ASK1 in peptidoglycan-induced COX-2 expression in RAW 264.7 macrophages: Involvement of C/EBPβ

Ming-Jen Hsu2, Chia-Kai Chang2, Bing-Chang Chen3, Chien-Huang Lin1
1Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, Taipei, Taiwan, 2Department of Pharmacology, Taipei Medical University, Taipei, Taiwan, 3School of Respiratory Therapy, Taipei Medical University, Taipei, Taiwan

In this study, we investigated the role of ASK1 in PGN-induced C/EBPβ activation and COX-2 expression in macrophages. The PGN-induced COX-2 expression was attenuated by ASK1DN, JNK1 DN, JNK2DN, a JNK inhibitor (SP600125), and an AP-1 inhibitor (curcumin). PGN caused ASK1 Ser967 dephosphorylation, dissociation of ASK1 and 14-3-3, and a subsequent increase in ASK1 activity in time-dependent manners. In addition, PGN increased the activity of protein phosphatase 2A (PP2A). Suppression of PP2A activity by the specific inhibitor, okadaic acid, markedly inhibited PGN-induced ASK1 Ser967 dephosphorylation and COX-2 expression. Treatment of cells with PGN caused the activation of JNK; this effect was inhibited by ASK1DN. PGN caused increases in c-jun phosphorylation, the formation of an AP-1-specific DNA-protein complex, and AP-1-luciferase activity. The PGN-mediated increase in AP-1-luciferase activity was inhibited by SP600125 and curcumin. Treatment of macrophages with PGN caused the increases in C/EBPβ expression and the binding activity of C/EBPβ to the C/EBP binding sequence of COX-2 promoter; these effects were inhibited by ASK1DN, SP600125, and curcumin. Furthermore, PGN-induced COX-2-luciferase activity was attenuated in cells transfected with the COX-2 reporter construct possessing the C/EBP binding site mutation. Our data demonstrate for the first time that PGN might activate ASK1 through PP2A activation to cause JNK/AP-1 activation, which in turn induces C/EBPβ expression and subsequent activation, and ultimately results in COX-2 expression in RAW 264.7 macrophages.