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

J Neurosci. 2002 Mar 1;22(5):1738-51. doi: 10.1523/JNEUROSCI.22-05-01738.2002.

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 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.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Acetophenones / pharmacology
  • Amino Acid Substitution
  • Animals
  • Apoptosis / drug effects
  • Benzopyrans / pharmacology
  • Caspase 3
  • Caspase 9
  • Caspase Inhibitors
  • Caspases / metabolism*
  • Cell Death
  • Cell Line
  • Cytochrome c Group / metabolism
  • DNA Fragmentation / drug effects
  • Dopamine / metabolism
  • Dose-Response Relationship, Drug
  • Enzyme Activation / drug effects
  • Enzyme Inhibitors / pharmacology
  • Feedback, Physiological / drug effects
  • Isoenzymes / genetics
  • Isoenzymes / metabolism*
  • Manganese Compounds / pharmacology
  • Mesencephalon / cytology
  • Mesencephalon / drug effects
  • Mesencephalon / metabolism
  • Organometallic Compounds / toxicity*
  • Oxidative Stress / physiology*
  • PC12 Cells / cytology
  • PC12 Cells / drug effects*
  • PC12 Cells / metabolism
  • Protein Kinase C / genetics
  • Protein Kinase C / metabolism*
  • Protein Kinase C-delta
  • Protein Subunits
  • Rats
  • Reactive Oxygen Species / metabolism
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Transfection

Substances

  • Acetophenones
  • Benzopyrans
  • Caspase Inhibitors
  • Cytochrome c Group
  • Enzyme Inhibitors
  • Isoenzymes
  • Manganese Compounds
  • Organometallic Compounds
  • Protein Subunits
  • Reactive Oxygen Species
  • Recombinant Fusion Proteins
  • 2-methylcyclopentadienyl manganese tricarbonyl
  • rottlerin
  • Prkcd protein, rat
  • Protein Kinase C
  • Protein Kinase C-delta
  • Casp3 protein, rat
  • Casp9 protein, rat
  • Caspase 3
  • Caspase 9
  • Caspases
  • Dopamine