FMLP-induced formation of F-actin in HL60 cells is dependent on PI3-K but not on intracellular Ca2+, PKC, ERK or p38 MAPK.
OBJECTIVE
To further understand the mechanisms of signal transduction pathways for the formation of F-actin (polymerization of actin) and the activation of NADPH oxidase in phagocytic cells, the effects of various inhibitors on them were studied.


METHODS
Differentiated HL60 cells were studied to examine their N-formyl-methionyl-leucyl-phenylalanine (fMLP)-stimulated formation of F-actin and activation of NADPH oxidase following treatment with various inhibitors. These included a protein kinase C (PKC) inhibitor (GF 109203X), a phosphatidylinositide 3 kinase (PI3-K) inhibitor (wortmannin), an extracellular response kinase (ERK) inhibitor (PD 98059), a p38 mitogen-activated protein kinase (MAPK) inhibitor (SB 203580) and an intracellular Ca2+ -chelator (BAPTA-AM).


RESULTS
The treatment with wortmannin suppressed the formation of F-actin, with less suppression of the activation of NADPH oxidase. BAPTA-AM and GF 109203X did not attenuate the formation of F-actin but completely inhibited the activation of NADPH oxidase. PD 98059 and SB 203580 partially inhibited the activation of NADPH oxidase without influence on the formation of F-actin. Furthermore, wortmannin but not BAPTA-AM and GF 109203X inhibited the fMLP-induced activation of Akt, which is known to regulate NADPH oxidase.


CONCLUSIONS
These results suggest that the formation of F-actin is dependent on PI3-K and independent of PKC, ERK and p38 MAPK as well as the increase in intracellular Ca2+, whereas the activation of NADPH oxidase is partly dependent on ERK, p38 MAPK, Akt regulated by PI3-K, and strongly dependent on the activation of PKC and the increase in intracellular Ca2+.
Cui
Y D
YD
Department of Environmental Veterinary Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo, Japan.
Inanami
O
O
Yamamori
T
T
Niwa
K
K
Nagahata
H
H
Kuwabara
M
M
D000199:Actins / Q000378:metabolism*; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism*; D002454:Cell Differentiation / Q000187:drug effects; D002614:Chelating Agents / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D020536:Enzyme Activators / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D018922:HL-60 Cells; D006801:Humans; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology*; D019255:NADPH Oxidases / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000378:metabolism; D048051:p38 Mitogen-Activated Protein Kinases
D000199:Actins; D002614:Chelating Agents; D020536:Enzyme Activators; D004791:Enzyme Inhibitors; D017382:Reactive Oxygen Species; D009240:N-Formylmethionine Leucyl-Phenylalanine; D019255:NADPH Oxidases; D019869:Phosphatidylinositol 3-Kinases; D011493:Protein Kinase C; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D002118:Calcium