Mechanism of inhibition of lipopolysaccharide-induced interferon-β production by 2-aminopurine

2-Aminopurine (2-AP) is widely used as an inhibitor for double stranded RNA-dependent protein kinase (PKR). Previously, we reported that 2-AP inhibits Toll-like receptor (TLR) ligand-induced nitric oxide production through the prevention of interferon (IFN)-beta production. In this study, we investigated the mechanisms for 2-AP inhibition of lipopolysaccharide (LPS)-induced IFN-beta production. A reporter gene assay showed that LPS-induced IFN-beta promoter, but not nuclear factor (NF)-kappa B, activation was significantly inhibited by 2-AP. IFN-beta promoter activation induced by the overexpression of Toll/interleukin-1 receptor domain-containing adaptor inducing IFN-I3 (TRIF) was significantly inhibited by 2-AP in a dose-dependent manner, while TRIF- or myeloid differentiation primary response gene 88-dependent NF-kappa B activation was not inhibited. IFN-beta promoter activation induced by expression of the downstream signaling molecules, tumor necrosis factor receptor-associated factor family member-associated NF-kappa B activator-binding kinase 1, inhibitor of NF-KB kinase i and a constitutively active mutant of interferon regulatory factor (IRF)-3, was also inhibited by 2-AP. Another PKR inhibitor harboring the imidazolooxindole structure, however, did not affect TRIF signaling molecules-induced IFN-beta promoter activation, suggesting that the inhibition of IFN-p transcription by 2-AP is independent of PKR inhibition. Further, we examined the effect of 2-AP on LPS-induced IRF-3 activation by immunoblotting. While 2-AP did not affect LPS-induced phosphorylation of IRF-3, nuclear translocation of IRF-3 was inhibited. Moreover, we revealed that LPS-induced phosphorylation of Akt, another key molecule involved in IRF-3 activation, was inhibited by 2-AP. These results suggest that 2-AP inhibits nuclear translocation of phosphorylated-IRF-3 by inhibiting Akt activation. (C) 2012 Elsevier Ltd. All rights reserved.