The Role And Mechanism Of Extracellular HMGB1 Regulated P53 Expression In T Lymphocytes

ObjectiveHigh mobility group Box 1 protein(HMGB1),as a late infltammatory factor,plays an important role in sepsis induced immune dysfunction.The transcription factor p53,also known as TP53,effects on cell proliferation by regulating cell cycle.It has been confirmed that intracellular fHMGB1 and p53 interacts with each other by a direct binding.However,the effect of extracellular HMGB1 on p53 activity has not been illustrated.This study aims to identify the effect of HMGB1 on T lymphocytes proliferation,and whether HMGB1 can promote p53 expression,and to reveal the role of p53 on HMGB1 induced T lymphocytes proliferation impairment,apoptosis and dysfunction.Furthermore,explore the underlying mechanism of HMGB1 activated p53 expression by detection MAPKs pathways.Finally,we focused on p38 MAPK and construct its mediating role on HMGB1-induced p53 activation.Methods1)Jurkat cells were incubated with HMGB1(100ng/ml)for 0,12,24,or 48 hours,or with HMGB1(0,10,100,100 ng/ml)for 24 hours.Afterwards,cell proliferations were detected by CCK-8.The total mRNA were extracted and subjected to Quantitative Real-time PCR(RT-PCR)for the detection of p53,p21,mdm2 mRNA levels,and the cell proteins were subjected to Western blot analysis of p-p53,p53,p21and mdm2 expression levels.2)Jurkat cells were transfected with p53 shRNA expressing or vector Lentivirals,and construct stable expression cell lined by puromycin selection.Stimulate both vector and p53 shRNA expressing cells with or without 100 ng/ml HMGB1 for 24 hours,and then exam cells proliferation,p53 mRNA,p-p53 and p53 protein levels as mentioned above.Cells were stained with Annexin V and 7-AAD,and analyzed by flow cytometry(Flow cytometry FCM)to detect cell apoptosis rates.Meanwhile cells were also subjected to Wersterm blot analysis of bcl-2,bax and caspase-3.Furthermore,nuclear proteins were extracted and subjected to NF-AT activity examination.The cytokines production including IL-2.IL-4.1FN-y were assessed by ELISA.3)Jurkat cells were stimulated with 100 ng/ml HMGB1 for 24 hours.Cells proteins were subjected to Western blot analysis of p-ERKl/2,ERK1/2,p-p38 MAPK,p38 MAPK,p-JNKl/2 and JNK1/2 levels.4)Jurkat cells were stimulated with HMGB1 alone or in the absence of SB203580 for 24 hours.Cells were subjected to analysis of p53,p21 and mdm2 mRNA levels by RT-PCR,and p-p53 and p53 protein levels by Western blot.Results1)HMGB1 inhibited Jurkat cells proliferation.Jurkat cells proliferation rate were significant depressed by 24 or 48 hours incubation with HMGB1(100ng/ml)(p<0.05).Medium and high concentration of HMGB1(1OOng/ml or 1OOOng/ml)inhibited cell proliferation in 24 hours(p<0.05).2)HMGB1 upregulated both p53 mRNA and protein levels.After 24 and 48 hours of HMGB1(100 ng/ml)stimulation,the expression of p53 mRNA levels were gradually upregulated.Consistently,phosphorylated p53 and p53 proteins were accumulated(p<0.05).In a dose-dependent manner,a low and medium concentration of HMGB1 induced an upregulation of p53mRNA,phospho-p53 and p53 protein expression.Furthermore,the upregulated p53 possesses transcription activity,indicating by an elevation of its target genes mRNA levels,including p21 and mdm2(p<0.05),However a high concentration HMGB1 did not altered p53 expression.3)p53 mediated HMGB1-induced cell proliferation impairment.P53 shRNA and vector expressed cells were constructed and subjected to HMGB1 stimulation.Proliferation were depressed significantly in vector-expressed cells,but not in p53 shRNA-expressed cells(p<0.05).4)p53 mediated HMGB1-induced cell apoptosis.HMGB1 stimulation promoted a significant apoptosis in vector-expressed cells,compared to which p53 shRNA-expressed cells showed a less apoptosis(p<0.05).In the meanwhile,related molecules expression altered along with the tendency of apoptosis,including bcl-2,bax and caspase-3(p<0.05).5)p53 mediated HMGB1-induced T cell dysfunction.In HMGB1 treated vector cells,NF-AT activity showed a dramatic decrement.In addition,cells presented a lower IL-2 production and a shift to Th2 phenotype indicating by a decreased IFN-?/IL-4 ratio.However,HMGB1 did not altered above parameters in p53 shRNA-expressed cells(p<0.05).6)HMGB1 induced p53 activation was regulated by p38 MAPK.Among three major MAPKs pathways,only p38 MAPK pathway was significantly activated by HMGB1.After blockade of p38 MAPK activation by using its specific inhibitor SB203580,HMGB1 induced p53 expression elevation were reversed including mRNA,phosphorylated and total protein.Consistently,its target genes,mdm2 and p21mRNA transcriptions were also blocked(p<0.05).Conclusions1)HMGB1 suppressed Jurkat T cells proliferation.2)HMGB1 activated p53 effectively.3)The effects of HMGB1 on T cells were mediated by p53,not only cell proliferationsuppression but also apoptosis and cell dysfunction.4)HMGB1 induced p53 activation might be regulated by p38 MAPK pathway.