5 cells infected with the PKCdeltaKD adenovirus, whereas caspase-3 activity was inhibited up to 65%. The activation of caspase-9-like proteases by all agents was also inhibited in parotid C5 cells expressing PKCdeltaKD. The ability of PKCdeltaKD to block the loss of mitochondrial membrane potential was similarly determined. Expression of PKCdeltaKD blocked the decrease in mitochondrial membrane potential observed in cells treated with etoposide, UV, brefeldin A, or paclitaxel in a dose-dependent manner. In contrast to the protective function of PKCdeltaKD, expression of PKCdeltaWT resulted in a potent induction of apoptosis, which could be inhibited by co-infection with PKCdeltaKD. These results suggest that PKCdelta is a common intermediate in mitochondrial-dependent apoptosis in salivary epithelial cells.
Insights
Protein kinase C delta (PKCdelta) is essential for mitochondrial-dependent apoptosis triggered by various toxins. Inhibiting PKCdelta prevents DNA fragmentation and caspase activation, highlighting its role in programmed cell death.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Apoptosis is a crucial process for cellular homeostasis and development.
- Mitochondrial-dependent apoptosis is triggered by various intrinsic and extrinsic stimuli.
- Protein kinase C (PKC) isoforms play diverse roles in cellular signaling, including cell death pathways.
Purpose of the Study:
- To investigate the role of the novel protein kinase C delta (PKCdelta) isoform in mitochondrial-dependent apoptosis.
- To determine if PKCdelta is required at or before the mitochondrial stage of apoptosis.
- To elucidate the specific mechanisms by which PKCdelta influences apoptotic signaling.
Main Methods:
- Adenoviral expression of a kinase-dead (KD) mutant of PKCdelta (PKCdeltaKD) in salivary epithelial cells.
- Treatment of cells with apoptosis-inducing agents: etoposide, UV-irradiation, brefeldin A, and paclitaxel.
- Assessment of apoptosis markers: DNA fragmentation, caspase-3 activity, caspase-9-like protease activation, and mitochondrial membrane potential.
Main Results:
- PKCdeltaKD expression dose-dependently inhibited apoptosis induced by all tested toxins.
- DNA fragmentation was reduced by up to 71% and caspase-3 activity by up to 65% in PKCdeltaKD-expressing cells.
- PKCdeltaKD expression blocked the activation of caspase-9-like proteases and the loss of mitochondrial membrane potential.
- Expression of wild-type PKCdelta (PKCdeltaWT) potently induced apoptosis, which was counteracted by PKCdeltaKD co-expression.
Conclusions:
- PKCdelta is a critical mediator in the mitochondrial-dependent apoptotic pathway.
- PKCdelta functions at or upstream of the mitochondria in response to diverse cellular toxins.
- Targeting PKCdelta may offer a therapeutic strategy for modulating apoptosis in salivary epithelial cells.