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Published online before print August 24, 2004

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© by The Society for Leukocyte Biology
Journal of Leukocyte Biology, doi:10.1189/jlb.0104048

Received for publication January 28, 2004.
Revised June 30, 2004.
Accepted for publication July 23, 2004.

Article

Short-term delay of Fas-stimulated apoptosis by GM-CSF as a result of temporary suppression of FADD recruitment in neutrophils: evidence implicating phosphatidylinositol 3-kinase and MEK1-ERK1/2 pathways downstream of classical protein kinase C

Yasuko Kotone-Miyahara ^*, Kouhei Yamashita ^*, Kyung-Kwon Lee , Shin Yonehara , Takashi Uchiyama ^*, Masataka Sasada , and Atsushi Takahashi ^*@

*Department of Hematology and Oncology, Graduate School of Medicine,Graduate School of Biostudies, andCollege of Medical Technology, Kyoto University, Japan

^@ To whom correspondence should be addressed. E-mail: atakahashi{at}ca.wakwak.com.

	Abstract

Granulocyte macrophage-colony stimulating factor (GM-CSF) inhibits Fas-induced apoptosis of neutrophils. However, the exact step in the apoptotic pathway blocked by GM-CSF remained unclear. Here, we found that pretreatment of neutrophils with GM-CSF inhibits the recruitment of Fas-associated protein with death domain (FADD) to Fas, abolishing the formation of the death-inducing signaling complex required for Fas-induced apoptosis. Two-dimensional electrophoresis revealed that GM-CSF modifies the ratio of FADD subspecies. These GM-CSF-triggered changes were abrogated, and Fas-induced apoptosis was restored by an inhibitor of classical protein kinase C (PKC), Gö6976, and by the combination of a phosphatidylinositol 3-kinase (PI-3K) inhibitor, LY294002, and an inhibitor of mitogen-activated protein kinase kinase (MEK)1/extracellular signal-regulated kinase (ERK), PD98059. Gö6976 blocked GM-CSF-elicited phosphorylation of Akt/PKB and ERK1/2. These results indicated that GM-CSF suppresses Fas-induced neutrophil apoptosis by inhibiting FADD binding to Fas, through redundant actions of PI-3K and MEK1-ERK1/2 pathways downstream of classical PKC.

Key Words: death-inducing signaling complex • Gö6976 • LY294002 • PD98059 • caspase-8