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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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Activation of caspase-3 by PKCδ-CF. (A) Twenty-four hours after transfection, phase contrast images and fluorescence images were taken to determine transfection efficiency. (B) Caspase-3 activity was measured as described in the ‘Materials and methods’ section (B). Values represent mean ± S.E.M. from 6 samples in each group. *P< 0.05 versus cells transfected with pmaxGFP alone; #P< 0.05 comparing the indicated groups. (C) and (D) Inhibition of mitochondria-mediated apoptosis by rottlerin. N27 cells were treated with 5.0 μM MG-132 for 120 min with or without rottlerin (2.5 μM) for a 40-min pre-treatment. Rottlerin treatment alone was also included in the experiment. Caspase-9 (C) and -3 activities (D) were assayed for the treated cells as described above. Data are presented as mean ± S.E.M. from 6 samples in each group. ***P< 0.001 comparing with vehicle-treated control cells. ###P< 0.001, comparison between the indicated groups. (E) Effect of MnTBAP on MG-132-induced caspase-3 activation. Cells were treated with either with 5.0 μM MG-132 alone or pre-treated with 10.0 μM MnTBAP 30 min prior to MG-132 treatment. The caspase-3 activity was determined as described previously. Data are presented as mean ± S.E.M. from 6 samples in each group. ***P< 0.001 comparing with vehicle-treated control cells.
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fig04: Activation of caspase-3 by PKCδ-CF. (A) Twenty-four hours after transfection, phase contrast images and fluorescence images were taken to determine transfection efficiency. (B) Caspase-3 activity was measured as described in the ‘Materials and methods’ section (B). Values represent mean ± S.E.M. from 6 samples in each group. *P< 0.05 versus cells transfected with pmaxGFP alone; #P< 0.05 comparing the indicated groups. (C) and (D) Inhibition of mitochondria-mediated apoptosis by rottlerin. N27 cells were treated with 5.0 μM MG-132 for 120 min with or without rottlerin (2.5 μM) for a 40-min pre-treatment. Rottlerin treatment alone was also included in the experiment. Caspase-9 (C) and -3 activities (D) were assayed for the treated cells as described above. Data are presented as mean ± S.E.M. from 6 samples in each group. ***P< 0.001 comparing with vehicle-treated control cells. ###P< 0.001, comparison between the indicated groups. (E) Effect of MnTBAP on MG-132-induced caspase-3 activation. Cells were treated with either with 5.0 μM MG-132 alone or pre-treated with 10.0 μM MnTBAP 30 min prior to MG-132 treatment. The caspase-3 activity was determined as described previously. Data are presented as mean ± S.E.M. from 6 samples in each group. ***P< 0.001 comparing with vehicle-treated control cells.

Mentions: Next, we examined the role of proteolytically cleaved PKCδ in apoptosis by using PKCδ catalytic fragment (PKCδ-CF) and PKCδ regulatory fragment (PKCδ-RF). N27 cells were transfected with PKCδ-CF or PKCδ-RF, and the transfection efficiency was estimated by the cotransfected GFP plasmids (Fig.4A). Measurement of caspase-3 activity revealed a significant increase in the caspase-3 activity in PKCδ-CF-transfected cells as compared to RF-transfected or GFP-transfected cells, suggesting that kinase active PKCδ-CF is responsible for its proapoptotic effect in dopaminergic cells (Fig.4B). Additionally, pre-treatment with the PKCδ-specific inhibitor rottlerin also significantly attenuated MG-132-induced caspase-9 and -3 activation (Fig.4C and D), indicating that PKCδ activation indeed contributes to caspase activation following exposure to the proteasome inhibitor MG-132. However, MnTBAP, a superoxide dismutase (SOD) mimetic, failed to attenuate caspase-3 activation following MG-132 exposure (Fig.4E). Further, we and others have recently demonstrated a positive feedback activation of caspase-3 and caspase-9 by proteolytically activated PKCδ during apoptotic cell death [21, 27]. Thus, a positive feedback loop would in part explain the observed inhibition of caspase-9 activation by rottlerin (Fig.4C). This finding suggests that the proapoptotic effect of PKCδ proceeds through the mitochondria-mediated apoptosis pathway.


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)

Activation of caspase-3 by PKCδ-CF. (A) Twenty-four hours after transfection, phase contrast images and fluorescence images were taken to determine transfection efficiency. (B) Caspase-3 activity was measured as described in the ‘Materials and methods’ section (B). Values represent mean ± S.E.M. from 6 samples in each group. *P< 0.05 versus cells transfected with pmaxGFP alone; #P< 0.05 comparing the indicated groups. (C) and (D) Inhibition of mitochondria-mediated apoptosis by rottlerin. N27 cells were treated with 5.0 μM MG-132 for 120 min with or without rottlerin (2.5 μM) for a 40-min pre-treatment. Rottlerin treatment alone was also included in the experiment. Caspase-9 (C) and -3 activities (D) were assayed for the treated cells as described above. Data are presented as mean ± S.E.M. from 6 samples in each group. ***P< 0.001 comparing with vehicle-treated control cells. ###P< 0.001, comparison between the indicated groups. (E) Effect of MnTBAP on MG-132-induced caspase-3 activation. Cells were treated with either with 5.0 μM MG-132 alone or pre-treated with 10.0 μM MnTBAP 30 min prior to MG-132 treatment. The caspase-3 activity was determined as described previously. Data are presented as mean ± S.E.M. from 6 samples in each group. ***P< 0.001 comparing with vehicle-treated control cells.
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fig04: Activation of caspase-3 by PKCδ-CF. (A) Twenty-four hours after transfection, phase contrast images and fluorescence images were taken to determine transfection efficiency. (B) Caspase-3 activity was measured as described in the ‘Materials and methods’ section (B). Values represent mean ± S.E.M. from 6 samples in each group. *P< 0.05 versus cells transfected with pmaxGFP alone; #P< 0.05 comparing the indicated groups. (C) and (D) Inhibition of mitochondria-mediated apoptosis by rottlerin. N27 cells were treated with 5.0 μM MG-132 for 120 min with or without rottlerin (2.5 μM) for a 40-min pre-treatment. Rottlerin treatment alone was also included in the experiment. Caspase-9 (C) and -3 activities (D) were assayed for the treated cells as described above. Data are presented as mean ± S.E.M. from 6 samples in each group. ***P< 0.001 comparing with vehicle-treated control cells. ###P< 0.001, comparison between the indicated groups. (E) Effect of MnTBAP on MG-132-induced caspase-3 activation. Cells were treated with either with 5.0 μM MG-132 alone or pre-treated with 10.0 μM MnTBAP 30 min prior to MG-132 treatment. The caspase-3 activity was determined as described previously. Data are presented as mean ± S.E.M. from 6 samples in each group. ***P< 0.001 comparing with vehicle-treated control cells.
Mentions: Next, we examined the role of proteolytically cleaved PKCδ in apoptosis by using PKCδ catalytic fragment (PKCδ-CF) and PKCδ regulatory fragment (PKCδ-RF). N27 cells were transfected with PKCδ-CF or PKCδ-RF, and the transfection efficiency was estimated by the cotransfected GFP plasmids (Fig.4A). Measurement of caspase-3 activity revealed a significant increase in the caspase-3 activity in PKCδ-CF-transfected cells as compared to RF-transfected or GFP-transfected cells, suggesting that kinase active PKCδ-CF is responsible for its proapoptotic effect in dopaminergic cells (Fig.4B). Additionally, pre-treatment with the PKCδ-specific inhibitor rottlerin also significantly attenuated MG-132-induced caspase-9 and -3 activation (Fig.4C and D), indicating that PKCδ activation indeed contributes to caspase activation following exposure to the proteasome inhibitor MG-132. However, MnTBAP, a superoxide dismutase (SOD) mimetic, failed to attenuate caspase-3 activation following MG-132 exposure (Fig.4E). Further, we and others have recently demonstrated a positive feedback activation of caspase-3 and caspase-9 by proteolytically activated PKCδ during apoptotic cell death [21, 27]. Thus, a positive feedback loop would in part explain the observed inhibition of caspase-9 activation by rottlerin (Fig.4C). This finding suggests that the proapoptotic effect of PKCδ proceeds through the mitochondria-mediated apoptosis pathway.

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