Abstract

In this study, we investigated the signaling pathway involved in cyclooxygenase-2 (COX-2) expression caused by peptidoglycan (PGN), a cell wall component of the Gram-positive bacterium Staphylococcus aureus, in RAW 264.7 macrophages. PGN caused dose- and time-dependent increases in COX-2 expression, which was attenuated by a Ras inhibitor (manumycin A), a Raf-1 inhibitor (GW 5074), and an MEK inhibitor (PD 098059). Treatment of RAW 264.7 macrophages with PGN caused time-dependent activations of Ras, Raf-1, and ERK. The PGN-induced increase in Ras activity was inhibited by manumycin A. Raf-1 phosphorylation at Ser-338 by PGN was inhibited by manumycin A and GW 5074. The PGN-induced increase in ERK activity was inhibited by manumycin A, GW 5074, and PD 098059. Stimulation of cells with PGN activated IκB kinase α/β (IKKα/β), IκBα phosphorylation, IκBα degradation, and κB-luciferase activity. Treatment of macrophages with an NF-κB inhibitor (pyrrolidine dithiocarbamate), an IκBα phosphorylation inhibitor (Bay 117082), and IκB protease inhibitors (L-1-tosylamido-2-phenylethyl chloromethyl ketone and calpain inhibitor I) all inhibited PGN-induced COX-2 expression. The PGN-mediated increase in the activities of IKKα/β and κB-luciferase were also inhibited by the Ras dominant negative mutant (RasN17), manumycin A, GW 5074, and PD 098059. Further studies revealed that PGN induced the recruitment of p85α and Ras to Toll-like receptor 2 in a time-dependent manner. Our data demonstrate for the first time that PGN activates the Ras/Raf-1/ERK pathway, which in turn initiates IKKα/β and NF-κB activation, and ultimately induces COX-2 expression in RAW 264.7 macrophages.

Original languageEnglish
Pages (from-to)20889-20897
Number of pages9
JournalJournal of Biological Chemistry
Volume279
Issue number20
DOIs
Publication statusPublished - May 14 2004

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Peptidoglycan
Macrophages
Cyclooxygenase 2
Chemical activation
Phosphorylation
Luciferases
Toll-Like Receptor 2
MAP Kinase Signaling System
Mitogen-Activated Protein Kinase Kinases
Gram-Positive Bacteria
Cellular Structures
Protease Inhibitors
Ketones
Cell Wall
Staphylococcus aureus
Bacteria
Phosphotransferases
Cells
manumycin

ASJC Scopus subject areas

  • Biochemistry

Cite this

@article{7b1ea6a9c26b4fa3a38fe38e6e5cd441,
title = "Peptidoglycan induces nuclear factor-κB activation and cyclooxygenase-2 expression via Ras, Raf-1, and ERK in RAW 264.7 macrophages",
abstract = "In this study, we investigated the signaling pathway involved in cyclooxygenase-2 (COX-2) expression caused by peptidoglycan (PGN), a cell wall component of the Gram-positive bacterium Staphylococcus aureus, in RAW 264.7 macrophages. PGN caused dose- and time-dependent increases in COX-2 expression, which was attenuated by a Ras inhibitor (manumycin A), a Raf-1 inhibitor (GW 5074), and an MEK inhibitor (PD 098059). Treatment of RAW 264.7 macrophages with PGN caused time-dependent activations of Ras, Raf-1, and ERK. The PGN-induced increase in Ras activity was inhibited by manumycin A. Raf-1 phosphorylation at Ser-338 by PGN was inhibited by manumycin A and GW 5074. The PGN-induced increase in ERK activity was inhibited by manumycin A, GW 5074, and PD 098059. Stimulation of cells with PGN activated IκB kinase α/β (IKKα/β), IκBα phosphorylation, IκBα degradation, and κB-luciferase activity. Treatment of macrophages with an NF-κB inhibitor (pyrrolidine dithiocarbamate), an IκBα phosphorylation inhibitor (Bay 117082), and IκB protease inhibitors (L-1-tosylamido-2-phenylethyl chloromethyl ketone and calpain inhibitor I) all inhibited PGN-induced COX-2 expression. The PGN-mediated increase in the activities of IKKα/β and κB-luciferase were also inhibited by the Ras dominant negative mutant (RasN17), manumycin A, GW 5074, and PD 098059. Further studies revealed that PGN induced the recruitment of p85α and Ras to Toll-like receptor 2 in a time-dependent manner. Our data demonstrate for the first time that PGN activates the Ras/Raf-1/ERK pathway, which in turn initiates IKKα/β and NF-κB activation, and ultimately induces COX-2 expression in RAW 264.7 macrophages.",
author = "Bing-Chang Chen and Chang, {Ya Sheng} and Kang, {Ju Chiun} and Ming-Jen Hsu and Joen-Rong Sheu and Ta-Liang Chen and Teng, {Che Ming} and Chien-Huang Lin",
year = "2004",
month = "5",
day = "14",
doi = "10.1074/jbc.M311279200",
language = "English",
volume = "279",
pages = "20889--20897",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "20",

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TY - JOUR

T1 - Peptidoglycan induces nuclear factor-κB activation and cyclooxygenase-2 expression via Ras, Raf-1, and ERK in RAW 264.7 macrophages

AU - Chen, Bing-Chang

AU - Chang, Ya Sheng

AU - Kang, Ju Chiun

AU - Hsu, Ming-Jen

AU - Sheu, Joen-Rong

AU - Chen, Ta-Liang

AU - Teng, Che Ming

AU - Lin, Chien-Huang

PY - 2004/5/14

Y1 - 2004/5/14

N2 - In this study, we investigated the signaling pathway involved in cyclooxygenase-2 (COX-2) expression caused by peptidoglycan (PGN), a cell wall component of the Gram-positive bacterium Staphylococcus aureus, in RAW 264.7 macrophages. PGN caused dose- and time-dependent increases in COX-2 expression, which was attenuated by a Ras inhibitor (manumycin A), a Raf-1 inhibitor (GW 5074), and an MEK inhibitor (PD 098059). Treatment of RAW 264.7 macrophages with PGN caused time-dependent activations of Ras, Raf-1, and ERK. The PGN-induced increase in Ras activity was inhibited by manumycin A. Raf-1 phosphorylation at Ser-338 by PGN was inhibited by manumycin A and GW 5074. The PGN-induced increase in ERK activity was inhibited by manumycin A, GW 5074, and PD 098059. Stimulation of cells with PGN activated IκB kinase α/β (IKKα/β), IκBα phosphorylation, IκBα degradation, and κB-luciferase activity. Treatment of macrophages with an NF-κB inhibitor (pyrrolidine dithiocarbamate), an IκBα phosphorylation inhibitor (Bay 117082), and IκB protease inhibitors (L-1-tosylamido-2-phenylethyl chloromethyl ketone and calpain inhibitor I) all inhibited PGN-induced COX-2 expression. The PGN-mediated increase in the activities of IKKα/β and κB-luciferase were also inhibited by the Ras dominant negative mutant (RasN17), manumycin A, GW 5074, and PD 098059. Further studies revealed that PGN induced the recruitment of p85α and Ras to Toll-like receptor 2 in a time-dependent manner. Our data demonstrate for the first time that PGN activates the Ras/Raf-1/ERK pathway, which in turn initiates IKKα/β and NF-κB activation, and ultimately induces COX-2 expression in RAW 264.7 macrophages.

AB - In this study, we investigated the signaling pathway involved in cyclooxygenase-2 (COX-2) expression caused by peptidoglycan (PGN), a cell wall component of the Gram-positive bacterium Staphylococcus aureus, in RAW 264.7 macrophages. PGN caused dose- and time-dependent increases in COX-2 expression, which was attenuated by a Ras inhibitor (manumycin A), a Raf-1 inhibitor (GW 5074), and an MEK inhibitor (PD 098059). Treatment of RAW 264.7 macrophages with PGN caused time-dependent activations of Ras, Raf-1, and ERK. The PGN-induced increase in Ras activity was inhibited by manumycin A. Raf-1 phosphorylation at Ser-338 by PGN was inhibited by manumycin A and GW 5074. The PGN-induced increase in ERK activity was inhibited by manumycin A, GW 5074, and PD 098059. Stimulation of cells with PGN activated IκB kinase α/β (IKKα/β), IκBα phosphorylation, IκBα degradation, and κB-luciferase activity. Treatment of macrophages with an NF-κB inhibitor (pyrrolidine dithiocarbamate), an IκBα phosphorylation inhibitor (Bay 117082), and IκB protease inhibitors (L-1-tosylamido-2-phenylethyl chloromethyl ketone and calpain inhibitor I) all inhibited PGN-induced COX-2 expression. The PGN-mediated increase in the activities of IKKα/β and κB-luciferase were also inhibited by the Ras dominant negative mutant (RasN17), manumycin A, GW 5074, and PD 098059. Further studies revealed that PGN induced the recruitment of p85α and Ras to Toll-like receptor 2 in a time-dependent manner. Our data demonstrate for the first time that PGN activates the Ras/Raf-1/ERK pathway, which in turn initiates IKKα/β and NF-κB activation, and ultimately induces COX-2 expression in RAW 264.7 macrophages.

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