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Proteasome inhibitor-induced apoptosis is mediated by positive feedback amplification of PKCdelta proteolytic activation and mitochondrial translocation.

Sun F, Kanthasamy A, Song C, Yang Y, Anantharam V, Kanthasamy AG - J. Cell. Mol. Med. (2008)

Bottom Line: PKCdelta was a key downstream effector of caspase-3 because the kinase was proteolytically cleaved by caspase-3 following exposure to proteasome inhibitors MG-132 or lactacystin, resulting in a persistent increase in the kinase activity.Notably MG-132 treatment resulted in translocation of proteolytically cleaved PKCdelta fragments to mitochondria in a time-dependent fashion, and the PKCdelta inhibition effectively blocked the activation of caspase-9 and caspase-3, indicating that the accumulation of the PKCdelta catalytic fragment in the mitochondrial fraction possibly amplifies mitochondria-mediated apoptosis.Collectively, these results demonstrate that proteolytically activated PKCdelta has a significant feedback regulatory role in amplification of the mitochondria-mediated apoptotic cascade during proteasome dysfunction in dopaminergic neuronal cells.

View Article: PubMed Central - PubMed

Affiliation: Parkinson's Disorder Research Laboratory, Iowa Center for Advanced Neurotoxicology, Department of Biomedical Sciences, Iowa State University, Ames, IA 50011, USA.

ABSTRACT
Emerging evidence implicates impaired protein degradation by the ubiquitin proteasome system (UPS) in Parkinson's disease; however cellular mechanisms underlying dopaminergic degeneration during proteasomal dysfunction are yet to be characterized. In the present study, we identified that the novel PKC isoform PKCdelta plays a central role in mediating apoptotic cell death following UPS dysfunction in dopaminergic neuronal cells. Inhibition of proteasome function by MG-132 in dopaminergic neuronal cell model (N27 cells) rapidly depolarized mitochondria independent of ROS generation to activate the apoptotic cascade involving cytochrome c release, and caspase-9 and caspase-3 activation. PKCdelta was a key downstream effector of caspase-3 because the kinase was proteolytically cleaved by caspase-3 following exposure to proteasome inhibitors MG-132 or lactacystin, resulting in a persistent increase in the kinase activity. Notably MG-132 treatment resulted in translocation of proteolytically cleaved PKCdelta fragments to mitochondria in a time-dependent fashion, and the PKCdelta inhibition effectively blocked the activation of caspase-9 and caspase-3, indicating that the accumulation of the PKCdelta catalytic fragment in the mitochondrial fraction possibly amplifies mitochondria-mediated apoptosis. Overexpression of the kinase active catalytic fragment of PKCdelta (PKCdelta-CF) but not the regulatory fragment (RF), or mitochondria-targeted expression of PKCdelta-CF triggers caspase-3 activation and apoptosis. Furthermore, inhibition of PKCdelta proteolytic cleavage by a caspase-3 cleavage-resistant mutant (PKCdelta-CRM) or suppression of PKCdelta expression by siRNA significantly attenuated MG-132-induced caspase-9 and -3 activation and DNA fragmentation. Collectively, these results demonstrate that proteolytically activated PKCdelta has a significant feedback regulatory role in amplification of the mitochondria-mediated apoptotic cascade during proteasome dysfunction in dopaminergic neuronal cells.

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Inhibition of the mitochondria-mediated apoptotic cascade by PKCδ siRNA. N27 cells were transfected with non-specific siRNA (siRNA-NS) or siRNA for PKCδ (siRNA-PKCδ). Twenty-four hours after transfection, cells were treated with 5.0 μM MG-132 for 120 min and then activities of caspase-3 (A) and caspase-9 (B) and DNA fragmentation (C) were assayed. Data are presented as mean ± S.E.M. from 5 samples in each group. ***P< 0.001 compared to vehicle-treated control cells. ##P <0.01, ###P <0.001, comparison between the indicated groups.
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fig07: Inhibition of the mitochondria-mediated apoptotic cascade by PKCδ siRNA. N27 cells were transfected with non-specific siRNA (siRNA-NS) or siRNA for PKCδ (siRNA-PKCδ). Twenty-four hours after transfection, cells were treated with 5.0 μM MG-132 for 120 min and then activities of caspase-3 (A) and caspase-9 (B) and DNA fragmentation (C) were assayed. Data are presented as mean ± S.E.M. from 5 samples in each group. ***P< 0.001 compared to vehicle-treated control cells. ##P <0.01, ###P <0.001, comparison between the indicated groups.

Mentions: To further confirm the critical role of PKCδ in mitochondria-mediated apoptosis during proteasome inhibition, we used a RNAi approach. siRNA duplex specifically targeting PKCδ[16, 24] was introduced into N27 cells using electroporation transfection, and then caspase-9, caspase-3, and DNA fragmentation were assayed. The results revealed a remarkable inhibitory effect of siRNA-PKCδ on the activation of caspase-9 (Fig.7A), caspase-3 (Fig.7B) and DNA fragmentation (Fig.7C). Nonspecific siRNA treatment did not alter these apoptotic markers. Again, a positive feedback loop would in part explain the observed inhibition of caspase-9 activation by PKCδ siRNA (Fig.7A).


Proteasome inhibitor-induced apoptosis is mediated by positive feedback amplification of PKCdelta proteolytic activation and mitochondrial translocation.

Sun F, Kanthasamy A, Song C, Yang Y, Anantharam V, Kanthasamy AG - J. Cell. Mol. Med. (2008)

Inhibition of the mitochondria-mediated apoptotic cascade by PKCδ siRNA. N27 cells were transfected with non-specific siRNA (siRNA-NS) or siRNA for PKCδ (siRNA-PKCδ). Twenty-four hours after transfection, cells were treated with 5.0 μM MG-132 for 120 min and then activities of caspase-3 (A) and caspase-9 (B) and DNA fragmentation (C) were assayed. Data are presented as mean ± S.E.M. from 5 samples in each group. ***P< 0.001 compared to vehicle-treated control cells. ##P <0.01, ###P <0.001, comparison between the indicated groups.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2957660&req=5

fig07: Inhibition of the mitochondria-mediated apoptotic cascade by PKCδ siRNA. N27 cells were transfected with non-specific siRNA (siRNA-NS) or siRNA for PKCδ (siRNA-PKCδ). Twenty-four hours after transfection, cells were treated with 5.0 μM MG-132 for 120 min and then activities of caspase-3 (A) and caspase-9 (B) and DNA fragmentation (C) were assayed. Data are presented as mean ± S.E.M. from 5 samples in each group. ***P< 0.001 compared to vehicle-treated control cells. ##P <0.01, ###P <0.001, comparison between the indicated groups.
Mentions: To further confirm the critical role of PKCδ in mitochondria-mediated apoptosis during proteasome inhibition, we used a RNAi approach. siRNA duplex specifically targeting PKCδ[16, 24] was introduced into N27 cells using electroporation transfection, and then caspase-9, caspase-3, and DNA fragmentation were assayed. The results revealed a remarkable inhibitory effect of siRNA-PKCδ on the activation of caspase-9 (Fig.7A), caspase-3 (Fig.7B) and DNA fragmentation (Fig.7C). Nonspecific siRNA treatment did not alter these apoptotic markers. Again, a positive feedback loop would in part explain the observed inhibition of caspase-9 activation by PKCδ siRNA (Fig.7A).

Bottom Line: PKCdelta was a key downstream effector of caspase-3 because the kinase was proteolytically cleaved by caspase-3 following exposure to proteasome inhibitors MG-132 or lactacystin, resulting in a persistent increase in the kinase activity.Notably MG-132 treatment resulted in translocation of proteolytically cleaved PKCdelta fragments to mitochondria in a time-dependent fashion, and the PKCdelta inhibition effectively blocked the activation of caspase-9 and caspase-3, indicating that the accumulation of the PKCdelta catalytic fragment in the mitochondrial fraction possibly amplifies mitochondria-mediated apoptosis.Collectively, these results demonstrate that proteolytically activated PKCdelta has a significant feedback regulatory role in amplification of the mitochondria-mediated apoptotic cascade during proteasome dysfunction in dopaminergic neuronal cells.

View Article: PubMed Central - PubMed

Affiliation: Parkinson's Disorder Research Laboratory, Iowa Center for Advanced Neurotoxicology, Department of Biomedical Sciences, Iowa State University, Ames, IA 50011, USA.

ABSTRACT
Emerging evidence implicates impaired protein degradation by the ubiquitin proteasome system (UPS) in Parkinson's disease; however cellular mechanisms underlying dopaminergic degeneration during proteasomal dysfunction are yet to be characterized. In the present study, we identified that the novel PKC isoform PKCdelta plays a central role in mediating apoptotic cell death following UPS dysfunction in dopaminergic neuronal cells. Inhibition of proteasome function by MG-132 in dopaminergic neuronal cell model (N27 cells) rapidly depolarized mitochondria independent of ROS generation to activate the apoptotic cascade involving cytochrome c release, and caspase-9 and caspase-3 activation. PKCdelta was a key downstream effector of caspase-3 because the kinase was proteolytically cleaved by caspase-3 following exposure to proteasome inhibitors MG-132 or lactacystin, resulting in a persistent increase in the kinase activity. Notably MG-132 treatment resulted in translocation of proteolytically cleaved PKCdelta fragments to mitochondria in a time-dependent fashion, and the PKCdelta inhibition effectively blocked the activation of caspase-9 and caspase-3, indicating that the accumulation of the PKCdelta catalytic fragment in the mitochondrial fraction possibly amplifies mitochondria-mediated apoptosis. Overexpression of the kinase active catalytic fragment of PKCdelta (PKCdelta-CF) but not the regulatory fragment (RF), or mitochondria-targeted expression of PKCdelta-CF triggers caspase-3 activation and apoptosis. Furthermore, inhibition of PKCdelta proteolytic cleavage by a caspase-3 cleavage-resistant mutant (PKCdelta-CRM) or suppression of PKCdelta expression by siRNA significantly attenuated MG-132-induced caspase-9 and -3 activation and DNA fragmentation. Collectively, these results demonstrate that proteolytically activated PKCdelta has a significant feedback regulatory role in amplification of the mitochondria-mediated apoptotic cascade during proteasome dysfunction in dopaminergic neuronal cells.

Show MeSH
Related in: MedlinePlus