Abstract
Inflammatory stimulants such as bacterial endotoxin (lipopolysaccharide (LPS)) are known to induce tissue damage and injury partly through the induction of reactive oxygen species (ROS). Although it is recognized that the induction of ROS in macrophages by LPS depends upon the expression and activation of NADPH oxidase, as well as the suppression of antioxidative enzymes involved in ROS clearance, the underlying molecular mechanisms are poorly defined. In this study, we examined the contribution of the interleukin-1 receptor-associated kinase 1 (IRAK-1) to LPS-induced generation of ROS. We observed that LPS induced significantly less ROS in IRAK-1(-/-) macrophages, indicating that IRAK-1 is critically involved in the induction of ROS. Mechanistically, we observed that IRAK-1 is required for LPS-induced expression of NOX-1, a key component of NADPH oxidase, via multiple transcription factors, including p65/RelA, C/EBPbeta, and C/EBPdelta. On the other hand, we demonstrated that IRAK-1 associated with and activated small GTPase Rac1, a known activator of NOX-1 oxidase enzymatic activity. IRAK-1 forms a close complex with Rac1 via a novel LWPPPP motif within the variable region of IRAK-1. On the other hand, we also observed that IRAK-1 is required for LPS-mediated suppression of peroxisome proliferator-activated receptor alpha and PGC-1alpha, nuclear factors essential for the expression of antioxidative enzymes such as GPX3 and catalase. Consequently, injection of LPS causes significantly less plasma lipid peroxidation in IRAK-1(-/-) mice compared with wild type mice. Taken together, our study reveals IRAK-1 as a novel component involved in the generation of ROS induced by LPS.
Highlights
Reactive oxygen species (ROS)2 play a critical role in the regulation of inflammatory processes causing the oxidation of lip
Based on studies done in other cell types [5, 6], it is conceivable that LPS may contribute to the activation of NOX-1 containing NADPH oxidase via the small GTPase Rac1 in macrophages [7]
LPS treatment decreases the levels of nuclear receptor family transcription factors such as PPAR␣ and PGC-1, which are responsible for the sustained expression of antioxidative enzymes, including glutathione peroxidase and catalase (8 –11)
Summary
Western Blot Analysis—Isolation of whole cell lysates was performed as described earlier [18]. Specific siRNA-transfected cells were analyzed by Western blotting to detect the expression of IRAK-1 (bottom panel). The intensities of the bands were quantified using the Fujifilm MultiGauge ment in LPS-mediated ROS production in macrophages has software and normalized against -actin levels. We tested the hypothesis that IRAK-1 may acti- treated or treated BMDM and MEF cells using TRIzol vate the transcription of NOX-1 via NFB and other related (Invitrogen) according to the manufacturer’s protocol. We tested whether IRAK-1 may facilitate LPS-mediated activation of the small GTPase Rac, a key factor involved in the activation of NOX-1-containing NADPH oxidase. The generation of ROS following LPS challenge was measured both in vivo and in vitro using wild type (WT) and IRAK-1Ϫ/Ϫ mice and cells. The relative levels of transcripts were calculated using the ⌬⌬Ct
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