Experimental Study On Regulation Of JAB1 On Transcriptional Activation And Function Of Glucocorticoid Receptor

Glucocorticoids (GC) are used in the treatment of inflammatory and autoimmune diseases. It is widely accepted that anti-inflammatory effects of GC are mediated by the intracellular glucocorticoid receptor (GR) that modulate inflammatory gene expression. Previous works have suggested that the expression and function of GR decreased, especially the translocation of GR from cytoplasm to nucleus reduced after severe trauma. These results indicated that the transcriptional activation of GR is decreased, but the molecular mechanism is not clear. The activated GR may also interact directly with other transcription factor, such as activator protein-1 (AP-1) and nuclear factor-?B (NF-?B), which may be important role in the induction of proinflammatory gene expression. Some of the interactions between GR and transcription factors are mediated by interaction with common coactivator molecule. JAB1 is a multi-functional protein, which interacts with and controls multiple intracellar signaling and potentiates the transactivation properties of most receptors. JAB1 potentiates the activity of a variety of transcription factors, such as nuclear receptors, AP-1 and NF-?B. Previous study showed that JAB1 interacts specifically with GR-LBD in COS-7 cells. JAB1 is common coactivator of GR, AP-1 and NF-?B, so it may play an important role in inflammatory development. But the specific effects of JAB1 on GR still remain obscure. The present study was undertaken to explore the effects of JAB1 on transcriptional activation and function of the GR and the role of JAB1 and GR in post-traumatic stress disorder, and investigate the function and molecular mechanism of diazepam-ketamine in stress and inflammatory, and eventually pave a new way for improve the disorder of stress at early stages after severe trauma. In the present study, we firstly used COS-7 cells transiently cotransfected with JAB1 and hGR expression plasmids and GRE-CAT reporter plasmids by DOTAP liposome, using CAT-ELISA technique observed the expression of the CAT reporter gene to examine the effects of JAB1 and LPS on the transcriptional activation of GR. Secondly, we explored the expression level and function of GR when the JAB1 level increased using transient transfection by electroporation technology in the murine macrophage cell line RAW 264.7 cells. The proinflammatory cytokine levels of TNF-a and IL-6 were detected in the supernatants derived from RAW264.7 cells culture by ELISA. Finally, we used burned mouse as the experimental traumatic animal model and investigated the expression change of JAB1 and GR in the liver, lung and peritoneal macrophages of mice at early stages after severe burn or treatment with diazepam (0.4mg/kg) and ketamine (10mg/kg), in order to clarify the relationship between JAB1 and GR during the traumatic stress. Main results are summarized as follows: 1. COS-7 cells were cultured in vitro, different doses of pCDNA3.1-JAB1 containing the full-length human cDNA encoding JAB1 were cotransfected with eukaryotic vector of pCMV-hGRa and pMAMneo-CAT CAT reporter plasmids containing the GRE by using the transfection reagent DOTAP, The expression of CAT was observed by CAT ELISA after the cotransfected COS-7 cells were cultured with dexamethasone for 36 hours. The concentration of CAT was enhenced with the increasing doses of pcDNA3.1-JAB1. Overexpression of JAB1 promoted the transcriptional activation of GR. This result indicated that JAB1 can enhance the transcriptional activation of GR in a dose-dependent manner. 2. The production of CAT in cotransfected COS-7 cells was significantly decreased after stimulation with LPS in the presence of dexamethasone, but partially reversed cotransfected with pcDNA3.1-JAB1. Thus, LPS could inhibit the transcriptional activation of GR. However, JAB1 overexpression could partially alleviate the repressive effects of LPS on transcriptional activation of GR. 3. RAW264.7 macrophages were incubated with LPS (100ng/ml), dexamethasone (100nM) or both agents for 24 hours and the expression of JAB1 and GR measured by RT-PCR and Western bolt assay. Cytokine levels of TNF-a and IL-6 in the supernatant of cultured RAW264.7 cells were determined by ELISA. We found that LPS-stimulated RAW264.7 macrophages were markedly down-regulated the mRNA and protein expression of JAB1. The protein level of JAB1 was significantly reduced by treatment withdexamethasone for 24h, but JAB1 mRNA did not change significantly. Treatment of RAW264.7 cells with 100nM dexamethasone for 24h led to a significant decrease in the expresson of GR protein. Compared to the control group, GR protein level was significantly increased by LPS treatment of RAW264.7 cells for 24h. Furthermore, the expression of GR protein in RAW264.7 cells costimulated with LPS and dexamethasone was significantly higher than that in the control group. The mean production of proinflammatory cytokines TNF-a and IL-6 in the supernatant of cultured RAW264.7 cells stimulated with LPS was significantly increased compared to that in control cells, and remained heightened through 24h. LPS is a potent inducer of macrophage-derived proinflammatory cytokines TNF-a and IL-6. 4. The expression of JAB1 in RAW264.7 cells was obviously increased after the cells were transfected with pFLAG-CMV2-JAB1 by electroporation procedure. Overexpression of JAB1 in transfected RAW264.7 cells significantly increased the expression of GR protein compared to the control group. ELISA analysis revealed that the prinflammatory cytokine levels of TNF-a and IL-6 were significantly decreased in the supernatant of transfected RAW264.7 cells costimulated with LPS and dexamethasone compared to the non-transfected RAW264.7 cells treated with LPS or LPS plus dexamethason. This analysis indicated that overexpression of JAB1 led to repression of LPS-induced proinflammatory cytokines production in RAW264.7 cells. These results demonstrated that JAB1 could enhance the expression and function of GR in the RAW264.7 cells, and plays a critical role in the suppression of LPS-induced proinflammatory cytokines TNF-a and IL-6 production, and the mechanism may be involved in up-regulation of the expression and function of GR protein by JAB1 overexpression. 5. RT-PCR and Western blot analysis revealed the expression levels of JAB1 and GR in the liver, lung and peritoneal macrophages of mice were markedly decreased after severe burn, and then increased at 12h, but were still lower than normal control group at 24h. The change tendency of protein expression of GR was consistent with the protein and mRNA expression change of JAB1 in mice after severe burn. This phenomenon implied the down-regulation of GR associated with low expression of JAB1 during traumatic stress and eventually resulted in the increase of the proinflammatory cytokine production in mice aftersevere burn. Treatment with diazepam-ketamine could inhibit the down-regulation of expression of JAB1 and GR in the liver, lung and peritoneal macrophages of mice at early stage after severe burn. Taken together, we concluded that: 1. In the presence of dexamethasone, JAB1 enhanced the transcriptional activation of GR in a dose-dependent manner. 2. LPS inhibited the transcriptional activation of GR In the presence of dexamethasone. However, JAB1 overexpression could partially relieve the inhibition of GR transcriptional activation by LPS. 3. The mRNA and protein expression levels of JAB1 were markedly down-regulated in LPS-stimulated RAW264.7 macrophages. The protein level of JAB1 was significantly reduced in RAW264.7 macrophages treated with dexamethasone, but JAB1 mRNA did not change significantly. 4. Dexamethasone led to a significant decrease in the expresson of GR protein in RAW264.7 macrophages. However, GR protein level was significantly increased by LPS. 5. The expression and function of GR in RAW264.7 macrophages was markedly increased by JAB1 overexpression. In murine RAW264.7 macrophages, LPS-induced proinflammatory cytokines production was inhibited by Overexpression of JAB1, this data may be indicated the function of GR was improved by overexpression of JAB1 in RAW264.7 macrophages stimulated with LPS. It was also concluded that overexpression of JAB1 played an important role in the anti-inflammatory response. 6. The expressin of mRNA and protein levels of JAB1 decreased in the liver, lung and peritoneal macrophages of mice at early stage after severe burn. Meanwhile, the expression level of GR also was down-regulated. Taked together above results, this phenomenon may be implied that the expression and function changes of GR were at least partially associated with the down-regulation of JAB1 expression after severe burn. 7. After severe burn injury, the expression levels of the JAB1 and GR in the liver, lung and peritoneal macrophages of mice were significantly elevated by diazepam-ketamine. Diazepam-ketamine partially improved the post-traumatic stress disorder induced by severe burn. This mechanism may be involved in the expression up-regulation of JAB1 and GR.