| Background Glucocorticoids(GCs) are the most potent anti-inflammatory agents known.GCs are widely prescribed to treat a range of inflammatory and immune diseases such as asthma and COPD in the clinic. In response to GCs, the GRα complex rapidly undergoes a conformational change and subsequently dissociates from the heat shock proteins. Subsequently, the ligand bound GRα translocates into the nucleus. Activated GR binds to glucocorticoid response elements in the host cell genome to activate gene transcription.These are thought to be significant changes in the cellular microenvironment including alterations in GR translocation and combination of GC with GR.Hypoxia is observed in many sites of inflammation in inflammatory disease such as asthma and COPD. Our previous study showed hypoxia inhibited the expression of GRα but the mechanism is unknown and whether hypoxia affected the translocation of GR was not clear.Objective The aim of our study was to reveal the pathway of GRα down-regulation under hypoxic condition and whether hypoxia influenced GR translocation. Asthma model was induced to observe the expression of GRα and Hif-1. In vitro the effects of hypoxia on GRα, TLR2, Hif-1 and MAPK signal pathway in human alveolar epithelial A549 cells were observed. TLR2, Hif-1 and MAPK were targeted respectively, then the expression of TLR2, Hif-1 and GRα were detected to reveal the underlying mechanism of GR down-regulation under hypoxia. Live-cell imaging was used to observe the GRα translocation under hypoxia and normxia.Methods 1.Asthma model was induced in C57 and TLR2-/- mice by ovalbumin. Mice were divided into three groups:(i) control,(ii) Asthma,(iii)TLR2-/-+Asthma. The expression of Hif-1 and GRα were examined by westernblot and lung histology. 2.A549 cells were exposed to chemical hypoxic conditions with COCL2 for 0h,2h, 4h, 6h,8h,12 h,24h. 3.Western blot were used to determine the expression of GRα, Hif-1, TLR2 and mitogen-activated protein kinases(MAPKs) signal proteins under hypoxia. Under hypoxic condition MAPK were targeting inhibited and the expression of GRα. and MAPK were detected. The Hif-1 and TLR2 signal pathway were blockaded by si RNA, and COCL2 of 1200umol/L were added in the medium. The expression of MAPK signal protein and Hif-1, TLR2 were detected by westenblot. 4. A549 cells were treated with lipoteichoic acid for different times. TLR2, Hif-1 and GRα were detected by westernblot. 5.Live-cell imaging was used to observe the GRα translocation under normoxic or hypoxic conditions. 6.Immunoprecipitation assay was used to observe the effect of hypoxia on GRα-HSP90 two dimer. The amount of GRα can be detected by HSP90 binding under hypoxia and normoxia. 7.Continuous variables were reported as means ± standard error. For selected comparisons between two group means, the independent t-test was used. A P value < 0.05 was considered to indicate statistical significance. One-way ANOVA was used to compare the differences between different groups.Results 1.The expression of GRα in asthma mice was down-regulated and Hif-1 was up-regulated compared with normal lung tissue in mice, the expression of GRα protein in the lung tissue of asthmatic mice were significantly lower than the corresponding normal tissues of mice, the expression of Hif-1 protein in asthmatic mice was significantly higher than that in corresponding normal tissues of mice. 2. Hypoxia caused a time-dependent decrease in protein expression levels for GRα and increase of hypoxia inducible factor(Hif). Hypoxia activated multiple signaling pathways including mitogen-activated protein kinases(MAPKs). 3. The changes of GRα expression were observed after targeted inhibition of MAPK signaling pathway. SB203580(a selective inhibitor of p38MAPK) but not SP600125(a selective inhibitor of JNK) and U0126(ERK inhibitor) could improve the expression of GRα under hypoxic condition. TLR2 and MAPK signal were also observed downregulated after targeting of Hif-1. There were no changes of HIF-1, MAPK and GRα after targeting TLR2.Targeting Hif-1 could upregulate the expression of GRα. There was no change of the expression of GRα after targeting TLR2. 4.LTA stimulation could cause the increase in TLR2 expression, and GRα expression was downregulated at 8h of LTA stimulation. 5.GRα translocation was inhibited significantly under hypoxia.We found under normxic condition GRα imported into nuclear fastly within 15 min after GC was added to cell medium otherwise GRα translocation was significantly inhibited under hypoxia condition. There was still a certain proportion of GRα did not import into the nucleus after hormone administration of 60 min. 6.The amount of GR could be detected by HSP90 binding under hypoxia was significantly higher than that under normoxia.Conclusions 1.The expression of GRα protein in the lung tissue of asthmatic mice were significantly lower than the corresponding normal tissues of mice, the expression of Hif-1 protein in asthmatic mice was significantly higher than that in corresponding normal tissues of mice. 2.Hypoxia activated MAPK signal pathway in time dependent manner. Targeting p38 could upregulate the expression of GR under hypoxia. 3.Hypoxia inhibited GR translocation significantly compared with normxia. 4.Targeting Hif-1 could upregulate the expression of GR and inhibit MAPK signal pathway. 5.LTA stimulation could cause the increase in TLR2 expression, and GR expression was downregulated at 8h of LTA stimulation. |
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