Abstract
Human neutrophils normally have a very short half-life and die by apoptosis. Cytokines such as granulocyte-macrophage colony-stimulating factor (GM-CSF) can delay this apoptosis via increases in the cellular levels of Mcl-1, an anti-apoptotic protein of the Bcl-2 family with a rapid turnover rate. Here we have shown that inhibition of the proteasome (a) decreases the rate of Mcl-1 turnover within neutrophils and (b) significantly delays apoptosis. This led us to determine whether GM-CSF could enhance neutrophil survival by altering the rate of Mcl-1 turnover. Addition of GM-CSF to neutrophils enhanced Mcl-1 stability and delayed apoptosis by signaling pathways requiring PI3K/Akt and p44/42 Erk/Mek, because inhibitors of these pathways completely abrogated the GM-CSF-mediated effect on both Mcl-1 stability and apoptosis delay. Conversely, induction of Mcl-1 hyperphosphorylation by the phosphatase inhibitor, okadaic acid, significantly accelerated both Mcl-1 turnover and apoptosis. Neither the calpain inhibitor, carbobenzoxy-valinyl-phenylalaninal, nor the pan caspase inhibitor, benzyloxycarbonyl-VAD-fluoromethylketone, had any effect on Mcl-1 stability under these conditions. These observations indicate that profound changes in the rate of neutrophil apoptosis following cytokine signaling occur via dynamic changes in the rate of Mcl-1 turnover via the proteasome.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Apoptosis / drug effects
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Apoptosis / physiology*
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Calpain / metabolism
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Caspases / metabolism
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Cycloheximide / pharmacology
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Cysteine Endopeptidases / metabolism
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Cysteine Proteinase Inhibitors / pharmacology
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Enzyme Activation / drug effects
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Enzyme Inhibitors / pharmacology
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Granulocyte-Macrophage Colony-Stimulating Factor / pharmacology
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Granulocyte-Macrophage Colony-Stimulating Factor / physiology*
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Humans
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Leupeptins / pharmacology
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Mitogen-Activated Protein Kinases / metabolism
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Multienzyme Complexes / antagonists & inhibitors*
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Multienzyme Complexes / metabolism
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Myeloid Cell Leukemia Sequence 1 Protein
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Neoplasm Proteins / metabolism*
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Neutrophils / cytology*
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Neutrophils / drug effects
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Neutrophils / metabolism
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Phosphatidylinositol 3-Kinases / metabolism
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Phosphoinositide-3 Kinase Inhibitors
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Phosphoric Monoester Hydrolases / antagonists & inhibitors
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Proteasome Endopeptidase Complex
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Protein Serine-Threonine Kinases / metabolism
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Protein Synthesis Inhibitors / pharmacology
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Proto-Oncogene Proteins / metabolism
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Proto-Oncogene Proteins c-akt
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Proto-Oncogene Proteins c-bcl-2*
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Signal Transduction
Substances
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Cysteine Proteinase Inhibitors
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Enzyme Inhibitors
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Leupeptins
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Multienzyme Complexes
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Myeloid Cell Leukemia Sequence 1 Protein
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Neoplasm Proteins
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Phosphoinositide-3 Kinase Inhibitors
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Protein Synthesis Inhibitors
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Proto-Oncogene Proteins
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Proto-Oncogene Proteins c-bcl-2
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Granulocyte-Macrophage Colony-Stimulating Factor
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Cycloheximide
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AKT1 protein, human
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Protein Serine-Threonine Kinases
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Proto-Oncogene Proteins c-akt
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Mitogen-Activated Protein Kinases
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Phosphoric Monoester Hydrolases
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Calpain
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Caspases
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Cysteine Endopeptidases
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Proteasome Endopeptidase Complex
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benzyloxycarbonylleucyl-leucyl-leucine aldehyde