Rac1 regulates peptidoglycan-induced nuclear factor-kappaB activation and cyclooxygenase-2 expression in RAW 264.7 macrophages by activating the phosphatidylinositol 3-kinase/Akt pathway.
Previously, we found that peptidoglycan (PGN), a cell wall component of the gram-positive bacterium Staphylococcus aureus, may activate the Ras/Raf-1/extracellular signal-regulated kinase (ERK) pathway, which in turn initiates IkappaB kinases alpha/beta (IKKalpha/beta) and nuclear factor-kappaB (NF-kappaB) activation, and ultimately induces cyclooxygenase-2 (COX-2) expression in RAW 264.7 macrophages. In this study, we further investigated the roles of Rac1, phosphatidylinositol 3-kinase (PI3K), and Akt in PGN-induced NF-kappaB activation and COX-2 expression in RAW 264.7 macrophages. PGN-induced COX-2 expression was attenuated by a Rac1 dominant negative mutant (RacN17), PI3K inhibitors (wortmannin and LY 294002), and an Akt inhibitor (1L-6-hydroxymethyl-chiro-inositol2-[(R)-2-O-methyl-3-O-octadecylcarbonate]). PGN-induced PGE(2) release was also inhibited by RacN17. Treatment of RAW 264.7 macrophages with PGN caused the activation of Rac and Akt. PGN-induced Akt activation was inhibited by RacN17, LY 294002, and the Akt inhibitor. Stimulation of RAW 264.7 macrophages with PGN resulted in an increase in IKKalpha/beta phosphorylation and p65 Ser536 phosphorylation; these effects were inhibited by RacN17, LY 294002, an Akt inhibitor, and an Akt dominant negative mutant (AktDN). The PGN-induced increases in kappaB-luciferase activity were also inhibited by RacN17, wortmannin, LY 294002, an Akt inhibitor, and AktDN. Treatment of macrophages with PGN induced the recruitment of p85alpha and Rac1 to Toll-like receptor 2 (TLR2) in a time-dependent manner. These results indicate that PGN may activate the Rac1/PI3K/Akt pathway through the recruitment of p85alpha and Rac1 to TLR2 to mediate IKKalpha/beta activation and p65 phosphorylation, which in turn induces NF-kappaB transactivation, and ultimately causes COX-2 expression in RAW 264.7 macrophages.
Chen
Bing-Chang
BC
School of Respiratory Therapy, College of Medicine, Taipei Medical University, Taipei, Taiwan.
Kang
Ju-Chiun
JC
Lu
Yen-Ta
YT
Hsu
Ming-Jen
MJ
Liao
Chiao-Chun
CC
Chiu
Wen-Ta
WT
Yeh
Fu-Lung
FL
Lin
Chien-Huang
CH
D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D002460:Cell Line; D002867:Chromones / Q000494:pharmacology; D051546:Cyclooxygenase 2 / Q000096:biosynthesis*; D015232:Dinoprostone / Q000096:biosynthesis; D006801:Humans; D051550:I-kappa B Kinase / Q000378:metabolism; D007294:Inositol / Q000031:analogs & derivatives / Q000494:pharmacology; D008264:Macrophages / Q000187:drug effects / Q000378:metabolism*; D051379:Mice; D009025:Morpholines / Q000494:pharmacology; D009154:Mutation; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010457:Peptidoglycan / Q000378:metabolism* / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010766:Phosphorylation; D051057:Proto-Oncogene Proteins c-akt / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051195:Toll-Like Receptor 2 / Q000378:metabolism; D000077191:Wortmannin; D020830:rac1 GTP-Binding Protein / Q000235:genetics / Q000502:physiology*
D000730:Androstadienes; D002867:Chromones; D051546:Cyclooxygenase 2; D015232:Dinoprostone; D051550:I-kappa B Kinase; D007294:Inositol; D009025:Morpholines; D016328:NF-kappa B; D010457:Peptidoglycan; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D051057:Proto-Oncogene Proteins c-akt; D051195:Toll-Like Receptor 2; D000077191:Wortmannin; D020830:rac1 GTP-Binding Protein; C477931:1L-6-hydroxymethyl-chiro-inositol 2(R)-2-O-methyl-3-O-octadecylcarbonate; C493486:Tlr2 protein, mouse; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; C514862:Akt1 protein, mouse