In this study, we investigated the effect of tunicamycin on the production of pro-inflammatory molecules in RAW264.7 macrophage cells in response to lipopolysaccharide ([PS) and Toll-like receptor (TLR) agonists. Tunicamycin caused a reduction in [PS-induced nitric oxide (NO) production and expression of inducible NO synthase (iNOS), cyclooxygenase-2 (COX-2), interleukin-1 beta (IL-1 beta) and tumor necrosis factor alpha (TNE-alpha). In contrast, other ER stress-inducing chemicals, such as A23187 and thapsigargin (TG), increased [PS-induced COX-2 expression and had no effect on [PS-induced iNOS, TNFa or IL-1 beta expression. Furthermore, the inhibitory effect of tunicamycin on [PS-induced inflammation was not influenced by salubrinal, an ER stress inhibitor, suggesting that the anti-inflammatory effect of tunicamycin is independent of ER stress. Tunicamycin also inhibited the expression of inflammatory molecule mRNAs induced by stimulation of TLR2 (with lipoteichoic acid) or TLR3 (with polyinosinic: polycytidylic acid), which do not require myeloid differentiation protein-2 (IV1D2) for their activation. Moreover, inhibition of [PS-induced iNOS expression was not inhibited by castanospermine, another N-glycosylation inhibitor, suggesting that the inhibitory effect of tunicamycin on [PS-induced iNOS induction is likely independent of IV1D2 N-glycosylation. Tunicamycin inhibited nuclear factor-kappaB (NE-kappa B) activity by suppressing [PS-induced nuclear translocation of p50 and subsequent DNA binding of p50 and 05 to the NE-kappa B site of the iNOS promoter. Tunicamycin also inhibited the transcriptional activity of a CAMP-response element (CRE) reporter, possibly by inhibiting c-Jun activation. Therefore, we conclude that tunicamycin represses TLR-induced inflammation through suppression of NE KB and CRE activity via a mechanism that is independent of ER stress and N-glycosylation. (C) 2013 Published by Elsevier B.V.
