Coordinate Regulation Of JAK/STAT3 And MAPK Parthways On Tumor Necrosis Factor Alpha Transcription Activity

At the early stage of sepsis,mitogen-activated protein kinase(MAPK)and signal transducer and activator of transcription(STAT3)pathways are activated directly or indirectly.MAPK is an important signal transduction pathway mediating extracellular signal to eukaryotic cells.JAK/STAT,which participates in the regulation of many inflammatory factors,is one of potential signal pathways in sepsis.During the development of sepsis,it is clear that cytoldnes including tumor necrosis factor alpha (TNF-α)and interleukins will be excessively produced after MAPK is activated by lipopolysaccharide(LPS).It has been demonstrated that TNF-αappears to be the pivotal early inflammatory eytokine.After JAK/STAT pathway is indirectly stimulated by TNF-α,activated STATs enter into the nucleus to take part in the gene expression induced by LPS.In this process,it is known that both activated STAT3 and increased high mobility group box-1 protein.(HMGB1)would augment upregulation of expression of TNF-α,but the mechanism of signal transduction is still not clear.It is suggested that complex interactions and cross-talk may exist among the signal transduetion pathways,in which there is a simple and highly conserved MAPK phosphorylation site in the STAT3 protein sequence.All the extracellular-signal regulatory protein kinase(ERK),c-Jun amino-terminal kinase(JNK)and p38 are capable of phosphorylating 727 serine site of STAT3.It seems that ERK1/2 and p38 MAPK might be closely related to STAT3 pathway during inflammatory respose. Therefore,a hypothesis is raised that there exists protein interaction between p38/ERK2 and STAT3,with the function of regulating LPS-indueed TNF-αgene expression.Also, the present study was performed to explore the potential sites that STAT3 and HMGB1 binding to TNF-αpromoter.Co-immunoprecipitation method is used to verify the hypothesis regarding binding of p38/ERK2 to STAT3 in vivo.It was found that there were interactions between p38/ERK2 and STAT3.Then further study was carried out to investigate the regulatory mechanism underlying LPS-mediated TNF-αgene expression.First of all,human p38 and ERK2 genes were amplified by specific primers,and were cloned to pcDNA3 vector tagged with HA.Flag-STAT3 and HA-p38 or HA-ERK2 plasmids were co-transfected into 293T cells respectively,then the cells were lysed and the supematant was co-immunoprecipitated.The results showed that positive STAT3 protein was found in the supernatant with the use of anti-Flag antibody conjugated beads.With anti-HA antibody,expression of p38/ERK2 proteins were detected, indicating that there were interactions between p38/ERK2 and STAT3 in vivo.After Flag-STAT3,HA-p38 and TNF-αpromoter full length reporter gene plasmids were co-transfected into 293T cells,TNF-αpromoter activity was coordinated by the binding of p38 and STAT3.The results revealed that p38 and STAT3 could not only regulate the activity alone,but also coordinate it prior to and after LPS stimulation.At the same time,after Flag-STAT3,HA-ERK2 and TNF-αpromoter full length reporter gene plasmids were co-transfected into 293T cells,TNF-αpromoter activity was coordinated by the binding of ERK2 and STAT3.The results suggested that ERK2 and STAT3 not only regulate the activity alone,but also coordinate it both before and after LPS stimulation.In the RNA interfering experiment,it was to explore whether obstruction of STAT3 pathway would decrease synergistic effect and reduce TNF-α.STAT3-RNAi, HA-p38/ERK2 and TNF-αpromoter reporter gene plasmids were co-transfected into 293T cells,and the results showed that STAT3 expression was inhibited by STAT3-RNAi and the synergistic effects of STAT3 and p38/ERK2 were suppressed.In order to investigate the STAT3 binding site on the TNF-αpromoter, dose-dependent response of STAT3 on TNF-αexpression was observed after Flag-STAT3 and full length TNF-αpromoter reporter gene were co-transfected into COS-7 cells.The results indicated that TNF-αgene expression was enhanced along with increased doses of STAT3 with or without LPS.Flag-STAT3(200 ng/ml)and TNF-αpromoter reporter gene of different length were co-transfected into COS-7 cells respectively,and LPS was added into cells 4 hours later.Compared to the controls,fold activity value of pTNF-α(95 bp)was found to be the raised 6.9-fold.Then,pTNF-α(70 bp),pTNF-α(75 bp),pTNF-α(80 bp)and pTNF-α(85 bp)deletion mutants were constructed and co-transfected with Flag-STAT3(200 ng/ml)to COS-7 cells induced by LPS.Data showed that fold activity value of pTNF-α(85 bp)was similar to that of pTNF-α(95 bp),and fold activity value of pTNF-α(80 bp)decreased to the control levels.81 base and 82 base.in the TNF-αpromoter were mutated and then the activity results proved that the two sites were important to the TNF-αgene expression induced by STAT3.The potential binding site of STAT3 on TNF-αpromoter was identified to be between 80 bp and 85 bp.was applied in further study.The results of EMSA howed that it was wild probe tagged withγ-p³²of the TNF-αpromoter 62-85 bp could bind to nucleic proein STAT3,but not mutant 62-85 bp probe.The search of the binding sites of HMGB1 on the TNF-αpromoter was carried out as follows.First,human HMGB1 gene was constructed to the pcDNA3 vector tagged with HA.Second,after HA-HMGB1 and full length TNF-αpromoter reporter gene Were co-transfected into COS-7 cells induced by LPS.It was found that TNF-αpromoter activity was increased,peaking at 300 ng/ml HMGB1,and then decreased at a dose exceeding 500 ng/ml HMGB1.when HA-HMGB1(300 ng/ml)and full length TNF-αpromoter reporter gene were co-transfected into COS-7 cells induced by LPS,it was found that a classic endotoxin site,NF-IL6,between 95 and 120 bp of TNF-αpromoter,might be the major site for HMGB1 to act on the TNF-αpromoter.From these data,it might be concluded as follows:1.STAT3 protein and p38/ERK2 protein could interact in vivo.2.There existed synergistic effect of STAT3 and p38/ERK2 interaction on TNF-αexpression induced by LPS.3.After STAT3 pathway was blocked,synergistic effect of STAT3 and p38/ERK2 interaction on TNF-αexpression was inhibited. 4.The effect of STAT3 on TNF-αgene expression did not depend on LPS.5.Potential binding site of STAT3 on TNF-αpromoter was between 80 and 85 base radical fragment.6.The effect of HMGB1 on TNF-αgene expression depended on LPS.7.A classic endotoxin site-NF-IL6 was found to be the potential HMGB1 site of action on TNF-αpromoter.