Abstract
A role for protein kinase C (PKC) isotypes is implicated in the activation of phagocytic cell functions. An antisense approach was used to selectively deplete beta-PKC, both betaI- and betaII-PKC, but not alpha-PKC, delta-PKC, or zeta-PKC in HL60 cells differentiated to a neutrophil-like phenotype (dHL60 cells). Depletion of beta-PKC in dHL60 cells elicited selective inhibition of O-2 generation triggered by fMet-Leu-Phe, immune complexes, or phorbol myristate acetate, an activator of PKC. In contrast, neither ligand-elicited beta-glucuronidase (azurophil granule) release nor adherence to fibronectin was inhibited by beta-PKC depletion. Ligand-induced phosphorylation of a subset of proteins was reduced in beta-PKC-depleted dHL60 cells. Phosphorylation of p47(phox) and translocation of p47(phox) to the membrane are essential for activation of the NADPH oxidase and generation of O-2. beta-PKC depletion had no effect on the level of p47(phox) in dHL60 cells but did significantly decrease ligand-induced phosphorylation of this protein. Furthermore, translocation of p47(phox) to the membrane in response to phorbol myristate acetate or fMet-Leu-Phe was reduced in beta-PKC-depleted cells. These results indicate that beta-PKC is essential for signaling for O-2 generation but not cell adherence or azurophil degranulation. Depletion of beta-PKC inhibited ligand-induced phosphorylation of p47(phox), translocation of p47(phox) to the membrane, and activation of O-2 generation.
Highlights
Protein kinase C (PKC)1 mediates signaling for multiple functions of phagocytic cells, neutrophils, monocytes, macrophages, and differentiated HL60 cells
An antisense strategy was used to probe a role for a specific PKC isotype in signaling for the ligandactivated responses of O2. generation and cell adherence in differentiated HL60 (dHL60) cells. -PKC was selectively depleted by an antisense strategy in dHL60 cells
Depletion of -PKC Isotypes by Antisense Treatment—To deplete -PKC, dHL60 cells were first treated with 1.3% Me2SO for 4 days to initiate differentiation, followed by two treatments with -PKC antisense oligonucleotide (AS) and the cationic lipid DMRIE-C (2.5 g/ml) at 24-h intervals
Summary
Protein kinase C (PKC) mediates signaling for multiple functions of phagocytic cells, neutrophils, monocytes, macrophages, and differentiated HL60 (dHL60) cells. Ligands such as the chemotactic peptide fMet-Leu-Phe and phagocytic stimuli (immune complexes) trigger responses that include O2. Generation, degranulation, adherence, and actin filament assembly [1,2,3,4,5,6,7,8] These functions are essential for the microbicidal activity of phagocytic cells and are proinflammatory. Depletion of -PKC reduced ligand-induced phosphorylation of a subset of proteins including p47phox and reduced translocation of p47phox to the membrane, concordant with a selective role for -PKC in signaling for assembly of an active NADPH oxidase
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