Expression of glucocorticoid receptors in the regenerating human skeletal muscle

Many stress conditions are accompanied by skeletal muscle dysfunction and regeneration, which is essentially a recapitulation of the embryonic development. However, regeneration usually occurs under conditions of hypothalamus-pituitary-adrenal gland axis activation and therefore increased glucocorticoid (GC) levels. Glucocorticoid receptor (GR), the main determinant of cellular responsiveness to GCs, exists in two isoforms (GRalpha and GRbeta) in humans. While the role of GRalpha is well characterized, GRbeta remains an elusive player in GC signalling. To elucidate basic characteristics of GC signalling in the regenerating human skeletal muscle we assessed GRalpha and GRbeta expression pattern in cultured human myoblasts and myotubes and their response to 24-hour dexamethasone (DEX) treatment. There was no difference in GRalpha mRNA and protein expression or DEX-mediated GRalpha down-regulation in myoblasts and myotubes. GRbeta mRNA level was very low in myoblasts and remained unaffected by differentiation and/or DEX. GRbeta protein could not be detected. These results indicate that response to GCs is established very early during human skeletal muscle regeneration and that it remains practically unchanged before innervation is established. Very low GRbeta mRNA expression and inability to detect GRbeta protein suggests that GRbeta is not a major player in the early stages of human skeletal muscle regeneration.     

Interindividual glucocorticoid sensitivity in young healthy subjects: the role of glucocorticoid receptor alpha and beta isoforms ratio.

Only few studies have addressed the interindividual variation and tissue specificity of glucocorticoid (GC) sensitivity in healthy individuals, a phenomenon observed in pathological conditions. Alternative splicing of the glucocorticoid receptor (GR) produces alpha and beta isoforms. GRbeta has dominant-negative effects on hormone-induced GRalpha effects, and an increased expression of the GRbeta has been associated with glucocorticoid resistance. We determined, using a simple, rapid, and accurate Real-Time PCR assay, the individual mRNAs expression of GRalpha and GRbeta in 26 normal subjects (mean+/-SE, age 30+/-6 years; 12 males and 14 females), in order to evaluate the role of these isoforms in glucocorticoid sensitivity in health. Glyceraldehydes-3-phosphate dehydrogenase (GAPDH) was used as a housekeeper gene. GRalpha/GAPDH, GRbeta/GAPDH and GRalpha/GRbeta ratios showed a normal distribution. We observed a higher expression of GRalpha compared to GRbeta and an interindividual variability in the GRalpha, GRbeta, and GAPDH gene expressions in the young healthy population. In addition, no correlation was observed between GRalpha/GRbeta ratio and the dexamethasone (DEX) doses needed to suppress plasma cortisol, GRalpha/GRbeta ratio and the concentration of DEX that caused inhibition of Con-A stimulated cell proliferation, and GRalpha/GRbeta ratio and the affinity of GR (Kd) of each subject. Therefore, the variability of GC sensitivity observed in normal subjects can not be ascribed to the variation in the GRalpha and GRbeta expression.     

Modulation of glucocorticoid receptor expression, inflammation, and cell apoptosis in septic guinea pig lungs using methylprednisolone

The use of glucocorticoids for treatment of sepsis has waxed and waned during the past several decades, and recent randomized controlled trials have evoked a reassessment of this therapy. Most glucocorticoid actions are mediated by its specific intracellular receptors (GRs). Thus we initially evaluated whether sepsis and high-dose corticosteroid therapy can regulate guinea pig pulmonary expression of GRs: active receptor, GRalpha, and dominant negative receptor, GRbeta. Sepsis induction by LPS injection (300 mug/kg ip) decreased mRNA and protein levels of GRalpha and increased protein expression of GRbeta in lungs. High-dose methylprednisolone (40 mg/kg ip), administered simultaneously with LPS, markedly potentiated the decrease in GRalpha expression but slightly affected the increase in GRbeta expression. Consequently, this led to a significant reduction in GRalpha nuclear translocation. Nevertheless, methylprednisolone treatment strongly eliminated LPS induction of NF-kappaB activity, as determined by NF-kappaB nuclear translocation and by gel mobility shift assays. Furthermore, the LPS-induced increase in inflammatory cells in bronchoalveolar lavage fluid was blunted by administration of the corticosteroid. On the other hand, immunofluorescent staining for cleaved caspase-3 showed a marked increase in this proapoptotic marker in lung sections, and terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling (TUNEL) represented an enhanced appearance of cell apoptosis in lungs and spleen when methylprednisolone was given together with LPS. Cell apoptosis is now considered to play a role in the pathogenesis of septic syndrome. We thus suggest that the action of glucocorticoids at high doses to accelerate sepsis-induced cell apoptosis may overwhelm their therapeutic advantages in septic shock.     

Elevation of human tumor necrosis factor-like weak inducer of apoptosis in peripheral blood mononuclear cells is correlated with disease activity and lupus nephritis in patients with systemic lupus erythematosus

Tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) is a recently identified proinflammatory cytokine of the TNF superfamily. Studies have indicated that TWEAK plays an important role in renal, vascular injury and immune disease. The aim of this study was to explore the expression of the TWEAK in peripheral blood mononuclear cells (PBMCs) and analyze the correlation between TWEAK and disease activity and renal damage of SLE. The expression of TWEAK in PBMCs was determined by RT-PCR and western blot. SLE disease activity was evaluated by Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) 2000 score. Next were analyzed the correlations of TWEAK mRNA and protein to serum IL-10, MCP-1 and some laboratory parameters of SLE disease activity. Subjects comprised 48 patients with SLE including 25 patients with renal damage and 23 without, 20 patients with rheumatoid arthrithis (RA) and 15 healthy controls. The results showed that TWEAK expressions in PBMCs from SLE patients were significantly higher than that in RA patients or healthy controls, especially higher in those patients with renal disease. Elevated production of TWEAK is correlated positively and significantly with SLEDAI, proteinuria, serum anti-dsDNA, IL-10 and MCP-1, but inversely associated with serum complements. Our results suggested that TWEAK in PBMCs is positively related to SLE disease activity and might be involved in the pathogenesis of SLE.     

Increased expression and phosphorylation of the two S100A9 isoforms in mononuclear cells from patients with systemic lupus erythematosus: a proteomic signature for circulating low-density granulocytes

Proteins differentially expressed in peripheral blood mononuclear cells (PBMCs) from systemic lupus erythematosus (SLE) patients versus Normal controls were identified by 2-DE and MALDI-MS. Thus, S100A9 expression was significantly increased in SLE PBMCs relative to Normal PBMCs at both mRNA and protein levels. Increased S100A9 levels in SLE PBMCs correlated positively with the abnormal presence of low-density granulocytes (LDGs) detected by flow-cytometry in the mononuclear cell fractions. Another set of proteins that were differentially expressed in SLE PBMCs formed S100A9-independent clusters, suggesting that these differences in protein expression are in fact reflecting changes in the abundance of specific cell types. In SLE PBMCs spots of the two S100A9 isoforms, S100A9-l and S100A9-s, and their phosphorylated counterparts were identified and confirmed to be phosphorylated at Thr¹¹³ by MS/MS analyses. In addition, the phorbol ester PMA alone or in combination with ionomycin induced a stronger increase in threonine phosphorylation of S100A9 in SLE than in Normal PBMCs, while the same stimuli caused the opposite effect on phosphorylation and activation of Erk1/2, suggesting the existence of an abnormal S100A9 signaling in SLE PBMCs. Therefore, the expansion and activation of LDGs in SLE seems to underlie this prominent S100A9 signature.     

趋化因子CXCL14在SLE患者外周血单个核细胞中的表达及启动子甲基化分析

目的：分析趋化因子CXCL14在系统性红斑狼疮（SLE）患者外周血单个核细胞（PBMC）中的表达及其启动子甲基化特征。方法：收集28例SLE患者和20名健康人外周血单个核细胞（PBMC）标本,抽提细胞中RNA,逆转录后,以GAPDH为内参,通过定量PCR检测PBMC中CXCL14的表达,统计分析CXCL14表达水平与SLE各种临床资料的相关性,探讨PBMC中CXCL14表达与SLE的关系,通过重亚硫酸盐修饰的全基因组DNA用以BSP测序来明确不同标本中CXCL14启动子中甲基化位点的甲基化率。结果：CXCL14在SLE患者和正常人PBMC中的表达量存在显著差异（P < 0.05）。与健康人PBMC中CXCL14的含量相比较,CXCL14在SLE患者PBMC中表达量显著降低。与进一步CXCL14表达水平与SLE各种临床资料的相关性分析显示,CXCL14表达水平与SSB抗体（干燥综合征B抗体）、蛋白尿及血小板计数相关。与SSB抗体阴性SLE患者比较,SSB抗体阳性患者CXCL14表达水平更低（P <0.05）;与蛋白尿阴性SLE患者相比,蛋白尿阳性患者CXCL14表达水平更低（P < 0.05）;而血小板升高患者的CX-CL14表达水平更高（P < 0.05）。CXCL14表达水平与SLE活动、肾损害指标、抗ds-DNA、C反应蛋白（CRP）、补体C3水平等指标未见显著相关性。CXCL14启动子区甲基化分析显示,SLE患者CpG岛甲基化明显高于正常对照。结论：PBMC中低表达的CXCL14与SLE发生发展相关,其启动子区CpG岛过度甲基化是SLE患者CXCL14低表达的重要机制。     

系统性红斑狼疮患者外周血单个核细胞Toll样受体9的表达及血清白介素-10水平的研究

目的:研究Toll样受体9(TLR-9)在系统性红斑狼疮(SLE)患者外周血单个核细胞(PBMCs)上的表达水平及SLE患者血清白介素-10水平,探讨发病机制。方法:从23例活动期、19例缓解期SLE患者和20例正常对照组中分离PBMCs,利用反转录-聚合酶链反应(RT-PCR)法检测PBMCs中TLR9 mRNA的表达水平,利用酶联免疫吸附试验法检测其血清白介素-10水平。结果:活动期SLE患者PBMCs的TLR-9mRNA表达高于缓解组(P<0.01)及正常对照(P<0.01),缓解期和正常对照组相比,差异无统计学意义(P>0.05)。SLE活动期患者血清IL-10水平显著高于缓解期患者(P<0.01),并均高于正常对照组(P<0.01)。结论:活动期SLE患者PBMC的TLR9 mRNA的表达水平增高;并且活动期及缓解期SLE患者血清IL-10水平升高可能与TLR9 mRNA表达的上调相关。     

New insights into the role and mechanism of macrophage migration inhibitory factor in steroid-resistant patients with systemic lupus erythematosus

Introduction

Glucocorticoid (GC) therapy remains important in improving the prognosis of patients with systemic lupus erythematosus (SLE). However, some patients do not achieve an effective response with GC treatment, creating an obstacle to the remission of SLE. Identification of the underlying mechanisms responsible for steroid resistance can be significant. Macrophage migration inhibitory factor (MIF) arouses our interest because of its reciprocal relationship with GCs. In the present study, we investigated for the first time whether MIF correlated with steroid resistance in SLE and explored potential mechanisms of action.

Methods

Sixty-two patients with SLE (40 steroid sensitive and 22 steroid resistant) and 21 normal controls were recruited. Serum levels of MIF were measured by ELISA. Cytosolic MIF and IκB expression in peripheral blood mononuclear cells (PBMCs) were determined by western blotting. The electrophoretic mobility shift assay was assessed by NF-κB in nuclear aliquots. Gene silencing was applied to reduce expression of MIF in PBMCs in steroid-resistant patients. PBMCs obtained from steroid-sensitive patients were treated with recombinant human MIF of different concentrations.

Results

MIF levels in serum and PBMCs were higher in steroid-resistant patients compared with steroid-sensitive patients and controls. In contrast to the steroid-sensitive group, NF-κB levels were significantly higher and IκB levels lower in steroid-resistant patients. After MIF gene silencing, IκB levels in cells from steroid-resistant patients were increased. In steroid-sensitive patients, a decrease in IκB levels and an increase in NF-κB expression from baseline were detected in PBMCs treated with a higher concentration of recombinant human MIF. Treatment with recombinant human MIF did not regulate expression of IκB and NF-κB in PBMCs from patients treated with an anti-MIF monoclonal antibody.

Conclusions

Our results indicated that MIF may play a role in the formation of steroid resistance in SLE by affecting the NF-κB/IκB signaling cascade. As a regulator of glucocorticoid sensitivity, MIF may be a potential target for steroid sparing.     

Differential distribution of glucocorticoid and estrogen receptor isoforms: localization of GRbeta and ERalpha in nucleoli and GRalpha and ERbeta in the mitochondria of human osteosarcoma SaOS-2 and hepatocarcinoma HepG2 cell lines

The localization of glucocorticoid and estrogen receptors alpha (GRalpha, ERalpha) and beta (GRbeta, ERbeta) in osteosarcoma SaOS-2 and hepatocarcinoma HepG2 cells was studied by immunofluorescence labelling and confocal laser scanning microscopy, as well as by subcellular fractionation and immunoblotting of the proteins of the fractions with respective antibodies. In HepG2 and SaOS-2 cells GRbeta and ERalpha were localized mainly in the nucleus, particularly concentrated in nuclear structures, which on the basis of their staining with antibody against C23-nucleolin, were characterized as nucleoli. A faint, diffuse GRbeta and ERalpha staining was also observed in the cytoplasm. GRalpha and ERbeta were specifically enriched at the site of cell mitochondria, which were visualized by labelling with the vital dye CMX. Immunoblotting experiments corroborated the immunofluorescence labelling distribution of glucocorticoid and estrogen receptor isoforms in the cell lines studied. These findings support the concept of a direct action of steroid/thyroid hormones on mitochondrial functions by way of their cognate receptors and also suggest a direct involvement of GRbeta and ERalpha in nucleolar-related processes in HepG2 and SaOS-2 cells.