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(Journal of Leukocyte Biology. 2001;69:1060-1066.)
© 2001 by Society for Leukocyte Biology

Phosphatidylcholine-specific phospholipase C regulates activation of RAW264.7 macrophage-like cells by lipopeptide JBT3002

Fahao Zhang, Guiling Zhao and Zhongyun Dong

Department of Cancer Biology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas

Correspondence: Dr. Zhongyun Dong, Department of Cancer Biology, Box 173, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030. E-mail: zdong{at}notes.mdacc.tmc.edu

Phospholipase activities are thought to be involved in the activation of macrophages by lipopolysaccharide (LPS). Because our previous studies showed that the synthetic lipopeptide JBT3002 might activate macrophages via signaling pathways similar to those used by LPS, we investigated whether phospholipase activities are required for activation of macrophages by JBT3002. Treatment of RAW264.7 murine macrophage-like cells with JBT3002 stimulated expression of both inducible nitric oxide synthase (iNOS) and tumor necrosis factor-{alpha} (TNF-{alpha}) in a dose-dependent manner. The JBT3002-induced production of nitric oxide and TNF-{alpha} was significantly inhibited by tricyclodecan-9-yl xanthogenate (D609), a selective inhibitor of phosphatidylcholine (PC)-specific phospholipase C (PC-PLC). JBT3002-induced expression of steady-state mRNA for both iNOS and TNF-{alpha} was inhibited by D609. Cells treated with JBT3002 had greater production of diacylglycerol (DAG) in 2 min, which lasted for at least 30 min and could be blocked by D609. Activation of RAW264.7 cells was not affected by butanol, a PC-specific phospholipase D inhibitor, and treatment with JBT3002 did not affect phosphatidic acid formation. RAW264.7 cells treated with DAG analogue 1-oleoyl-2-acetyl-sn-glycerol, in the presence of interferon-{gamma}, produced TNF-{alpha}. These results suggested that activation of RAW264.7 cells by JBT3002 requires PC-PLC activity.

Key Words: signal transduction • immunomodulator • nitric oxide • tumor necrosis factor {alpha}




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