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The glucocorticoid receptor (GR) plays a critical role in signal transduction, expression of related genes and apoptosis of cancer cells. Hepatocellular carcinoma is characterized by high aggressiveness and mortality. In the exploration of the relationship between GR and hepatocellular carcinoma, numerous studies have shown that GR has an inhibitory effect on the growth of hepatocellular carcinoma cells, which provides new ideas and methods for the clinical application of GR in the treatment of hepatocellular carcinoma.
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Objective To evaluate the regulatory effects of lycopene on the key signaling receptors in human cutaneous squamous cell carcinoma cell line COLO16.Methods Cultured COLO16 cells were divided into 6 groups to be treated with lycopene at different concentrations of 0,5,10,15,20,and 25 μmol/L,respectively,for 24 hours (control group and 5,10,15,20,25 μmol/L lycopene groups),followed by estimation of the cell viability by lactate dehydrogenase (LDH) assay.Lycopene at a safe concentration was selected based on the LDH assay,and used for the determination of expression of signaling receptors,and Western blot analysis was performed to measure the expression of key signaling receptor proteins,including epidermal growth factor receptor (EGFR),glucocorticoid receptor (GR),retinoic acid receptor-alpha (RAR-α),retinoid X receptor-alpha (RXR-α),androgen receptor (AR) and progesterone receptor (PR).Statistical analysis was carried out by one-way analysis of variance (ANOVA),Tukey multiple comparison teat and Brown-Forsythe test with the SPSS software.Results After 24-hour treatment with lycopene at different concentrations,there were significant differences in the rate of cell death among these groups (F =13.116,P ＜ 0.05),and the rate of cell death in the 25 μmol/L lycopene group significantly differed from that in the control group (P ＜ 0.05).Therefor,lycopene at concentrations of 5,10 and 20 μmol/L were selected to treat COLO16 and HaCaT cells as well as human epidermal keratinocyte (HEK) for 24 hours in the following experiment.The treatment with lycopene significantly decreased the phosphorylation level of EGFR (P ＜ 0.05),but significantly increased the expression of GR protein (P ＜ 0.05),and showed no significant effects on the protein expression of RAR-α,RXR-α,AR,and PR in COLO16 cells.After 24-hour treatment with lycopene at concentrations of 5,10 and 20 μmol/L,there were no significant changes in the phosphorylation level of EGFR protein or the expression of GR protein in HaCaT cells and HEK (all P ＞ 0.05) compared with those without lycopene treatment.Conclusion Lycopene can decrease the viability of COLO16 cells,inhibit the activation of EGFR protein,and up-regulate the expression of GR,and these effects may be specific for tumor cells.
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Chronic central serous chorioretinopathy (CSC) usually demonstrates frequent recurrence,diffuse leakage and persistent subretinal fluid,which cannot be absorbed,thus lead to photoreceptor damage and poor visual acuity.As glucocorticoids have been implicated in the pathogenesis of chronic CSC,various anti-glucocorticoids oral drugs were used in the clinic to promote retinal fluid absorption and reduce the central retinal thickness of the macula and improve the vision outcomes.In addition,the 5α-reductase-specific inhibitor finasteride,the P450-3A4 inducer rifampicin,circadian rhythmic regulator melatonin,and systemic anti-inflammatory drug methotrexate have also been put into clinical trials for chronic CSC,and achieved certain effects.However,most of the clinical studies on these oral drugs were case reports,but not multi-center randomized clinical trials.The long-term effects of these oral drugs need to be observed and studied further.
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Glucocorticoid receptor (GR),a member of the steroid receptor superfamily,can mediate the signal pathway of ligands like glucocorticoids,regulate the transcription of target genes,and exert biological activity.GR is expressed in different degrees in three major kinds of urological malignant tumors including renal cancer,bladder cancer and prostate cancer.It also affects the metabolism of tumor cells,and is closely related to the occurrence,development and prognosis of tumors.GR provides an important clue for targeted therapy and endocrine therapy of urological malignant tumors.
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Objective:To observe the effect of moxibustion on learning and memory abilities, corticosterone and glucocorticoid receptor (GR) in subacute aging rats. Methods:Twenty four Sprague-Dawley (SD) rats were randomly divided into a normal group, a model group and a moxibustion group, 8 rats in each group. Rats in the model group and the moxibustion group were subcutaneously injected with 25% D-galactose [125 mg/(kg·bw)] for 40 d continuous; rats in the normal group were injected with saline at the same position for 40 d continuous. Rats in the moxibustion group were given mild moxibustion at bilateral Shenshu (BL 23) at the same time of modeling; rats in the normal group and the model group were only identically grabbed without moxibustion for 40 d. The learning and memory abilities of rats were observed using the Morris water maze at the end of the experiment. Abdominal aorta blood and thymus were collected after water maze experiment. Enzyme-linked immunosorbent assay (ELISA) was used to detect serum corticosterone level, and immunohistochemical method was used to detect the expression of thymus GR. Results:Compared with the normal group, rats in the model group showed that a significantly longer escape latency time (P<0.01) on the third and the fourth days; number of times crossing the platform in 70 s significantly reduced (P<0.01); activity times in the fourth quadrant significantly decreased (P<0.05); serum corticosterone levels increased (P<0.01); thymus GR expression decreased (P<0.05). Compared with the model group, rats in the moxibustion group showed that the escape latency times were significantly shorter on the third, the fourth and the fifth days (P<0.01,P<0.05); number of times crossing the platform in 70 s significantly increased (P<0.05); activity times in the fourth quadrant significantly increased (P<0.05); serum corticosterone levels decreased (P<0.05); thymus GR expression increased (P<0.05). Conclusion:Moxibustion could improve the learning and memory abilities of subacute aging rats, down-regulate serum corticosterone levels, and increase thymus GR content.
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[ABSTRACT]OBJECTIVETo study the mRNA expression of glucocorticoid receptor(GR) in peripheral blood mononuclear cells in patients with obstructive sleep apnea hypopnea syndrome(OSAHS) and its clinical significance. METHODSReal-time fluorescent quantitative PCR was used to detect GRα mRNA and GRβ mRNA exprssion in PBMC of 30 male patients with moderate to severe OSAHS and 27 healthy male subjects. The relationships between the expression of glucocorticoid receptor mRNA and the apnea hypopnea index, body mass index, neck circumference, waist circumference, the lowest oxygen saturation, the average oxygen saturation, the Epworth sleepiness scale(ESS) score, fasting blood glucose, heart rate and blood pressure were analyzed.RESULTSCompared with the control group, the expression level of GRα mRNA was lower in the OSAHS group(t=2.25,P0.05). We had not found the significant correlation between the expression of GRα mRNA and the clinical parameters above in OSAHS patients.CONCLUSIONThe expression of GRα mRNA in PBMC in moderate to severe OSAHS male patients have a downward trend compared with healthy group, but the mechanism remains unclear.
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Objective To explore the effects of hyperbaric oxygen treatment on chronic stress and glucocorticoid receptor (GR) expression in the hippocampus.Methods A total of 60 male Wistar rats were randomly divided into a restraint group,an HBO (hyperbaric oxygen) group,an HBO-restraint group and a control group using a random number table,each group with 15 animals.All the rats in the restraint and HBO groups were constrained by immobilizing their fore-and hind-limbs on a self-made frame for 3h daily for 21 days,and those in the HBO group received HBO treatment once daily for the same 21 days.The HBO-restraint group was immobilized in the morning and treated with HBO in the afternoon.The control group was reared without any special intervention.On the 1st,11th and 21st day of treatment,rats from the different groups were assessed using the open field test.On the 21st day,all the animals were sacrificed and their brains were harvested to detect GR expression.Results In the open field test on the 11 th day,the restraint group scored (131.0 ± 20.6) in terms of motor level and (26.5 ± 4.6) for exploratory behavior,both significantly higher than before restraint and significantly higher than those in the HBO-restraint group at the same time point.Immunofluorescence assay showed that GR expression in the hippocampus of the restraint group was significantly decreased compared with the control group.There was no significant difference,however,between the HBO-restraint group and the control group.Conclusion Chronic restraint stress induces changes in behavior and GR expression in rats which can be alleviated by hypbaric oxygen treatment.
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Objective To investigate the association of glucocorticoid receptor (GR) polymorphisms (BclI)with the prognosis of myasthenia gravis (MG).Methods We totally enrolled 74 patients diagnosed as MG from the Department of Neurology,Beijing Shijitan Hospital between 2002 and 2014.Of them,54 patients started with ocular MG and 20 patients started with general MG.MG patients were divided into recurrence group and non-recurrence group according to the progression at two years after onset.Patients with simple ocular symptom at disease onset were further divided into generalized MG (GMG) group and single ocular MG (OMG) group according to disease progression or not.The GMG group was divided into two groups (≤6 months,7-24 months) according to the progression time of generalization.The GMG group was further divided into three groups (limbs,throat,both limbs and throat) according to the first symptom of generalization.The genotypes of GR were determined by polymerase chain reaction and nucleotide sequence determination.Results The frequencies of three genotypes (GG,CG,CC) in BclI were 57.7％,34.6％,7.7％ in recurrence MG and 64.6％,31.3％,4.1％ in non-recurrence MG respectively.The difference in distribution of the genotypes between the two groups was not statistically significant (x2 =0.570,P =0.750).The frequencies of G and C allele were 75.0％ and 25.0％ in recurrence MG,and 80.2％ and 19.8％ in non-recurrence MG.The difference in distribution of the alleles between the two groups was not statistically significant (x2 =0.540,P =0.462).The frequencies of three genotypes GG,GC and CC were 55.9％,35.3％,8.8％ in GMG and 2/6,4/6,0/6 in OMG respectively.The frequencies of G and C allele were 73.5％ and 26.5 ％ in GMG,and 8/12,4/12 in OMG.The difference in distribution of the genotypes and alleles between the two groups was not statistically significant (x2 =2.278,P =0.320;x2 =0.241,P =0.624).The frequencies of three genotypes GG,GC,CC were respectively 61.9％,28.6％,9.5％ and 3/6,3/6,0/6 in ≤6 months,7-24 months of GMG group.The frequencies of G and C allele were 76.2％,23.8％ and 9/12,3/12 in the two groups.The difference in distribution of the genotypes and alleles between two of the three groups was not statistically significant (x2 =1.326,P =0.515;x2 =0.007,P =0.932).The frequencies of three genotypes GG,GC and CC were respectively 2/8,4/8,2/8;11/13,2/13,0/13 and 3/6,3/6,0/6 in limbs,throat,both limbs and throat of GMG group.The frequencies of G and C alleles were 8/16,8/16;92.3％,7.7％ and 9/12,3/12 in the three groups.The difference in distribution of the genotypes and alleles between two of the three groups was statistically significant (x2 =8.813,P =0.028;x2 =9.706,P =0.008).The genotype frequencies in every group were all in Hardy-Weinberg equilibrium.Conclusions BclI polymorphism may predict the first generalized symptom of OMG.BclI polymorphisms of GR might have no relationship with the recurrence of MG,generalization and generalized time of OMG during the first two years after MG onset.
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Objective To investigate the mRNA level of glucocorticoid receptor α (GRα) and heat shock protein 90 (HSP90) in peripheral blood mononuclear cells (PBMCs) and the plasma protein level of macrophage migration inhibitory factor (MIF) in patients with systemic lupus erythematosus (SLE) and to analyze their association with glucocorticoid (GC) resistance.Methods One hundred and six patients with SLE and thirty-eight healthy controls were enrolled in this study.Transcription levels of GRα and HSP90 were determined by real-time polymerase chain reaction.Enzyme-linked immunosorbent assay was used to detect the protein level of plasma MIF.The association between these parameters and GC resistance was analyzed by Spearman correlation analysis.The multivariate logistic regression model was used to analyze the risk factors for GC resistance.Results The mRNA level of GRα and HSP90 in GC resistance group was significantly lower than that in GC sensitive group [10.18(3.12,17.20) vs 16.83(12.01,24.18), P =0.001;18.46(14.77,26.45) vs 25.84 (17.97,35.90), P =0.005].MIF protein level in GC resistance group was significantly higher than that in GC sensitive group [(23.21 ±7.98) μg/L vs (18.34 ±6.29)μg/L;P =0.013].The mRNA level of HSP90 in the high MIF group was significantly lower than that in the low MIF group [23.67 (13.84,28.32) vs 26.64 (23.61,47.16);P =0.001], as well as HSP90/GRαratio(P =0.008).Additionally, the plasma protein level of MIF was negatively correlated with HSP90 (r =-0.275, P =0.004) and HSP90/GRα ratio(r =-0.341, P ＜ 0.001).SLE activity index score in GC resistance group was significantly higher than that in GC sensitive group [(12.23 ±2.86) μg./L vs (9.63 ± 3.48) μg/L;P =0.003].Logistic regression model indicated that disease activity was an independent risk factor for GC resistance (OR =17.481, 95％ CI 1.747-174.903, P =0.015).Conclusions Our preliminary findings suggest that low mRNA level of GRα and HSP90 and high protein level of MIF are associated with GC resistance.Elevated MIF level in SLE patients may play an important role in the development of GC resistance through down-regulating HSP90 and destabilizing the balance of HSP90/ Grα.Disease activity is the risk factor for GC resistance, which might be the viable evidence of therapy response.
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Objective To investigate the correlation of multidrug resistance gene 1 (MDR1),NR3C1 gene polymorphisms and clinical risk factors with efficacy,dependence,and resistance of glucocorticoid (GC) in patients with inflammatory bowel disease (IBD).Methods Anti coagulation blood samples of 196 healthy controls and 105 IBD patients received GC therapy were collected.There were 62 ulcerative colitis (UC) and 43 Crohn's disease (CD) in the IBD patients.The number of GC sensitive,GC dependent and GC resistant of UC patients were 36,13 and 13,respectively,and those of CD patients were 24,11 and eight.GC refractoriness included GC dependence and resistance.The genotype of MDR1 C3435T and NR3C1 Bcl Ⅰ of all the subjects was detected by the restriction fragment length polymorphism-polymerase chain reaction (RFLP-PCR).The correlation between each genotype frequency,clinical features of patients with IBD and the efficacy of GC treatment was analyzed by Chisquare test,Fisher exact probability method or t test.Results Among UC patients,the disease course of GC refractory group and GC resistant group was longer than that of GC sensitive group ((6.660±1.523)years,(6.500±1.111) yearsvs (3.350±0.697) years,t=2.211,P=0.031; t=2.930,P=0.005).The serum level of C reaction protein (CRP) of GC refractory group was higher than that of GC sensitive group ((47.628±13.913) mg/Lvs (16.854±4.121) mg/L,t=2.121,P=0.047).The chronic relapse type was more common in GC refractory UC patients (Fisher exact probability method,P=0.035),and severe patients were more common in UC with GC resistance (Fisher exact probability method,P=0.021).The white blood cell count of GC resistant and GC refractory CD patient was lower than that of GC sensitive CD patients ((5.710 ± 0.604) ×109/L,(5.878±0.405) × 109/L vs (7.814 ±0.670) × 109/L,t=2.334,P=0.028; t=2.045,P=0.018).Patients with extraqntestinal manifestations was more common in CD with GC resistance (Fisher exact probability method,P=0.035).There was no statistically significant difference in the frequencies of MDR1 C3435T,NR3C1 Bcl Ⅰ genotypes,allelic genes and gene carrier among control group and GC sensitive dependent and resistant group of IBD patients.However,the frequency of MDR1 C3435T gene carrier was significantly different between GC sensitive group and GC refractory group,especially between GC sensitive group and GC resistance group (68.33％ vs 48.89％,x2 =4.051,P=0.044; 68.33％ vs 42.86％,x2 =4.274,P =0.039).Conclusions GC sensitivity of IBD patients with MDR1 C3435T loci T gene carrier was higher than that of IBD patients without T gene carrier.NR3C1 gene polymorphisms was not related with GC resistance and GC dependence.Compared with GC sensitive IBD patients,in GC resistant and GC dependent IBD pantient UC patients with long disease course,chronic relapse type,severe type,high level of CRP and CD patients with low white blood cell count and extra-intestinal manifestations were more common.
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Objective To study the effects of glucocorticoid receptor (GR) signal pathway and downstream cytokineson androgen-independent growth of prostate cancer (PC) cells.Methods The human androgen-dependent PC (ADPC) cell line LNCaP and androgen-independent PC (AIPC) cell line DU145 were cultured in vitro.Immunocytochemistry was used to examine the expressions of the androgen receptor (AR),GR,HSPg0 and interleukin-6 (IL-6).The GR antagonist RU486 was used to treat cultured cells,and the effects of RU486 on the proliferation of both cell lines were analyzed by MTT assay.Expressions of HSP90 and IL-6 mRNA and protein were assessed by RT-PCR and Western blotting respectively.Results LNCaP cells were AR-positive and GR-negative,whereas DU145 cells were GR-positive and AR-negative.The expressions of HSP90 and IL-6 were significantly stronger in DU145 cells than in LNCaP cells (P＜0.01).RU486 had no obviously effects on the growth of LNCaP cells,but exerted a significant time-and dose-dependent growth inhibition on DU145 cells.RU486 treatment in DU145 cells also resulted in a dose-dependent decrease in the expressions of HSP90 and IL-6 mRNA and protein.Conclusions GR signal pathway may be the main survival pathway for DU145 cells.Abnormal hyperactivation of GR signal pathway and its promoting the expressions of HSP90 and IL-6 may contribute to the progression of ADPC to AIPC after androgen ablation.
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AIM:To explore the promoting action of chloroquine on the anti-proliferation effect of dexametha-sone on acute lymphoblastic leukemia cells .METHODS:CCK-8 assay was used to assess the viability of the dexametha-sone-resistant human acute lymphoblastic leukemia CEM-C1 cell line treated with the combination of chloroquine and dexa-methasone .Western blotting , quantitative real-time PCR and LysoTracker Red staining were utilized to examine the mecha-nism.RESULTS:Combination of chloroquine and dexamethasone significantly inhibited the proliferation of CEM -C1 cells compared with control group (P<0.01).The combination of chloroquine and dexamethasone increased the abundance of glucocorticoid receptor and inhibited lysosomal function , while lysosomal inhibitor bafilomycin A 1 also increased glucocorti-coid signaling .CONCLUSION:Dexamethasone combined with chloroquine triggers an anti-proliferation effect on CEM-C1 cells via a lysosome-mediated pathway .
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ObjectiveTo study the effects of small-dose glucocorticoid (GC) on glucocorticoid receptor (GR) and cellular immune function in critical patients.MethodsForty ICU critical patients admitted in Shanghai Changzheng Hospital from March 2007 to March 2009 were enrolled in the study and were divided into GC group and non-GC group according to the use or absence of GC.Blood samples were collected at days 1,7 and 10 after GC treatment to detect GR binding affinity of mononuclear leukocytes (MNLs) and polymorphonuclear leukocytes (PMLs) in the peripheral blood and the CD4/CD8 ratio in the T lymphocytes.The method of GC use was that the hydrocortisone was given intravenously at a dose of 100 mg every eight hours.ResultsGR binding capacity of MNLs at day 1 and 7 showed no statistical difference between the GC and non-GC groups.GR binding capacity of MNLs in the GC group was lower at day 1 and was much lower at day 7 (P ＜ 0.05 ).However,in the non-GC group,it was lower at day 1,but showed significant improvement at day 7 ( P ＜ 0.05 ).The change of GR binding capacity of PMLs was similar to that of MNLs.There was no significant difference of CD4/CD8 ratio between the GC and non-GC group at day 1.The ratio of CD4/CD8 in the non-GC group was significantly higher than that in the GC group at day 10 (P ＜0.05).CD4/CD8 ratio in the GC group showed a slight reduction at day 10,with no significant difference from that at day 1.While,the non-GC group showed a significant increase of CD4/CD8 ratio at day 10 as compared with that at day 1 (P ＜ 0.05 ).ConclusionLow-dose GC plays some role in the negative feedback regulation of GR binding capacity of peripheral blood leukocytes and in the inhibition of cellular immune function.
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Objective To investigate the role of spinal glucocorticoid receptors (GR) in phosphoinositide 3-kinase/protein kinase B (PI3K/Akt) signal pathway in rats with morphine tolerance.Methods Forty healthy male SD rats aged 8-10 weeks weighing 300-350 g in which intrathecal (IT) catheters were successfully implanted without complication were randomly divided into 4 groups (n =10 each):control group(group C) received IT injection of normal saline 10 μl twice a day for 7 consecutive days; morphine tolerance group(group M) received IT injection of morphine 10 μg twice a day for 7 consecutive days; dexamethasone (a GR agonist) group( group DEX)received IT injection of dexamethasone 4 μg 30 min before IT injection of morphine,twice a day for 7 consecutive days;RU38486(a GR blocker)group (group R) received IT injection of RU38486 2 μg 30 min before IT injection of morphine,twice a day for 7 consecutive days.Tail-flick test was measured once a day after first IT administration and 1 d after the end of IT administration,and the percentage of maximum possible antinociceptive effect (MPAE)was caculated.After the last measurem of tail-flick test,the spinal dorsal horns were removed for determination of PI3K,Caspase-3 expression and Akt activity.Results Morphine tolerance developed in groups M,DEX and R,but did not develop in group C.Compared with group C,Akt activity was decreased,PI3K expression was downregulated and Caspase-3 expression was up-regulated in group M (P ＜ 0.05).Compared with group M,MPAE and Akt activity were decreased,PI3K expression was down-regulated and Caspase-3 expression was up-regulated in group DEX,and MPAE and Akt activity were inecreased,PI3K expression was up-regulated and Caspase-3 expression was down-regulated in group R (P ＜ 0.05).Conclusion Spinal cord GR is involved in morphine tolerance by inhibiting PI3K/Akt signal pathway.
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Objective To evaluate the role of extracellular signal-regulated kinase-cyclic AMP response element binding protein(ERK-CREB) signal pathway in glucocorticoid receptors-mediated chronic morphine tolerance in rats.Methods Male SD rats aged 2 months weighing 280-320 g were used in this study.A catheter was placed in subarachnoid space via foramen magnum according to Yaksh.Thirty-six rats in which intrathecal (IT) catheters were successfully implanted were randomly divided into 6 groups ( n =6 each):control group ( group C),chronic morphine tolerance group (group M),morphine + dexamethasone group (group MD),morphine + RU38486 group (group MR),dexamethasone group (group D),RU38486 group (group R).Normal saline 10 μl,morphine 10 μg,morphine 10 μg + dexamethasone 4 μg,morphine 10 μg + RU38486 2 μg,dexamethasone 4μg,RU38486 2 μg was administered IT twice a day(8:00 and 20:00)for 6 consecutive days in groups C,M,MD,MR,D,R respectively.Tail flick latency (TFL) was measured at 1 d before IT drug administration(baseline)and at 30 min after first IT drug administration during 1,3,5 d and at 1 d after last IT drug administration (T1～4).Maximum analgesic effect (MPE) was calculated.The animals were sacrificed after last TEL measurement.The L3～5 segment of the spinal cord was isolated for determination of the expression of phosphorylated ERK(pERK)and phosphorylated CREB(pCREB) by immunofluorescence staining.Results MPE was significantly T1 lower at T3,4 than at T1 in groups M and MD.Compared with group C,MPE was significantly increased,the expression of pERK and pCREB up-regulated in group M,but no significant change was found in the parameters mentioned above in groups R and D.Compared with group M,MPE was significantly increased,the expression of pERK and pCREB up-regulated in group MR,and MPE was significantly increased,the expression of pERK and pCREB down-regulated in group MD.Conclusion The mechanism by which glucocorticoid receptors-mediated chronic morphine tolerance may be associated with the inhibition of ERK-CREB pathway.
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Objective To study the relationship of expression of central cortex glucocorticoid receptor (GR) at protein level with GR expression in the liver at protein level and with changes of serum cortisol and adrenocorticotropic hormone (ACTH) following severe closed traumatic brain injury (TBI) in mice. Methods Severe TBI was established in awake mice by using a BIM-Ⅲ biomechanical machine. At 0.5, 2, 8, 24, 48 and 72 hours after TBI, the total cytosolic GR in the cortex and liver were detected with Western blotting. Levels of serum ACTH and cortisol were measured by ELISA technique and radio-immunological assay (RIA) respectively. Results The expression of GR both in the cortex and liver were obviously down-regulated at protein levels at 2-72 hours after TBI and increased slowly eight hours after injury. The GR in the liver showed no recovery at 72 hours after injury and that in the cortex was decreased continually at 24 hours after injury. Serum ACTH and cortisol levels were increased markedly compared with control group, when there were two different peaks in the observation curve.Conclusion There is glucocorticoid resistance both in the central and peripheral tissues after severe closed TBI in the awake mice, which changes in a time-dependent manner.
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Objective To investigate the effect of tacrolimus on the expression of nuclear factor-κB (NF-κB) in HaCaT cells stimulated by tumor necrosis factor-α(TNF-α),and on the expression of glucocorticoid receptor (GR)α and β in untreated HaCaT cells in vitro.Methods Cultured Ha CaT cells were treated with TNF-α(10μg/L) only,combination of TNF-α(10μg/L) and various concentrations (10-8mol/L, 10-7mol/L,10-6moL/L) of tacrolimus or tacrolimus of different concentrations only.After additional 12-,24-, 36- or 48-hour cnlture, Westem blot and immunofluorescenee-confocal laser scanning microscopy were used to detect the expressions of NF-κB,GRα and GRβ in HaCaT cells.Those untreated HaCaT cells served as the control.Results The relative protein expression level of NF-κB was increased in HaCaT cells after treatment with TNF-α for 24 and 48 hours zompared with untreated ceils (0.73±0.0316 and 0.8925±0.0171 vs 0.4988±0.03506,both P<0.05);however,the increase in NF-κB expression was inhibited by the combination treatment with tacrolimus,and the relative expression level of NF-κB protein was 0.6825±0.0263.0.6200±0.0163 and 0.5575±0.0299 in HaCaT cells treated with TNF-α plus tacrolimus of 10-8mol/L 10-7mol/L and 10-6mol/L,respectively;the difference was significant etween TNF-α-treated cells and those dealt with the combination of NF-α and tacrolimus of 10-7 or 10-6 mol/L (both P<0.05).No significant difference was observed in the expression of NF-κB by HaCaT cells between different time oints treated with tacrolimus of 10-8,10-7 or 10-6 mol/L.Also,there was no zignificant difference in the expression of GRα or GRβ between untreated HaCaT cells and those treated with tacrolimus of 10-8, 10-7 or 10-6 mol/L at any time point.Conclusions Tacrolimus ould inhibit the expression of NF-κB by TNF-α-stimulated HaCaT cells,but does not affect the expression of GRα or GRβ,in untreated HaCaT cells.
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Increased expression of a number of proinflammatory genes, including IL-8, is associated with inflammatory conditions such as asthma. Glucocorticoid receptor (GR)beta, one of the GR isoforms, has been suggested to be upregulated in asthma associated with glucocorticoid insensitivity and to work as a dominant negative inhibitor of wild type GRalpha. However, recent data suggest that GRbeta is not a dominant negative inhibitor of GRalpha in the transrepressive process and has its own functional role. We investigated the functional role of GRbeta expression in the suppressive effect of glucocorticoids on tumor necrosis factor (TNF)-alpha-induced IL-8 release in an airway epithelial cell line. GRbeta expression was induced by treatment of epithelial cells with either dexamethasone or TNF-alpha. GRbeta was able to inhibit glucocorticoid-induced transcriptional activation mediated by binding to glucocorticoid response elements (GREs). The suppressive effect of dexamethasone on TNF-alpha-induced IL-8 transcription was not affected by GRbeta overexpression, rather GRbeta had its own weak suppressive activity on TNF-alpha-induced IL-8 expression. Overall histone deacetylase activity and histone acetyltransferase activity were not changed by GRbeta overexpression, but TNF-alpha-induced histone H4 acetylation at the IL-8 promoter was decreased with GRbeta overexpression. This study suggests that GRbeta overexpression does not affect glucocorticoid-induced suppression of IL-8 expression in airway epithelial cells and GRbeta induces its own histone deacetylase activity around IL-8 promoter site.
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Humans , Acetylation , Cell Line, Tumor , Dexamethasone/pharmacology , Epithelial Cells/metabolism , Gene Expression Regulation , Histones/metabolism , Interleukin-8/genetics , Receptors, Glucocorticoid/genetics , Transcriptional Activation , Transfection , Tumor Necrosis Factor-alpha/antagonists & inhibitorsABSTRACT
Increased expression of a number of proinflammatory genes, including IL-8, is associated with inflammatory conditions such as asthma. Glucocorticoid receptor (GR)beta, one of the GR isoforms, has been suggested to be upregulated in asthma associated with glucocorticoid insensitivity and to work as a dominant negative inhibitor of wild type GRalpha. However, recent data suggest that GRbeta is not a dominant negative inhibitor of GRalpha in the transrepressive process and has its own functional role. We investigated the functional role of GRbeta expression in the suppressive effect of glucocorticoids on tumor necrosis factor (TNF)-alpha-induced IL-8 release in an airway epithelial cell line. GRbeta expression was induced by treatment of epithelial cells with either dexamethasone or TNF-alpha. GRbeta was able to inhibit glucocorticoid-induced transcriptional activation mediated by binding to glucocorticoid response elements (GREs). The suppressive effect of dexamethasone on TNF-alpha-induced IL-8 transcription was not affected by GRbeta overexpression, rather GRbeta had its own weak suppressive activity on TNF-alpha-induced IL-8 expression. Overall histone deacetylase activity and histone acetyltransferase activity were not changed by GRbeta overexpression, but TNF-alpha-induced histone H4 acetylation at the IL-8 promoter was decreased with GRbeta overexpression. This study suggests that GRbeta overexpression does not affect glucocorticoid-induced suppression of IL-8 expression in airway epithelial cells and GRbeta induces its own histone deacetylase activity around IL-8 promoter site.
Subject(s)
Humans , Acetylation , Cell Line, Tumor , Dexamethasone/pharmacology , Epithelial Cells/metabolism , Gene Expression Regulation , Histones/metabolism , Interleukin-8/genetics , Receptors, Glucocorticoid/genetics , Transcriptional Activation , Transfection , Tumor Necrosis Factor-alpha/antagonists & inhibitorsABSTRACT
OBJECTIVE The purpose of our study was to measure the glucocorticoid receptor(GR)protein level in the nuclei of nasal polyp cells and compare the results in nasal polyposis patients with chronic rhinosinusitis alone and with chronic rhinosinusitis accompanied with asthma, before and after glucocorticoid(Glu)therapy. METHODS We used enzyme-linked immunosorbentassay(ELISA) techniques to quantitatively measure the activated- GR protein level in the nuclei of the nasal polyp cells. Nasal polyp tissues were obtained from patients with chronic rhinosinusitis alone and with chronic rhinosinusitis accompanied with bronchial asthma. In the latter, polyp tissues were obtained before and after Glu therapy seperatively. RESULTS Our data showed no significant differences between the activated- GR protein level of the nasal polyposis patients with chronic rhinosinusitis alone and with chronic rhinosinusitis accompanied with bronchial asthma before Glu therapy. However, the activated-GR protein level was significantly upregulated after Glu therapy in the patients with chronic rhinosinusitis accompanied with bronchial asthma. CONCLUSION The result of our research clearly showed that Glus upregulated the activated-GR protein level in the nuclei of nasal polyp cells and that the upregulation is essential for Glus anti-inflammatory action.