Impact of cholesterol depletion on shape changes, actin reorganization, and signal transduction in neutrophil-like HL-60 cells.
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-beta-cyclodextrin (MbetaCD) 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 MbetaCD. Membrane recruitment of PKCalpha induced by chemotactic peptide or phorbol ester was suppressed, whereas that of PKCbetaII was only partially affected. Membrane association of PKCdelta 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 PKCalpha.
Niggli
Verena
V
Department of Pathology, University of Bern, CH-3010 Bern, Switzerland. verena.niggli@pathology.unibe.ch
Meszaros
Anna Veronique
AV
Oppliger
Christoph
C
Tornay
Sophie
S
D000199:Actins / Q000378:metabolism / Q000648:ultrastructure; D048429:Cell Size; D002784:Cholesterol / Q000378:metabolism / Q000502:physiology*; D003505:Cyclodextrins; D003599:Cytoskeleton / Q000378:metabolism* / Q000648:ultrastructure; D018922:HL-60 Cells; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism; D021962:Membrane Microdomains / Q000502:physiology; D009504:Neutrophils / Q000166:cytology / Q000378:metabolism* / Q000648:ultrastructure*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D015398:Signal Transduction; D047392:beta-Cyclodextrins
D000199:Actins; D002784:Cholesterol; D003505:Cyclodextrins; D007527:Isoenzymes; D019869:Phosphatidylinositol 3-Kinases; D011493:Protein Kinase C; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin