Abstract
Stimulation of neutrophils with chemotactic peptide induces actin reorganization, formation of actin-rich protrusions, and development of polarity. Shape changes and actin polymerization can also be induced by phorbol ester-mediated direct activation of protein kinase C (PKC). We have investigated the role of cholesterol in stimulus-dependent motile events and in activation of signaling pathways in neutrophil-like differentiated HL-60 cells. Depletion of plasma membrane cholesterol using methyl-β-cyclodextrin (MβCD) prevented chemotactic peptide and phorbol ester-induced shape changes and increases in cytoskeletal actin. Cholesterol depletion almost completely suppressed chemotactic peptide-mediated activation of p42/44 mitogen-activated protein kinase (MAPK). Phosphorylation of protein kinase B on Thr-308, which is indicative of activation of phosphatidylinositol 3-kinase, was in contrast only partially inhibited. Stimulus-mediated membrane recruitment of different PKC isoforms was differentially affected by treatment of cells with MβCD. Membrane recruitment of PKCα induced by chemotactic peptide or phorbol ester was suppressed, whereas that of PKCβII was only partially affected. Membrane association of PKCδ was almost insensitive to cholesterol depletion. In summary, our results implicate an important role of cholesterol-containing lipid microdomains (rafts) especially in chemotactic peptide-induced activation of MAPK pathways and in chemotactic peptide- and phorbol ester-mediated activation of PKCα.
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