Caspase-3-dependent proteolytic cleavage of protein kinase Cδ is essential for oxidative stress-mediated dopaminergic cell death after exposure to methylcyclopentadienyl manganese tricarbonyl

Vellareddy Anantharam, Masashi Kitazawa, Jarrad Wagner, Siddharth Kaul, Anumantha G. Kanthasamy

Research output: Contribution to journalArticle

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Abstract

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 Cδ (PKCδ, 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 PKCδ, indicating that cleavage is mediated by caspase-3. Furthermore, inhibition of PKCδ activity with a specific inhibitor, rottlerin, significantly inhibited caspase-3 activation in a dose-dependent manner along with a reduction in PKCδ cleavage products, indicating a possible positive feedback activation of caspase-3 activity by PKCδ. The presence of such a positive feedback loop was also confirmed by delivering the catalytically active PKCδ fragment. Attenuation of ROS generation, caspase-3 activation, and PKCδ activity before MMT treatment almost completely suppressed DNA fragmentation. Additionally, overexpression of catalytically inactive PKCδK376R (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 PKCδ plays a key role in oxidative stress-mediated apoptosis in dopaminergic cells after exposure to an environmental neurotoxic agent.

Original languageEnglish
Pages (from-to)1738-1751
Number of pages14
JournalJournal of Neuroscience
Volume22
Issue number5
StatePublished - 1 Mar 2002

Fingerprint

Manganese
Caspase 3
Protein Kinase C
Oxidative Stress
Cell Death
Reactive Oxygen Species
Manganese Compounds
Apoptosis
Caspase Inhibitors
Cysteine Proteases
Caspase 9
Caspase 8
Environmental Exposure
Pheochromocytoma
DNA Fragmentation
Cytochromes
Cytoplasm

Keywords

  • Apoptosis
  • Dopaminergic degeneration
  • Environmental factors
  • Manganese
  • Oxidative stress
  • Parkinson's disease

Cite this

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title = "Caspase-3-dependent proteolytic cleavage of protein kinase Cδ is essential for oxidative stress-mediated dopaminergic cell death after exposure to methylcyclopentadienyl manganese tricarbonyl",
abstract = "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 Cδ (PKCδ, 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 PKCδ, indicating that cleavage is mediated by caspase-3. Furthermore, inhibition of PKCδ activity with a specific inhibitor, rottlerin, significantly inhibited caspase-3 activation in a dose-dependent manner along with a reduction in PKCδ cleavage products, indicating a possible positive feedback activation of caspase-3 activity by PKCδ. The presence of such a positive feedback loop was also confirmed by delivering the catalytically active PKCδ fragment. Attenuation of ROS generation, caspase-3 activation, and PKCδ activity before MMT treatment almost completely suppressed DNA fragmentation. Additionally, overexpression of catalytically inactive PKCδK376R (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 PKCδ plays a key role in oxidative stress-mediated apoptosis in dopaminergic cells after exposure to an environmental neurotoxic agent.",
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Caspase-3-dependent proteolytic cleavage of protein kinase Cδ is essential for oxidative stress-mediated dopaminergic cell death after exposure to methylcyclopentadienyl manganese tricarbonyl. / Anantharam, Vellareddy; Kitazawa, Masashi; Wagner, Jarrad; Kaul, Siddharth; Kanthasamy, Anumantha G.

In: Journal of Neuroscience, Vol. 22, No. 5, 01.03.2002, p. 1738-1751.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Caspase-3-dependent proteolytic cleavage of protein kinase Cδ is essential for oxidative stress-mediated dopaminergic cell death after exposure to methylcyclopentadienyl manganese tricarbonyl

AU - Anantharam, Vellareddy

AU - Kitazawa, Masashi

AU - Wagner, Jarrad

AU - Kaul, Siddharth

AU - Kanthasamy, Anumantha G.

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N2 - 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 Cδ (PKCδ, 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 PKCδ, indicating that cleavage is mediated by caspase-3. Furthermore, inhibition of PKCδ activity with a specific inhibitor, rottlerin, significantly inhibited caspase-3 activation in a dose-dependent manner along with a reduction in PKCδ cleavage products, indicating a possible positive feedback activation of caspase-3 activity by PKCδ. The presence of such a positive feedback loop was also confirmed by delivering the catalytically active PKCδ fragment. Attenuation of ROS generation, caspase-3 activation, and PKCδ activity before MMT treatment almost completely suppressed DNA fragmentation. Additionally, overexpression of catalytically inactive PKCδK376R (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 PKCδ plays a key role in oxidative stress-mediated apoptosis in dopaminergic cells after exposure to an environmental neurotoxic agent.

AB - 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 Cδ (PKCδ, 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 PKCδ, indicating that cleavage is mediated by caspase-3. Furthermore, inhibition of PKCδ activity with a specific inhibitor, rottlerin, significantly inhibited caspase-3 activation in a dose-dependent manner along with a reduction in PKCδ cleavage products, indicating a possible positive feedback activation of caspase-3 activity by PKCδ. The presence of such a positive feedback loop was also confirmed by delivering the catalytically active PKCδ fragment. Attenuation of ROS generation, caspase-3 activation, and PKCδ activity before MMT treatment almost completely suppressed DNA fragmentation. Additionally, overexpression of catalytically inactive PKCδK376R (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 PKCδ plays a key role in oxidative stress-mediated apoptosis in dopaminergic cells after exposure to an environmental neurotoxic agent.

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