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Proinsulin C-peptide causes multiple molecular and physiological effects, and improves renal and neuronal dysfunction in patients with diabetes. However, whether C-peptide controls the inhibitor kappaB (IkappaB)/NF-kappaB-dependent transcription of genes, including inflammatory genes is unknown. Here we showed that 1 nM C-peptide increased the expression of cyclooxygenase-2 (COX-2) mRNA and its protein in Swiss 3T3 fibroblasts. Consistently, C-peptide enhanced COX-2 gene promoter-activity, which was inhibited by GF109203X and Go6976, specific PKC inhibitors, and BAY11-7082, a specific nuclear factor-kappaB (NF-kappaB) inhibitor, accompanied by increased phosphorylation and degradation of IkappaB. These results suggest that C-peptide stimulates the transcription of inflammatory genes via activation of a PKC/IkappaB/NF-kappaB signaling pathway.

Manganese (Mn) exposure causes manganism, a neurological disorder similar to Parkinson's disease. However, the cellular mechanism by which Mn induces dopaminergic neuronal cell death remains unclear. In the present study, we sought to investigate the key downstream apoptotic cell signaling events that contribute to Mn-induced cell death in mesencephalic dopaminergic neuronal (N27) cells. Mn exposure induced a dose-dependent increase in neuronal cell death in N27 cells. The cell death was accompanied by sequential activation of mitochondrial-dependent proapoptotic events, including cytochrome c release, caspase-3 activation, and DNA fragmentation, but not caspase-8 activation, indicating that the mitochondrial-dependent apoptotic cascade primarily triggers Mn-induced apoptosis. Notably, Mn treatment proteolytically activated protein kinase Cdelta (PKCdelta), a member of a novel class of protein kinase C. The caspase-3 specific inhibitor benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone (Z-DEVD-FMK) significantly blocked PKCdelta cleavage and its kinase activity, indicating that caspase-3 mediates the proteolytic activation. Cotreatment with the PKCdelta inhibitor rottlerin or the caspase-3 inhibitor Z-DEVD-FMK almost completely blocked Mn-induced DNA fragmentation. Additionally, N27 cells expressing a catalytically inactive PKCdelta(K376R) protein (PKCdelta dominant negative mutant) or a caspase cleavage resistant PKCdelta(D327A) protein (PKCdelta cleavage resistant mutant) were found to be resistant to Mn-induced apoptosis. To further establish the proapoptotic role of PKCdelta, RNA interference-mediated gene knockdown was performed. Small interfering RNA suppression of PKCdelta expression protected N27 cells from Mn-induced apoptotic cell death. Collectively, these results suggest that caspase-3-dependent proteolytic activation of PKCdelta plays a key role in Mn-induced apoptotic cell death.

Chronic inorganic manganese exposure causes selective toxicity to the nigrostriatal dopaminergic system, resulting in a Parkinsonian-like neurological condition known as Manganism. Apoptosis has been shown to occur in manganese-induced neurotoxicity; however, the down-stream cellular target of caspase-3 that contributes to DNA fragmentation is not established. Herein, we demonstrate that proteolytic activation of protein kinase Cdelta (PKCdelta) by caspase-3 plays a critical role in manganese-induced apoptotic cell death. Treatment of PC12 cells with manganese caused a sequential activation of mitochondrial-dependent pro-apoptotic events, including mitochondrial membrane depolarization, cytochrome c release, caspase-3 activation, and DNA fragmentation. Overexpression of Bcl-2 in PC12 cells remarkably attenuated each of these events, indicating that the mitochondrial-dependent apoptotic cascade contributes to manganese-induced apoptosis. Furthermore, PKCdelta was proteolytically cleaved by caspase-3, causing a persistent activation of the kinase. The manganese-induced proteolytic cleavage of PKCdelta was significantly blocked by Bcl-2-overexpression. Administration of active recombinant PKCdelta induced DNA fragmentation in PC12 cells, suggesting a pro-apoptotic role of PKCdelta. Furthermore, expression of catalytically inactive mutant PKCdelta(K376R) via a lentiviral gene delivery system effectively attenuated manganese-induced apoptosis. Together, these results suggest that the mitochondrial-dependent caspase cascade mediates apoptosis via proteolytic activation of PKCdelta in manganese-induced neurotoxicity.

We previously demonstrated that the organochlorine pesticide dieldrin, a potential chemical risk factor for development of Parkinson's disease (PD), impairs mitochondrial function and promotes apoptosis in dopaminergic PC12 cells. We further demonstrated that caspase-3-dependent proteolytic activation of a member of the novel PKC family, protein kinase Cdelta (PKCdelta), contributes to apoptotic cell death in dopaminergic cells. In the present study, we report that the proapoptotic function of PKCdelta can be regulated by overexpression of the mitochondrial anti-apoptotic protein Bcl2 in dieldrin-treated dopaminergic cells. Exposure to dieldrin (30 or 100 micro M) for 3 h produced a dose-dependent increase in caspase-3 activation and DNA fragmentation in vector-transfected PC12 cells. Overexpression of human Bcl-2 in PC12 cells completely suppressed dieldrin-induced caspase-3 activation and DNA fragmentation. Furthermore, dieldrin-induced proteolytic activation of PKCdelta was also remarkably reduced in Bcl-2-overexpressed cells. Together, these results suggest that the proapoptotic function of PKCdelta can be regulated by mitochondrial redox modulators during neurodegenerative processes.

Protein kinase Cdelta (PKCdelta), a member of the novel PKC family, is emerging as a redox-sensitive kinase in various cell types. Oxidative stress activates the PKCdelta kinase by translocation, tyrosine phosphorylation, or proteolysis. During proteolysis, caspase-3 cleaves the native PKCdelta (72-74 kDa) into 41-kDa catalytically active and 38-kDa regulatory fragments to persistently activate the kinase. The proteolytic activation of PKCdelta plays a key role in promoting apoptotic cell death in various cell types, including neuronal cells. Attenuation of PKCdelta proteolytic activation by antioxidants suggests that the cellular redox status can influence activation of the proapoptotic kinase. PKCdelta may also amplify apoptotic signaling via positive feedback activation of the caspase cascade. Thus, the dual role of PKCdelta as a mediator and amplifier of apoptosis may be important in the pathogenesis of major neurodegenerative disorders, such as Parkinson's disease, Alzheimer's disease, and Huntington disease.

1-Methyl-4-phenylpyridinium (MPP+), the neurotoxic metabolite of MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine), induces apoptosis in dopaminergic neurons; however, the cellular mechanisms underlying the degenerative process are not well understood. In the present study, we demonstrate that caspase-3 mediated proteolytic activation of protein kinase C delta (PKC delta) is critical in MPP+-induced oxidative stress and apoptosis. MPP+ exposure in rat dopaminergic neuronal cells resulted in time-dependent increases in reactive oxygen species generation, cytochrome c release, and caspase-9 and caspase-3 activation. Interestingly, MPP+ induced proteolytic cleavage of PKC delta (72-74 kDa) into a 41-kDa catalytic and a 38-kDa regulatory subunit, resulting in persistently increased kinase activity. The caspase-3 inhibitor Z-DEVD-fmk effectively blocked MPP+-induced PKC delta cleavage and kinase activity, suggesting that the proteolytic activation is caspase-3 mediated. Similar results were seen in MPP+-treated rat midbrain slices. Z-DEVD-fmk and the PKC delta specific inhibitor rottlerin almost completely blocked MPP+-induced DNA fragmentation. The superoxide dismutase mimetic, MnTBAP also effectively attenuated MPP+-induced caspase-3 activation, PKC delta cleavage, and DNA fragmentation. Furthermore, rottlerin attenuated MPP+-induced caspase-3 activity without affecting basal activity, suggesting positive feedback activation of caspase-3 by PKC delta. Intracellular delivery of catalytically active recombinant PKC delta significantly increased caspase-3 activity, further indicating that PKC delta regulates caspase-3 activity. Finally, over-expression of a kinase inactive PKC delta K376R mutant prevented MPP+-induced caspase activation and DNA fragmentation, confirming the pro-apoptotic function of PKC delta in dopaminergic cell death. Together, we demonstrate for the first time that MPP+-induced oxidative stress proteolytically activates PKC delta in a caspase-3-dependent manner to induce apoptosis and up-regulate the caspase cascade in dopaminergic neuronal cells.

We previously reported that dieldrin, one of the potential environmental risk factors for development of Parkinson's disease, induces apoptosis in dopaminergic cells by generating oxidative stress. Here, we demonstrate that the caspase-3-dependent proteolytic activation of protein kinase Cdelta (PKCdelta) mediates as well as regulates the dieldrin-induced apoptotic cascade in dopaminergic cells. Exposure of PC12 cells to dieldrin (100-300 microM) results in the rapid release of cytochrome C, followed by the activation of caspase-9 and caspase-3 in a time- and dose-dependent manner. The superoxide dismutase mimetic Mn(III)tetrakis(4-benzoic acid)porphyrin chloride significantly attenuates dieldrin-induced cytochrome C release, indicating that reactive oxygen species may contribute to the activation of pro-apoptotic factors. Interestingly, dieldrin proteolytically cleaves native PKCdelta into a 41 kDa catalytic subunit and a 38 kDa regulatory subunit to activate the kinase. The dieldrin-induced proteolytic cleavage of PKCdelta and induction of kinase activity are completely inhibited by pretreatment with 50-100 microM concentrations of the caspase inhibitors benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD-FMK) and benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone (Z-DEVD-FMK), indicating that the proteolytic activation of PKCdelta is caspase-3-dependent. Additionally, Z-VAD-FMK, Z-DEVD-FMK or the PKCdelta specific inhibitor rottlerin almost completely block dieldrin-induced DNA fragmentation. Because dieldrin dramatically increases (40-80-fold) caspase-3 activity, we examined whether proteolytically activated PKCdelta amplifies caspase-3 via positive feedback activation. The PKCdelta inhibitor rottlerin (3-20 microM) dose-dependently attenuates dieldrin-induced caspase-3 activity, suggesting positive feedback activation of caspase-3 by PKCdelta. Indeed, delivery of catalytically active recombinant PKCdelta via a protein delivery system significantly activates caspase-3 in PC12 cells. Finally, overexpression of the kinase-inactive PKCdelta(K376R) mutant in rat mesencephalic dopaminergic neuronal cells attenuates dieldrin-induced caspase-3 activity and DNA fragmentation, further confirming the pro-apoptotic function of PKCdelta in dopaminergic cells. Together, we conclude that caspase-3-dependent proteolytic activation of PKCdelta is a critical event in dieldrin-induced apoptotic cell death in dopaminergic cells.

In the present study, we characterized oxidative stress-dependent cellular events in dopaminergic cells after exposure to an organic form of manganese compound, methylcyclopentadienyl manganese tricarbonyl (MMT). In pheochromocytoma cells, MMT exposure resulted in rapid increase in generation of reactive oxygen species (ROS) within 5--15 min, followed by release of mitochondrial cytochrome C into cytoplasm and subsequent activation of cysteine proteases, caspase-9 (twofold to threefold) and caspase-3 (15- to 25-fold), but not caspase-8, in a time- and dose-dependent manner. Interestingly, we also found that MMT exposure induces a time- and dose-dependent proteolytic cleavage of native protein kinase Cdelta (PKCdelta, 72-74 kDa) to yield 41 kDa catalytically active and 38 kDa regulatory fragments. Pretreatment with caspase inhibitors (Z-DEVD-FMK or Z-VAD-FMK) blocked MMT-induced proteolytic cleavage of PKCdelta, indicating that cleavage is mediated by caspase-3. Furthermore, inhibition of PKCdelta activity with a specific inhibitor, rottlerin, significantly inhibited caspase-3 activation in a dose-dependent manner along with a reduction in PKCdelta cleavage products, indicating a possible positive feedback activation of caspase-3 activity by PKCdelta. The presence of such a positive feedback loop was also confirmed by delivering the catalytically active PKCdelta fragment. Attenuation of ROS generation, caspase-3 activation, and PKCdelta activity before MMT treatment almost completely suppressed DNA fragmentation. Additionally, overexpression of catalytically inactive PKCdelta(K376R) (dominant-negative mutant) prevented MMT-induced apoptosis in immortalized mesencephalic dopaminergic cells. For the first time, these data demonstrate that caspase-3-dependent proteolytic activation of PKCdelta plays a key role in oxidative stress-mediated apoptosis in dopaminergic cells after exposure to an environmental neurotoxic agent.

We previously demonstrated that the organochlorine pesticide dieldrin, a potential chemical risk factor for development of Parkinson's disease (PD) impairs mitothondrial function and promotes apoptosis in dopaminergic PC12 cells. We further demonstrated that caspase-3-dependent proteolytic activation of a member of the novel PKC family, protein kinase Cδ (PKCδ), contributes to apoptotic cell death in dopaminergic cells. In the present study, we report that the proapoptotic function of PKCδ can be regulated by overexpression of the mitochondrial anti-apoptotic protein Bcl-2 in dieldrin-treated dopaminergic cells. Exposure to dieldrin (30 or 100 μM) for 3 h produced a dose-dependent increase in caspase-3 activation and DNA fragmentation in vector-transfected PC12 cells. Overexpression of human Bcl-2 in PC12 cells completely suppressed dieldrin-induced caspase-3 activation and DNA fragmentation. Furthermore, dieldrin-induced proteolytic activation of PKCδ was also remarkably reduced in Bcl-2-overexpressed cells. Together, these results suggest that the proapoptotic function of PKCδ can be regulated by mitochondrial redox modulators during neurodegenerative processes. [ABSTRACT FROM AUTHOR]