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Inhibition of PKCalpha induces a PKCdelta-dependent apoptotic program in salivary epithelial cells
A A Matassa1, R L Kalkofen, L Carpenter
1Department of Craniofacial Biology, School of Dentistry, University of Colorado Health Sciences Center, Denver 80262, USA.
Abstract:
We have used expression of a kinase dead mutant of PKCalpha (PKCalphaKD) to explore the role of this isoform in salivary epithelial cell apoptosis. Expression of PKCalphaKD by adenovirus-mediated transduction results in a dose-dependent induction of apoptosis in salivary epithelial cells as measured by the accumulation of sub-G1 DNA, activation of caspase-3, and cleavage of PKCdelta and PKCzeta, known caspase substrates. Induction of apoptosis is accompanied by nine-fold activation of c-Jun-N-terminal kinase, and an approximately two to three-fold increase in activated mitogen-activated protein kinase (MAPK) as well as total MAPK protein. Previous studies from our laboratory have shown that PKCdelta activity is essential for the apoptotic response of salivary epithelial cells to a variety of cell toxins. To explore the contribution of PKCdelta to PKCalphaKD-induced apoptosis, salivary epithelial cells were cotransduced with PKCalphaKD and PKCdeltaKD expression vectors. Inhibition of endogenous PKCdelta blocked the ability of PKCalphaKD to induce apoptosis as indicated by cell morphology, DNA fragmentation, and caspase-3 activation, indicating that PKCdelta activity is required for the apoptotic program induced under conditions where PKCalpha is inhibited. These findings indicate that PKCalpha functions as a survival factor in salivary epithelial cells, while PKCdelta functions to regulate entry into the apoptotic pathway.
Insights
Protein kinase C alpha (PKCalpha) acts as a survival factor in salivary cells. Inhibiting PKCalpha triggers apoptosis, requiring PKCdelta to initiate the cell death pathway.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Protein kinase C (PKC) signaling pathways regulate diverse cellular processes, including cell survival and apoptosis.
- The specific roles of individual PKC isoforms, such as PKCalpha and PKCdelta, in salivary epithelial cell apoptosis are not fully elucidated.
Purpose of the Study:
- To investigate the role of Protein Kinase C alpha (PKCalpha) in salivary epithelial cell apoptosis.
- To determine the involvement of PKCdelta in PKCalpha-mediated apoptotic pathways.
Main Methods:
- Adenovirus-mediated expression of a kinase-dead mutant of PKCalpha (PKCalphaKD) in salivary epithelial cells.
- Assessment of apoptosis using DNA content analysis (sub-G1 DNA), caspase-3 activation, and Western blotting for caspase substrates.
- Evaluation of c-Jun-N-terminal kinase (JNK) and mitogen-activated protein kinase (MAPK) activation.
- Co-transduction experiments with PKCalphaKD and PKCdelta kinase-dead mutant (PKCdeltaKD) to inhibit endogenous PKCdelta.
Main Results:
- Expression of PKCalphaKD induced apoptosis in a dose-dependent manner, evidenced by sub-G1 DNA accumulation and caspase-3 activation.
- PKCalphaKD expression led to the cleavage of PKCdelta and PKCzeta, and increased activation of JNK and MAPK signaling.
- Inhibition of endogenous PKCdelta by co-expression of PKCdeltaKD abrogated PKCalphaKD-induced apoptosis, blocking cell morphology changes, DNA fragmentation, and caspase-3 activation.
Conclusions:
- PKCalpha functions as a survival factor in salivary epithelial cells.
- PKCdelta is essential for mediating the apoptotic pathway initiated by PKCalpha inhibition.
- These findings reveal a functional antagonism between PKCalpha and PKCdelta in regulating salivary epithelial cell fate.