IκB Kinase Interacting Protein Negatively Regulates Production Of Proinflammatory Cytokines By Targeting IKKβ

In the human body, innate immunity plays a very important role in defending against microbial infection. Recognition of microbial conserved pathogen-associated molecule patterns (PAMP) by corresponding pattern recognition receptor (PRR) initiates signal transduction pathways, leading to the production of various kinds of cytokines. There are several families of PRRs such as Toll-like receptors (TLRs), NOD-like receptors (NLRs), RIG-I-like receptors (RLRs) et al. TLRs play a pivotal in sensing bacteria.TLRs could sense ligands such as LPS, LTA, PGN et al.Then initiates a’cascade of signaling pathway by recruiting many adaptors such as MyD88,TRAF6, IKKβ to activate the transcripters including NF-κB, which bind to response elements to produce a large amount of proinflammatory cytokines. TLR4and TLR2acts as important component of innate immunity that can be stimulated by LPS and PGN respectively, leading to the activation of NF-κB to produce proinflammatory cytokines, chemokines and type I interferons (IFNs), including IL-6and TNF-a.These responses activate innate immunity to eliminate viral and bacterial infection. However, uncontrolled activation may result in autoimmune and inflammatory diseases. Therefore, it is very important to strictly control NF-κB activation and secretion of proinflammatory cytokines, chemokines and type I interferons.IKIP (I kappaB kinase interacting protein) that is a novel human gene, IKIP was found to bind to IKKβ by yeast two-hybrid.There are three IKIP transcripts which are generated by different splicing and alternative exon usages. The expression of IKIP can be enhanced by X-irradiation dependent on p53. Moreover, IKIP could promote apoptosis when transfected into endothelial cells. However, we have no idea of the precise functions of IKIP in the regulation of NF-κB immune response, so we investigated the function and the underlying mechanism of IKIP in TLR signaling in this study.Objectives:1. Measuring the expression of IKIP in macrophages stimulated with LPS and PGN.2. Investigating the role of IKIP in production of proinflammatory cytokines upon LPS and PGN stimulation.3. Revealing the underlying mechanism of IKIP to regulate NF-κB activation.Methods:1. The expression of IKIP in LPS, PGN stimulated macrophages.Firstly, peritoneal macrophages were prepared and stimulated by LPS and PGN for0、2、4、8、12、24h, extract the mRNA of the cells,then IKIP was measured by semi-quantitative PCR.With the same methods to get the macrophages, stimulate the macrophages by LPS and PGN for different times, isolate total protein, then IKIP was measured by western-blot.2. Investigate the role of IKIP in the production of pro inflammatory cytokines2.1Test the efficiency of the siRNA of IKIPFlag-IKIP vectors were transfected into HEK293cells, after24hours, siRNA was transfected into HEK293cells, after24hours, isolate total protein, western-blot were performed to detect the efficiency of the siRNA of IKIP to knockdown IKIP experssion.2.2Measure the expression of Proinflammatory cytokinesIKIP siRNA was transfected into macrophages, followed stimulation with LPS and PGN for0、4、8h. Proinflammatory cytokines such as IL-6, TNF-α, IFN-βwere measured by RT-PCR. Flag-IKIP vectors were transfected into Hela and HEK293-TLR2cerlls, followed stimulation with LPS and PGN for0、4、8h respectively. extract the mRNA.Pro inflammatory cytokines such as IL-6, TNF-α, IFN-β were measured by RT-PCR.3. Analyze the mechanism that IKIP affects NF-κB activation.3.1Check the effect of IKIP on the activation of NF-κB through Dual Luciferase Reporter Gene Assay.Various adaptors such as MyD88, TRIF, IKKβ were transfected into HEK293cells together with NF-κB-luc,24hours later, analyze the activation of NF-κB by Dual Luciferase Reporter Gene Assay.IFN-β-luc was also transfected into HEK293cells together with MyD88, TRIF,24hour later, analyze the activation of IFN-β by Dual Luciferase Reporter Gene Assay.3.2Identify the effect of IKIP in the NF-κB signaling.IKIP siRNA was transfected into macrophages, then stimulated with LPS for0、30min,60min. Extract the protein and the expression of pIκBα, pP65, IκBα P65were measured by western-blot.4Determine the undeflying mechanism of IKIP in NF-κB signaling.4.1Determine the interaction between IKIP and IKKβHA-IKKβ and Flag-IKIP vectors were transfected into Hela cells,24hour later, stimulated with LPS, The interaction was measured with IP followed by western-blot.4.2Determine the effect of IKIP on the IKK kinase formation.Flag-IKIP, HA-IKKβ, HA-IKKa and HA-IKKy were transfected into Hela cells, for24h, followed stimulation with LPS. The interaction between IKKy and IKK-β was measured with IP followed by western-blot.Results:1. The increased expression of IKIP in LPS and PGN activated macrophage.Compared with controls, The expression of IKIP was significantly increased after LPS and PGN stimulation in macrophages for0、2、4、8、12、24h at both mRNA level and protein level as measured by RT-PCR and western-blot,respectively.2. IKIP inhibited production of proinflammatory cytokines2.1IKIP siRNA knockdown the expression of IKIP efficiently.Flag-IKIP vectors were transfected into HEK293cells,24hours later, IKIP siRNA was transfected into HEK293cells, for another24hours, then extract total protein,IKIP expression was measured by western-blot. IKIP siRNA could inhibit the expression of IKIP efficiently.2.2IKIP inhibits LPS and PGN-induced production of Proinflammatory cytokinesIKIP siRNA was transfected into macrophages,then stimulated with LPS and PGN for0、4、8h. Production of proinflammatory cytokines was measured by RT-PCR. Compared to the control, production of IL-6and TNF-a were greatly increased after siRNA transfection.But, expression of IFN-β was not changed.Flag-IKIP vectors were transfected into Hela and HEK293-TLR2cells, Stimulated with LPS and PGN for4,8h respectively, Production of proinflammatory cytokines was measured RT-PCR.Compared to the control, production of IL-6and TNF-a were greatly decreased by Flag-IKIP transfection.But, expression of IFN-β was not changed.3. IKIP inhibited NF-κB activation3.1IKIP inhibits MyD88and IKK-β-induced NF-κB activationNF-κB-luc, IFN-β-luc reporter plasmid and various adaptors such as MYD88, IKK-β,TRIF were transfected into HEK293cells together with IKIP expression plasmid,the activation of NF-κB and IFN-P were measured by dual luciferase assays. NF-κB activation was inhibited by IKIP, while the activation of IFN-β was not affected.3.2IKIP affected the phosphorylation of IκBα and p65IKIP siRNA was transfected into macrophages, then stimulate with LPS for0、30min、60min. pIκBα and pP65are detected with western-blot. The level of pIκBα and pP65were substantially increased upon siRNA knockdown of IKIP expression.4. IKIP regulated NF-κB signaling by inhibiting IKKy binding to IKKβ 4.1IKIP bound to IKKβHA-IKKβ and Flag-IKIP were transfected into HELA, then stimulate with LPS for2hours, western-blot showed that IKIP bound to IKKp.4.2IKIP inhibited NF-κB activation by competion IKKy binding to IKKβ.Flag-IKIP, HA-IKKβ, HA-IKKa and HA-IKKγ were transfected into Hela for24h with stimulation of LPS. IP and western blot showed that IKIP bound to IKKβ specifically.Then ever-increasing dosage of IKIP and the same dosage of IKKy, IKK-β were transfected into Hela, IKKy binding to IKKβ was inhibited by IKIP.Innovation and significances:1. For the first time, we demonstrated that:1) IKIP is a negative regulator for NF-κB signaling.2) IKIP inhibits NF-κB signaling by competing IKKβ binding with IKKy2. Our studies provided new experimental evidence for negative regulation of NF-κB signaling, and may provide novel idea for diagnosis and therapy of inflammation diseases.