| Background and purpose:LPS?endotoxin/lipopolysaccharide?and PGN?peptidoglycan?or other pathogen-associated molecular patterns?PAMPs?,derived from gram-negative or positive bacteria,viruses and other pathogenetic microbials,is recognized by transmenbrane pattern-recognition receptors?PRRs?such as toll-like receptors?TLRs?.Combination of LPS with TLR4,activates a series of intracellular signaling pathways,ending with production of various cytokines and chemokines,ultimately leading to systemic inflammatory response syndrome?SIRS?and sepsis.LPS,a common factor leading to sepsis,is comprised of lipid A,core polysaccharide and O-antigen.LPS binds to TLR4,recruits MyD88-dependent and TRAM/TRIF-dependent signaling pathways,eventually activates NF-?B?nuclear factor kappa B?and IRF-3?interferon regulatory factor 3?,resulting in synthesis and release of TNF-??tumor necrosis factor?,IL-6?interleukin-6?.Anti-malaria drug CQ?chloroquine?,as a alkaline drug,suppresses acidification and maturation of endosomes and lysosomes.We have researched its anti-inflammation since several years ago.Our earlier researches have found that CQ significantly improved overall survival rate of sepsis mice modal,as well as negative control the release of TNF-?and IL-6 in LPS-stimulated hPBMC?human peripheral blood mononuclear cell?and mouse macrophages ANA-1 cells.This is supported by other research papers which also confirmed that CQ increased overall survival rate of sepsis mouse,reduced renal injury and spleen cell apoptosis,which indicate us CQ has a relative broad spectrum of anti-inflammatory activity.In addition,we have found that CQ affected internalization and dissociation of LPS-TLR4complex,altered expressions of transmembrane receptor TLR4.Furthermore,we also found that CQ dose-dependently increased SENP6 protein expressions via miR-669n,which played a role in the CQ-inhibited LPS-induced release of pro-inflammatory mediators like TNF-?,IL-6 and IFN-?.SUMO?small ubiquitin-related modifier?,a ubiquitin-like molecule,binds to residue lysine of the substrate in sequence of various enzymes one by one,the process which is called SUMOylation.SUMOylation,similar to ubiquitination,as one of the post-translational modifications,regulates protein subcellular distributions and spatial structure,involved in the NF-?B and STAT signal transduction.SUMOylation is a reversible reaction,thus SUMO can be removed from the substrate protein under the catalysis of ULP?ubiquitin-like modifier protease?/deSUMOylases.SENP6 is an enzyme which mediates the deSUMOylation of IRF3 and IRF7 in order to suppress transcription of IFNs.We found that CQ increased SENP6 protein expression,which induced deSUMOylation of NF-?B,to negatively regulate the release of TNF-?and IFN-?.Given that SUMO and ubiquitin has a higher degree of homology in terms of molecular construction and similarity in their modification process,though they compete for binding site,plus that CQ inhibits LPS-TLR4 pathways through up-regulation of SENP6 expressions,it seems to be reasonable that CQ might attenuate LPS-TLR4 pathways via deubiquitinases?DUBs??Ubiquitin is a small protein consisting of only 76 amino acids,covalently attached to lysine residues of substrate protein under the actions of E1,E2 and E3,which executes diverse biological functions.Ubiquitin-proteasome system?UPS?is made up of ubiquitin,E1,E2,E3 and 26S proteasome,the main lysosome-independent protein degradation pathway in eukaryotic cells,which is necessary for inflammation,signal transduction,apoptosis and other biological activities.There exists eight different topological structure and functions of poly-ubiquitin chains due to various E2 and E3.Like SUMOylation,ubiquitination is also a reversible reaction,its reverse process of deubiquitination through a family of DUBs is the dissociation of ubiquitin chains from substrate proteins,which plays a critical role in many signaling pathways and terminates excessive immune responses.The human genome encode five types of DUBs,namely OTU?ovarian tumor-type proteases?,USP?ubiquitin-specific proteases?,UCH?ubiquitin C-terminal hydrolases?,MJD?Machado-Joseph disease proteases?and metalloproteases,the first two got more attention.In addition,internalization and transportation of cell surface receptors is also participated in the intracellular signaling transduction.Combination of LPS with TLR4,entered into the early endosome,late endosome gradually and finally fused with lysosome,accompanied with acidification and maturation.During this dynamic process,endosomes contain a special half scabbard named ESCRT?endosomal sorting complex required for transport,ESCRT?,which is responsible for selecting ubiquitin-marked protein for lysosome degradation.Other studies have confirmed that interaction of ESCRT with E3 or DUB affects internalization and transportation of surface receptors,further regulates intracellular signal transduction.Lysosomes are mainly involved in the catabolism and recycle of internalized materials,while proteasomes is vital for degradation of abnormal proteins.Since CQ has a broad spectrum of anti-inflammatory activity,coupled with we have observed such a phenomenon earlier that LPS-TLR4 co-localization disappeared as well as a decreased expression of membrane TLR4 expression in CQ-pretreated and LPS-stimulated Raw264.7 cells,considering CQ is an acidification inhibitor,it seems its anti-inflammatory property has nothing to do with lysosomes.Thus we guess whether CQ suppresses LPS-induced macrophage activation via ubiquitin-proteasome pathways?Above all,the present research project takes the mechanism of CQ inhibits LPS-induced macrophage activation as a pointcut,from the effect of ubiquitin-proteasome pathway on blockade of inflammatory results,to demonstrate a possible molecular mechanism of CQ negative regulation of the LPS-TLR4 pathways.Therefore,we firstly screened the expression changes of different DUBs,and further explored the possible mechanism of zinc finger protein A20 and ubiquitin-specific protease USP25 influenced TLR4-mediated downstream pathways through altering the activity of middle molecules.Methods:1.Role of CQ in OTU and USP deubiquitinases mRNA expressions.Raw264.7 cells were pretreated with 20ug/ml CQ for 1h,then stimulated with 100ng/ml LPS for 2h.Total RNA was extracted,and real-time PCR was performed according to the manufacturer's instructions.The comparative Ct method with arithmetic formulae(2-Ct)was used to determine relative quantification of gene expression.2.Zinc finger protein A20 regulated inhibition of LPS-TLR4 pathways induced by CQ.2.1 CQ significantly increased A20 expressions in Raw264.7 cells.Raw264.7 cells were treated with gradient concentrations of CQ for 24h;total RNA or whole cell extracts were extracted and real-time PCR and western blot?WB?were performed to investigate A20 mRNA and protein expression respectively.Later,cells were treated with LPS with or without CQ.Total RNA or whole cell extracts were extracted and analysed to determine the role of CQ on A20 expressions.2.2 SiRNA-mediated A20 silencing resulted in blockade of attenuation of LPS-TLR4pathways mediated by CQ.?1?Three different siRNA designed and synthesized by company were transfected into Raw264.7 cells for 24-48 h,PCR and WB were used to evaluate A20 expressions in order to select the optimal siRNA and build the maximal interference model.?2?CQ pretreated for 1h and LPS stimulated for 4h in siRNA-transfected cells,TNF-?and IL-6 mRNA expressions were detected by PCR,and the phosphorylation level of p38was detected by WB.?3?After A20 expression was reduced by siRNA,cells were pretreated with LPS for 1h in the presence or absence of CQ,and translocation of NF-?B p65 and IRF3 were detected by laser scanning confocal microscope.3.Ubiquitin specific protease USP25 down-regulated CQ-induced attenuation of LPS-TLR4 pathways.3.1 CQ increased USP25 expressions in monocytes/macrophages as well as LPS-activated monocytes/macrophages.Mouse macrophage cell line Raw264.7 and human monocyte cell line THP-1 cells were treated with gradient concentrations of CQ for 24h,USP25 mRNA expression was analysed.Simultaneously,USP25 protein expression was assayed using WB in Raw264.7cells.In addition,20 ug/ml CQ was added in LPS-challenged cells and USP25 expressions were measured by PCR and WB respectively.3.2 The specific siRNA-mediated USP25 interference reduced CQ-induced inhibition of LPS-TLR4 pathways.?1?Specific siRNA bought from company were transfected into Raw264.7 cells for24-48 h,PCR and WB were used to evaluate USP25 expressions.?2?SiRNA was transfected into Raw264.7 cells,subsequently CQ was pretreated for1h and LPS was stimulated for 4 or 24 hours,TNF-?,IL-6 and IFN-?mRNA expressions and release levels in the supernatant were detected by PCR and ELISA.?3?After transfection with siRNA into Raw264.7 cells,CQ were pretreated for 1h and LPS were stimulated for 4h,TLR4 downstream signaling molecules like I?B or NF-?B and MAPK pathway p38,ERK and JNK as well as phosphorylation levels of these protein were measured using WB.?4?After USP25 expression was reduced by siRNA,cells were challenged with LPS for 4 or 12 hours in the presence or absence of CQ.The activation levels of NF-?B p65 and p50 subunits were detected by appropriate NF-?B ELISA kits.?5?Raw264.7 cells were transfected with USP25 siRNA,then were stimulated with LPS for 1 h with or without CQ.The subcellular distributions of NF-?B p65 and IRF3 were detected through confocal imaging method.4.Synergy or antagonist interactions of A20 with USP25 regulated attenuation of LPS-induced macrophage activation mediated by CQ.?1?Raw264.7 cells were co-transfected with A20 and USP25 siRNA,then cells were pretreated with CQ for 1 h,later activated with LPS for 24 hours.The TNF-?,IL-6 and IFN-?levels in the supernatant were analysed using ELISA.?2?Raw264.7 cells were co-transfected with A20 and USP25 siRNA,then cells were challenged with LPS for 4 hours with or without CQ.The phosphorylation of MAPK p38were detected by WB.?3?Raw264.7 cells were co-transfected with A20 and USP25 siRNA,followed by activating with LPS for 1 h in the presence or absence of CQ.The nuclear translocation of NF-?B p65 and IRF3 was assayed through confocal imaging method.5.Statistical analysisAll experiments were performed at least three times and data were presented as meanąstandard deviation?S.D.?.Statistical significance between groups was determined by an analysis of variance?ANOVA?test followed by Bonferroni test,with a value of p<0.05considered to be statistically significant.Results:1.CQ increased some OTU and USP deubiquitinases mRNA expressions.PCR results showed that CQ significantly up-regulated A20 and OTULIN mRNA expressions.In addition,CQ also enhanced mRNA levels of some USPs deubiquitinases.2.A20 regulated inhibition of LPS-TLR4 pathways induced by CQ.?1?CQ dose-dependently increased A20 mRNA and protein expressions in Raw264.7cells whether they are stimulated by LPS or not.?2?PCR and WB results showed that siRNA1 of the three siRNA sequences had the optimal interference,so we chose siRNA1 to perform subsequent experiments.?3?The increased TNF-?and IL-6 mRNA expressions,enhanced phosphorylation of p38,as well as nuclear accumulation of NF-?B p65 and IRF3 was seen in A20 siRNA-transfected cells.3.USP25 down-regulated CQ-induced attenuation of LPS-TLR4 pathways.?1?CQ increased USP25 mRNA and protein expressions in inactivated or activated Raw264.7 and THP-1 cells in a dose-dependent manner.?2?PCR and WB results revealed that siRNA bought from company could result in a decreased USP25 expressions.?3?After USP25 siRNA was transfected into Raw264.7 cells,mRNA expressions and release level of TNF-?,IL-6 and IFN-?were all increased.?4?SiRNA-mediated USP25 interference increased MAPKs phosphorylation,enhanced I?B degradation as well as augmented phosphorylation level of I?B and NF-?B.?5?USP25 silencing enhanced NF-?B p65 and p50 early and late activation.?6?USP25 interference induced nuclear accumulation of NF-?B and IRF3 in macrophages upon LPS infection.4.A20 and USP25 might display antagonism in MyD88-dependent pathway,while synergy in TRAM/TRIF-dependent pathway:A20 and USP25 siRNA co-transfection decreased TNF-?and IL-6 release levels,but increased IFN-?release.A20 and USP25co-silencing resulted in reduced phosphorylation of p38.A20 and USP25 both interference led to a significantly decreased NF-?B and an unapparent reduced IRF3 nuclear translocation.Conclusion:1.We determined that CQ pretreatment increased most of the USPs,A20 and CYLD mRNA expressions.2.We found that CQ increased A20 and USP25 expressions,which might to some extent play a role in CQ-induced attenuation of LPS-TLR4 pathways,thus inhibited NF-?B activation and the release of pro-inflammatory mediators.A20 or USP25 interference caused blockade of CQ inhibition of LPS-TLR4 pathways,while A20 and USP25co-interference seemed to increase inhibition of NF-?B activation but rather abate suppression of TRIF-IRF3 pathway.This indicates A20 and USP25 might be differently involved in MyD88-dependent and TRAM/TRIF-dependent pathways.3.CQ-mediated inhibition of LPS-induced macrophage activation might have something to do with the ubiquitin-proteasome system,but the UPS itself is complicated,thus CQ might attenuate LPS-TLR4 pathways through intersectional impacts of multiple DUBs. |
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