Study On The Regulation Of Inflammatory Cytokines By JAB1

In our previous reports we found that the function of Glucocorticoid receptor(GR) was repressed seriously after severe trauma so that the anti-inflammatory effect of GR was restricted.At the same time,the transcriptional factor NF-κB activated significantly and enhenced the production of cytokines.The inhibited GR,activated NF-κB and increased cytokine release formed a cascade amplification,which correlated closely to the development and progression of systemic inflammatory response syndrome(SIRS) and even to the development of multi-organ dysfunction syndrome(MODS).In this process,the production of cytokines played an important role in the development of systemic damage resulted from trauma at early stage.It will facilitate the recovery from trauma if the control of cytokine production is effective.Document retrieval indicated that GR and NF-κB were the up stream nuclear factors of several cytokines and they showed opposite functions in regulating the production of cytokines.GR operate its anti-inflammation action by inhibit the release of many cytokines (e.g.IL-6,TNF-αetc.),while NF-κB could notably evoke the inflammation.Inhibiting the activation of NF-κB could subsequently decrease the expression and release of cytokines significantly.JAB1(c-Jun-activation-domain binding protein 1) is the fifth subunit of COP9 signalosome regulatory complex.As a co-activators of some proteins,JAB1 can interaction with various proteins and act as an important element in signal transduction linkage.JAB1 was considered as the assistant activator of NF-κB and could up-regulate its ability of transcriptional activation.In our previous study,molecules that could interact with GR were screened by yeast two-hybrid system and JAB1 was among the possible candidates.Testing the relationship between transcriptional activation ability of GR and JAB1 expression in vitro by reporter gene confirmed that the expression level of exogenous JAB1 was paralleled to the expression level of target gene that transcriptionally activated by GR.These results suggest that JAB1 may be not only the co-activator of NF-κB but also the co-activator of GR.Up to date,no convincing reports reveal the function of JAB1 in inflammatory reaction.RNA interference(RNAi) is an important method to inhibit the expression of specific gene.RNAi can down-regulate the expression of target gene conveniently,effectively and specifically and it is widely used to investigate the function of interested genes.In order to elucidate the main function and regulation pathway of JAB1 in cells stimulated by endotoxin(LPS),mouse macrophage cell line RAW264.7 with JAB1 knock-down was exploited and the expression of GR and NF-κB as well as the cytokine production under this condition were examined.Results and conclusions are summarized as follows:1,The construction of recombinant plasmid pPUR/U6-siJAB1We used the mouse,according to the general principle for selecting siRNA sequence and using the information from internet,sequences for RNAi to target JAB1 gene (AF065386) was chosen.Based on this sequence,oligonucleotides which could be transcripted and form hairpin RNA structure inside the cells were designed and synthesized. For recombination,restriction enzyme digestion sites Apa I and EcoR I were added to the ends.After the oligonucleotides annealed to double strains,ligation were proceeded between the double strain fragment and the linearlized plasmid pPRU/U6 digested by Apa I and EcoR I.The product of ligation was transformed to the E.coli DH5α,positive clones were chosen.The extracted plasmids were then identified by restriction enzyme digestion and sequencing.The correct recombinant plasmid pPUR/U6-siJAB1 was obtained.2,Screen and identify cells of low expression of JAB1To test the sensitivity of cell line RAW264.7 to puromycin,MTT assay and observation directly under the microscope were applied to evaluate the effect of different concentrations of hygromycin.The proper concentration for the selection of puromycin resistant colonies in 4ug/ml was confirmed,which would be use to screen the transfected cells.With DOTAP as the transfection reagent and a routine transfection method,plasmids pPUR/U6 and pPUR/U6-siJAB1 were transfected respectively to RAW264.7.Puromycin was added after 24 h.About 4 weeks later,the stable puromycin-resistant cell clones were established and amplified.Cells with different plasmids were collected and total proteins were extracted. The expression of JAB1 was determined by western blot.The results indicated that there were no significant deviation between vector-control group and normal group,while the expression of JAB1 in knock-down group was only 29%of normal group.The result demonstrated that the plasmid pPUR/U6-siJAB 1 can inhibit the expression of JAB1 in the RAW264.7.3,Cell proliferation and the release of inflammatory factors of the JAB1 knock-down cells when stimulated with LPSCells from control group and JAB1 knock-down group were counted with hemacytometer to determine the cell proliferation.Under the conventional culture condition, cells achieved the exponential phase of growth 24h after replating and no obvious changes in cell growth in both groups.When LPS was added,the number of the cells with the low expression of JAB1 was higher than that of the normal cells at the different time points. After 24 hours,cells with the low expression of JAB1 got to the log phase while the growth of the normal cells were inhibited significantly and it took them 24 hours more to get into the log phase.The results indicated that cells with JAB1 knock-down were much more tolerable to the LPS stimulation than the normal cells were.The inflammatory factors TNF-αand IL-6 presented in culture supernatant of cells from different group stimulated by LPS were test by ELISA.At each time points set in experiment,lower expressions of the inflammatory factors in the supernatant of JAB1 knock-down cells were observed all the times.In the detection of the TNF-α,significant difference between cells in two groups were detected at 6h,12h,24h,48h and 72h respectively.IL-6 was not detectable 6h after LPS stimulation in JAB1 knock-down group and kept in significant low concentration at the 12h,24h,48h and 72h comparing to normal one.It was confirmed that less release of inflammatory factors by JAB1 knock-down cells when stimulated by the LPS.Conclusion1.RNAi expression vector targeting the mRNA sequence of the Jab1 mRNA of mouse was constructed successfully and was transfected into the mouse peritoneal macrophage cell line RAW264.7 in order to down-regulate the expression of JAB1.The efficiency of the interfering JAB1 expression was 71%and the stable cell clones with low expression of JAB1 facilitated further research.2.In our experiments,compared to the normal cells,no significant difference was found in cells expressing low JAB1 under the routine culture conditions;After the LPS stimulation,the JAB1 knock-down cells proliferated better than normal cells,which indicated their higher tolerance to the stimulation of LPS.3.The significant decrease of TNF-αand IL-6 release by JAB1 knock-down cells when stimulated with LPS demonstrated that down-regulation of the expression of JAB1 in cells could depress stress reaction that usually evoke the production of inflammatory factors TNF-αand IL-6.JAB1 can act as a co-activator of some proteins and an important signal transduction molecule.JAB1 can augment the activities of inflammatory factor as well as anti-inflammatory factor.Data from our study suggests that JAB1 positively regulate pro-inflammatory factors in the inflammatory reaction caused by LPS.Other molecules involved in this process need to be identified.