Peptidoglycan Induces Nuclear Factor-κB Activation and Cyclooxygenase-2 Expression via Ras, Raf-1, and ERK in RAW 264.7 Macrophages

Bing-Chang Chen,Ya-Sheng Chang,Ju-Chiun Kang,Ming-Jen Hsu,Joen-Rong Sheu,Ta-Liang Chen,Che-Ming Teng,Chien-Huang Lin

doi:10.1074/jbc.m311279200

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 IkappaB kinase alpha/beta (IKKalpha/beta), IkappaBalpha phosphorylation, IkappaBalpha degradation, and kappaB-luciferase activity. Treatment of macrophages with an NF-kappaB inhibitor (pyrrolidine dithiocarbamate), an IkappaBalpha phosphorylation inhibitor (Bay 117082), and IkappaB 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 IKKalpha/beta and kappaB-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 p85alpha 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 IKKalpha/beta and NF-kappaB activation, and ultimately induces COX-2 expression in RAW 264.7 macrophages.

Highlights

Wall component of Gram-negative bacteria, PGN induces most of the clinical manifestations of bacterial infections, including inflammation, fever, septic shock, etc. [3]
PGN-induced COX-2 Expression vation is mediated through Tolllike receptor 2 (TLR2)-dependent multiple signaling molecules including myeloid differentiation protein (MyD88), IL-1 receptor-associated kinase (IRAK), tumor necrosis factor receptor-associated factor 6 (TRAF6), nuclear factor-␬B (NF-␬B)-inducing kinase, and the IKK signaling pathway [4, 5], but little information is available about the role of Ras in regulating NF-␬B signaling following PGN stimulation
The findings of this study showed that PGN induces COX-2 expression through activation of the Ras/Raf-1/extracellular signal-regulated kinases (ERKs)/IKK␣/␤/ NF-␬B signal pathway in RAW 264.7 macrophages

Summary

Introduction

Wall component of Gram-negative bacteria, PGN induces most of the clinical manifestations of bacterial infections, including inflammation, fever, septic shock, etc. [3]. Treatment of RAW 264.7 macrophages with PGN caused time-dependent activations of Ras, Raf-1, and ERK. Our data demonstrate for the first time that PGN activates the Ras/Raf-1/ERK pathway, which in turn initiates IKK␣/␤ and NF-␬B activation, and induces COX-2 expression in RAW 264.7 macrophages.

Results

Conclusion