The Effect And Possible Mechanism Of TLR2on Cell Proliferration Of MMC Cells Induced By HMGB1

Objective: In this study, the mouse mesangial cells (MMC) andrecombinant murine HMGB1were used. The aim of this study was toexplore the effect and possible mechanism of TLR2on cell proliferration ofMMC cells induced by HMGB1Systemic lupus erythematosus (SLE) is a chronic inflammatoryautoimmune disease characterized by multiple organ involvement, productionof autoantibodies to nuclear components, and immune complex deposition.Lupus nephritis (LN) is regarded as one of the most severe organmanifestations of SLE, affecting approximately35%to50%of patients withlupus, and glomerulonephritis is a significant cause of death and disability[1].Mesangial cell proliferation is the main pathological change and it can bedetected in nearly every type, especially in type IV and V lupus nephritis (LN)High-mobility group box1protein (HMGB1)[2], a nuclear protein foundin all mammalian cells, is known as a DNA-binding protein participating inchromatin structure and transcriptional regulation. Extracellular HMGB1hasbeen identified as a proinflammatory mediator and contribute to thepathogenesis of multiple chronic inflammatory and autoimmune diseases byits proinflammatory and immunostimulatory properties[3]. HMGB1is activelysecreted from activated immune cells such as macrophages and monocytesand is passively released from injured or necrotic cells[4]. Moreover, HMGB1could induce other cytokines such as tumor necrosis factor(TNF) andinterleukin-1(IL-1), IL-6and IL-8. At same time, it is an activator ofendothelial cells leading to the upregulation of adhesion molecules. Elevatedserum levels of HMGB1have been found in different inflammatoryconditions such as sepsis, rheumatoid arthritis, anti-neutrophilic cytoplasmaticantibody (ANCA)-associated vasculitis, and chronic kidney disease as well as in SLE[5]. Recent studies have found that the levels of HMGB1increased inpatients with active LN, compared with patients with active non-renaldisease[6]. In our previous study, we detected the expression of HMGB1andits the possible receptor TLR2,4in renal tissue of patients with lupusnephritis and BXSB mice (a model of LN), the results showed that HMGB1and TLR2,4expression in renal tissue of LN were higher than that in controlgroup following with the proliferation of mesangial cells. So we conclude thatHMGB1may involve in the abnormal proliferation of mesangial cells oflupus, promoting the proliferative glomerulonephritis through its receptor,however, the exact mechanism is not clear.Methods: Mouse renal mesangial cells line (MMC) were grown asdescribed previously. Briefly, they were cultured in Dulbecco’s modifiedEagle medium-F12(GIBCO BRL, Gaithersburg, MD, USA) supplementedwith10%fetal bovine serum. Cells were made quiescent by cultured inserum-free medium for24h.1) In order to determine the effect of HMGB1oncell proliferation of MMC cells, cells were randomly divided into normalcontrol group,50μ g/L HMGB1stimulated group,100μ g/L HMGB1stimulated group and200μg/LHMGB1stimulated group, and cells werecollected at2,4,8and12h. BrdU assy was used to detect cell proliferationlevel.2) To determine the time-dependent effect of HMGB1on the expressionof TLR2, PCNA, Cyclin D1, CDK4and p16，MMC cells were randomlydivided into normal control group,100μg/LHMGB1stimulated group, andwere collected at2,4,6,8and12h respectively. Real-time PCR and WesternBlot were used for the detction of TLR2, PCNA, CyclinD1, CDK4and p16expressions.3) We performed an RNA interference (RNAi) experiment toassess the effect of TLR2on HMGB1-stimulated cell proliferation. Cells weredivided into four groups: control, untransfected HMGB1-treated, controlvector sh-Scramble-transfected HMGB1-treated and shTLR2-transfectedHMGB1-treated. Cells were collected after8h, and Western Blot were used todetect the silence effect of pYr-3.1-shTLR2and the expression of PCNA,CyclinD1, CDK4and p16protein. Results:1HMGB1upregulated the level of mouse mesangial cell proliferationBrdU assay showed that the proliferation level of MMC cells inHMGB1-stimulated group increased from2h, and reached the peak at8h, thengradually decreased to a lower level at12h. Compared with8h, the differencewas not statistically significant. Moreover, the level of proliferation in100μg/L and200μg/L HMGB1-stimulated groups was significantly higherthan that in50μg/L group respectively, but there was no significantdifference between100μg/L and200μg/L group (P>0.05).2HMGB1induced TLR2upregulation of MMC cells intime-dependent mannerThe result of Real-time PCR showed that HMGB1significantlyincreased the TLR2mRNA expression，peaked at6h(2.2±0.16) comparedwith the normal control group. There were statistically significant differences（P<0.05）. But the expression gradually decreased to a lower level at12h,and still higher than that in the normal group.By western Blot, HMGB1time-dependently upregulated the expressionof TLR2protein, reaching the maximum at8h (0.55±0.02), and decreasedslightly at12h（0.51±0.02）, but still higher than that in normal control group(0.1±0.01, P<0.05).3HMGB1upregulated the expression of PCNA mRNA and protein inmouse mesangial cellsHMGB1induced the expression of PCNA mRNA and protein in atime-dependent manner. The mRAN expression reached its peak at6h(1.9±0.13), while the PCNA protein peaked at8h (0.56±0.02), graduallydecreased to a lower level at12h compared with the normal control group（0.28±0.01）.There were statistically significant differences（P<0.05）.4HMGB1increased the expression of CyclinD1, CDK4mRNA andprotein in mouse mesangial cellsCompared with the normal control group, HMGB1up-regulated theexpression of CyclinD1,CDK4mRNA, peaked at6h(1.8±0.14and1.6± 0.12), and there were statistically significant differences（P<0.05）, whichgradually decreased to a lower level at12h.Western Blot results showed that HMGB1up-regulated the expression ofCyclinD1,CDK4protein, peaked at8h （0.43±0.02and0.23±0.01）compared with the normal control group（0.14±0.01and0.04±0.01）. Therewere statistically significant differences（P<0.05）, then the expression ofCyclinD1and CDK4proteins gradually decreased to a lower level at12h, butstill higher than that in the normal control group.5HMGB1down-regulated the p16expression time-dependently inmouse mesangial cellsCompared with the normal control group, the expression of p16mRNAand protein in mouse mesangial cells was significantly decreased at2,4,6,8and12h in a time-dependent manner, and there were statistically significantdifferences（P<0.05）.6The shTLR2vector effectively prevented HMGB1-induced cellproliferation, decreased cyclin D1/CDK4/p16pathway and PCNA expressionNo u-regulation of cyclin D1protein was observed in cells transfectedwith the pshTLR2vector in medium containing HMGB1. However, theup-regulation of cyclinD1protein could be easily seen in blankvector-transfected MMC cells and untransfected cells treated with HMGB1.Similarly, shTLR2vector reduced the HMGB1-induced CyclinD1, CDK4andPCNA protein overexpression and up-regulated the expression of p16protein.There was statistically significant differences（P<0.05）.Conclusion: HMGB1induced the expression of TLR2in MMC cells,following with the increase of proliferation level and the expression of PCNA,Cyclin D1/CDK4up-regulation. From this results, we presume that HMGB1might combined with its surface receptor TLR2, and then up-regulated theexpression of cell cycle regulatory protein CyclinD1, CDK4, anddown-regulated the expression of the cyclin-dependent kinase inhibitor p16through activating signaling pathways to induce cell proliferation. But this isonly a preliminary study of the cellular level, the exact role of TLR2in HMGB1-mediated mesangial cell proliferation is needed to be furtherlyexamined in the future.