Interleukin-2 Receptor Protein Research Articles

Anti-Inflammatory Sesquiterpenes from Fruiting Bodies of Schizophyllum commune.

Schizophyllum commune, a fleshy fungus, is an important medicinal and food-homologous mushroom in China. In this work, eight undescribed sesquiterpenes schizomycins A-H (1-8) and one new meroterpenoid schizomycin I (9) together with three known analogues (10-12) were isolated from fruiting bodies of S. commune. Their planar structures were established by extensive spectroscopic and mass spectrometric data. The absolute configurations of compounds 1, 2, and 4 were determined by single crystal X-ray diffraction, and compounds 3 and 5-9 were confirmed by electronic circular dichroism calculations. Anti-inflammatory activities of all isolated compounds were evaluated for their inhibitory effects on IL-6 and IL-1β production in RAW 264.7 cells. Among them, compound 7 exhibited significant IL-6 inhibitory activity with an IC50 value of 3.6 μM. The results of molecular docking showed that compound 7 interacts with amino acid residues (Gly117, Lys118, Asp120, Thr166, and Try168) of the IL-6 receptor protein through hydrogen bonding.

Human retinal endothelial cells express functional interleukin-6 receptor

BackgroundInterleukin (IL)-6 is an inflammatory cytokine present in the eye during non-infectious uveitis, where it contributes to the progression of inflammation. There are two major IL-6 signaling pathways: classic signaling and trans-signaling. Classic signaling requires cellular expression of the IL-6 receptor (IL-6R), which exists in membrane-bound (mIL-6R) and soluble (sIL-6R) forms. Prevailing dogma is that vascular endothelial cells do not produce IL-6R, relying on trans-signaling during inflammation. However, the literature is inconsistent, including with respect to human retinal endothelial cells.FindingsWe examined IL-6R transcript and protein expression in multiple primary human retinal endothelial cell isolates, and assessed the effect of IL-6 on the transcellular electrical resistance of monolayers. Using reverse transcription-polymerase chain reaction, IL-6R, mIL-6R and sIL-6R transcripts were amplified in 6 primary human retinal endothelial isolates. Flow cytometry on 5 primary human retinal endothelial cell isolates under non-permeabilizing conditions and following permeabilization demonstrated intracellular stores of IL-6R and the presence of mIL-6R. When measured in real-time, transcellular electrical resistance of an expanded human retinal endothelial cell isolate, also shown to express IL-6R, decreased significantly on treatment with recombinant IL-6 in comparison to non-treated cells across 5 independent experiments.ConclusionsOur findings indicate that human retinal endothelial cells produce IL-6R transcript and functional IL-6R protein. The potential for classic signaling in human retinal endothelial cells has implications for the development of therapeutics targeted against IL-6-mediated pathology in non-infectious uveitis.

IL-6 upregulates the expression of IL-6R through the JAK2/STAT3 signalling pathway to promote progression of hepatocellular carcinoma.

The progression of hepatocellular carcinoma (HCC) involves multifactor, multistep interactions. High expression of interleukin-6 receptor (IL-6R) plays an important role in the occurrence and development of tumours, but the regulatory mechanism of IL-6R expression and its function in HCC have not been fully defined. Western blot was used to evaluate the phosphorylation of key kinases in the JAK2/STAT3 pathway and the protein expression levels of related proliferation molecules, migration molecules and apoptotic molecules. The antiapoptosis, migration and proliferation of cells of each group were analysed with JC-1 to judge the cell apoptosis rate, the EdU method to determine the proliferation vitality of the cells, clone formation experiments and Transwell experiments. High expression of IL-6R in cell lines, lower protein levels of the apoptotic molecules c-Caspase7 and c-Caspase3 and higher protein levels of the proliferative molecules p-P70S6K and migration molecules MMP9 and MMP2 were consistent with stronger antiapoptosis, proliferation and migration. Interestingly, IL-6 upregulated the expression of IL-6R by activating the JAK2/STAT3 signalling pathway. Also, the expression of IL-6R protein was downregulated after lentivirus knockdown of STAT3. In nude mice bearing subcutaneous tumours, upregulation of IL-6R expression after activation of the JAK2/STAT3 signalling pathway by IL-6 significantly increased tumour growth. Moreover, the expression of IL-6R protein was downregulated, and the terminal tumour volume was significantly downregulated in the lentiviral STAT3 knockdown group. IL-6 regulated the transcription of IL-6R through the activation of the JAK2/STAT3 signalling pathway.

Provisional Designation of the Interleukin-6 Receptor (IL-6R) as a Novel Marker Gene for Exercise Tolerance

Objectives: Genomic markers linked to exercise-associated health benefits and/or sporting performance are increasingly used to guide decision-making in healthcare and sport/exercise science. This project investigated whether the IL-6R SNP “rs2228145” might be provisionally designated a novel physical activity/exercise marker. rs2228145 results in an Aspartate358/Alanine358 change adjacent to the site where the IL-6R protein is cleaved into two fragments, resulting in ~two-fold increases in blood-borne levels of soluble IL-6R [‘sIL-6R’].Methods: Cohorts of staff/students at Cardiff Metropolitan University donated/completed: [i] finger-prick capillary blood samples (subjected to ELISAs for sIL-6R, the associated signalling protein sgp130, and the IL-6/sIL-6R complex); [ii] cheek-swab samples containing buccal epithelial cell DNA (subjected to PCR-based IL-6R/rs2228145 genotyping assays); [iii] International Physical Activity Questionnaires (to estimate physical activity levels in the week preceding sample donation).Results: As expected, we observed significant genotype-dependent differences in blood-borne sIL-6R levels (CC (44.1±21.7ng/mL) vs. AC (28.6±7.3ng/mL) vs. AA (19.9±6.5ng/mL; P<0.05)); Importantly, AA homozygotes undertook significantly more physical activity than AC heterozygotes (6318±899 v. 3904±2280 MET-mins/week; P<0.01). Genotype was significantly associated with physical activity levels (P<0.05), and sIL-6R (P=0.197) and sgp130 (P=0.160) showed non-significant correlations with physical activity levels.Conclusions: These data suggest that IL-6R/rs2228145 genotype may influence participation in physical activity/exercise, perhaps by impacting on abilities to tolerate activity without experiencing adverse-effects. Although more research is required to confirm these preliminary findings, designation of IL-6R/rs2228145 as a novel marker, and determination of participants’ IL-6R/rs2228145 genotypes, may in future be useful tools to aid exercise-providers in designing personalised exercise programmes matched to clients/patients.

Circulating miR-451a levels as a potential biomarker to predict the prognosis of patients with multiple myeloma.

The natural course of multiple myeloma (MM) varies greatly between patients. The Revised MM International Staging System (R-ISS) identifies high-risk patients, but it is unsuitable for assessing minimal residual disease (MRD). Furthermore, the focal location of myeloma cells and clonal evolution often produce false negative results in flow cytometry. Extracellular microRNA (miRNA/miR) expression levels are stable in bodily fluids, and are retrievable and measurable from fresh or archived serum or plasma samples. Therefore, the present study aimed to investigate the clinical utility of circulating miRNA levels in patients with MM, particularly miR-451a, which is commonly downregulated in MM, and whether it could predict the prognosis and relapse of patients with MM. In total, 66 patients with MM, stratified using the R-ISS criteria, were recruited, while 10 healthy subjects (transplantation donors) were enrolled as controls. Reverse transcription-quantitative PCR was used to evaluate miR-451a expression in bone marrow (BM) and in the circulation. IL-6 levels were measured using ELISA, while western blotting was conducted to analyze the protein expression levels of the IL-6 receptor (IL-6R). During follow-up, MRD was assessed via multiparameter flow cytometry (MFC). miR-451a was identified to target IL-6R using a dual-luciferase reporter assay. Circulating miR-451a levels were low in patients with MM, and was found to be 0.39 times that of the control group (U=4.00; P<0.001). Among the 66 patients with MM, the median level of miR-451a was 0.73 and 0.41 times that of the control group in R-ISS stage I MM (15 patients) and R-ISS stage II stage (17 patients), respectively; patients with R-ISS stage III MM (34 patients) had the lowest level, at 0.24 times the value of the control group. Circulating miR-451a levels had a strong positive correlation with miR-451a levels in BM, but negatively correlated with IL-6 and IL-6R levels. After two courses of consolidation chemotherapy, 19 patients achieved complete remission, 10 of whom presented steady circulating miR-451a levels during follow-up; the other nine patients had an abrupt decrease in circulating miR-451a levels. The turning points in the trend appeared 4–8 weeks before positive results were obtained via MFC, and 4–16 weeks before clinical relapse. Moreover, miR-451a overexpression notably downregulated the expression of the IL-6R mRNA and protein. Collectively, circulating miR-451a levels potentially represent a novel biomarker to monitor MRD and predict relapse.

MicroRNA-30b participates in the pathological process of hyperuricemia by regulating interleukin-6 receptor

The present study aimed to examine the expression of hyperuricemia (HUA)-related factors in the body fluids of HUA patients and in renal tissues and body fluids of HUA mice to elucidate the underlying mechanism of HUA and provide theoretical basis for the diagnosis, prevention and treatment of this disease. A total of 51 HUA patients (HUA group), and 36 healthy subjects (control group) were included in the present study. The peripheral blood and urine were collected from all patients and healthy subjects. A total of 20 male Kunming mice were used to construct HUA model, and another 20 mice were used as controls. The kidney tissues, peripheral blood and urine were collected from all mice. ELISA was performed to determine the levels of interleukin-6 receptor (IL-6R) proteins in the serum and urine of human or mice, while western blotting was employed to determine the protein expression in the kidney tissues of mice. Quantitative real-time polymerase chain reaction was used to measure the expression of mRNA and miR-30b in all sample types. Dual luciferase reporter assay was performed to identify the direct interaction between 3′-untranslated region of IL-6R mRNA and miR-30b. The expression of IL-6R mRNA and protein was increased in serum and urine of HUA patients, while the expression of miR-30b was reduced in HUA patients when compared with healthy subjects. The contents of uric acid, urea nitrogen and creatinine in the blood of HUA mice model were significantly elevated. Similarly, the expression of IL-6R mRNA and protein was increased in kidney, serum and urine of HUA mice model, while the expression of miR-30b was reduced in kidney tissues, serum and urine of HUA mice model. Dual luciferase reporter assay showed that miR-30b was able to bind with 3′-UTR seed region of IL-6R mRNA to regulate its expression. These findings demonstrated that the expression of IL-6R in patients and mouse with HUA is elevated, which is related with the down-regulation of miR-30b. Therefore, miR-30b might participate in the pathological process of HUA by regulating IL-6R.

Transforming growth factor-β dampens interleukin-6 signaling in chondrocytes by decreasing the interleukin-6 receptor

Purpose: Interleukin-6 (IL-6) is a pro-inflammatory cytokine present in increased levels in serum and synovial fluid from patients with osteoarthritis (OA) and is even considered to be predictive for disease progression. Previous studies implicate IL-6 in articular cartilage destruction, which is the main hallmark of OA. In contrast, transforming growth factor-β (TGF-β) is an important homeostatic regulator of cartilage that can decrease the catabolic effects of pro-inflammatory cytokines in chondrocytes. Multiple mechanisms of cross-talk between TGF-β and IL-6 are reported in other tissues. In this study, we investigated whether TGF-β is able to control IL-6 signaling in chondrocytes. Methods: Human primary chondrocytes and the human G6 chondrocyte cell line were stimulated with rhTGF-β1 (1.0 ng/ml). We determined mRNA expression levels of IL-6 and IL-6 receptor (IL-6R) using qPCR, measured IL-6 protein release using Luminex multianalyte technology and IL-6R protein levels by Western Blot. rhIL-1β was used as a positive control for IL-6 induction. Induction of signal transducer and activator of transcription (STAT)3 phosphorylation by TGF-β was determined using Western Blot, in presence of the IL-6R inhibitor Tocilizumab to study IL-6R dependency. Induction of suppressor of cytokine signaling (SOCS)3 gene expression was used as a read-out for IL-6/STAT3 mediated transcriptional activity. Results: TGF-β increased both IL-6 mRNA expression and protein release in G6 and primary chondrocytes. Moreover, we observed activation of the main IL-6R downstream signaling protein STAT3 after TGF-β exposure, which could be completely prevented by blockade of the IL-6R, suggesting IL-6 dependency. We further explored whether TGF-β mediated IL-6/p-STAT3 signaling resulted in activation of target genes, and measured expression of SOCS3, which is a well-known IL-6 target gene as well as an important regulator of IL-6 signaling. Surprisingly however, TGF-β did not increase SOCS3 expression, despite the increased IL-6 and p-STAT3 levels we observed in these cells. This unexpected finding suggests regulation of STAT3 mediated transcriptional activity by TGF-β. The effect of exogenously added rhIL-6 was also inhibited by TGF-β, as 6h pre-treatment with TGF-β prevented rhIL-6-induced SOCS3 expression. At the level of STAT3, TGF-β decreased the response to rhIL-6 as well, although p-STAT3 levels were not reduced to baseline level. This points towards a crucial role for TGF-β in the regulation of IL-6 effects upstream of STAT3. Interestingly, we observed decreased expression of the IL-6R in both primary and G6 chondrocytes after exposure to TGF-β, which might explain the decreased intracellular IL-6 signaling. This effect was not dependent on IL-6 itself, as IL-1β did not affect IL-6R expression, despite being a strong inducer of IL-6. Finally, we showed that rescuing IL-6R levels using soluble IL-6R dose-dependently restored induction of SOCS3 and p-STAT3 by rhIL-6 in the presence of TGF-β. This confirms that TGF-β inhibition of IL-6 effects is indeed dependent on reduced IL-6R levels. Conclusions: Our study reveals a novel, protective effect of TGF-β that could help preserve cartilage homeostasis under inflammatory conditions by dampening IL-6 signaling via IL-6R down regulation.

Decreased expression of microRNA-30b promotes the development of pulpitis by upregulating the expression of interleukin-6 receptor.

The present study aimed to examine the expression of interleukin-6 receptor (IL-6R) mRNA and protein in pulp tissues, blood and saliva from patients with pulpitis. It also investigated the association between IL-6R and microRNA (miR)-30b, as well as their effects on pulpitis. A total of 28 patients with pulpitis were recruited into the experimental group and 16 subjects with no pulpitis who also underwent tooth extraction were recruited into the control group. Pulp tissues, plasma and saliva were collected from all participants. Reverse transcription-quantitative polymerase chain reaction was used to determine the expression of IL-6R mRNA and miR-30b in all sample types. Western blot analysis was performed to examine the protein expression of IL-6R in pulp tissues, while ELISA was used to determine the contents of IL-6R protein in the plasma and saliva samples. A dual luciferase reporter assay was performed to verify the interactions between IL-6R and miR-30b. The expression of IL-6R mRNA in the pulp tissues, plasma and saliva was significantly increased in patients with pulpitis compared with the control group. Similarly, the IL-6R protein expression in the samples from patients with pulpitis were also significantly increased compared with the control group. Conversely, the expression of miR-30b was significantly reduced in the samples from patients with pulpitis compared with the control group. The dual luciferase reporter assay revealed that miR-30b may bind with the 3'-untranslated seed region of IL-6R mRNA to regulate its expression. The present study demonstrated that the upregulated expression of IL-6R in pulp tissues, plasma and saliva from patients with pulpitis was associated with the downregulation of miR-30b expression. In addition, miR-30b may affect the progression of pulpitis via IL-6R and may be a potential genetic marker for the diagnosis of pulpitis.

MicroRNA-497 regulates the proliferation of clear cell renal cell carcinoma via interleukin-6 receptor

This study determined the expression of microRNA-497 (miR-497) and interleukin-6 receptor (IL-6R) in tumour, blood and urine from patients with clear cell renal cell carcinoma (ccRCC), and examined their relationship with the proliferation of ccRCC. Twenty-eight ccRCC patients and 32 healthy subjects gave peripheral blood and urine samples. Tumour and tumour-adjacent tissues were collected from ccRCC patients. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to measure the expression of IL-6R mRNA and miR-497; Western blotting, to determine IL-6R protein expression in tumour tissues or ccRCC Caki-1 cells; enzyme-linked immunosorbent assay, to measure the content of IL-6R protein in blood or urine; dual luciferase reporter assay, to test the direct interaction between miR-497 and the 3′-untranslated region (UTR) of IL-6R mRNA; MTT (methyl thiazolyl tetrazolium) assay, to determine the proliferation of cells. The expression of IL-6R mRNA and protein was significantly higher in tumour tissues than in tumour-adjacent tissues. Similarly, the expression of IL-6R mRNA and protein in blood and urine was significantly higher in ccRCC patients than in healthy subjects. The levels of miR-497 in tumour tissues, blood and urine from ccRCC patients were decreased. miR-497 bound with the 3′-UTR of IL-6R mRNA to regulate the expression of IL-6R. Overexpression of miR-497 inhibited the proliferation of ccRCC Caki-1 cells by down-regulating the expression of IL-6R. These results demonstrate that up-regulation of IL-6R expression in tumour tissues, blood and urine from ccRCC patients is related with the down-regulation of miR-497 expression. miR-497 might regulate the proliferation of ccRCC via IL-6R.

Genome-wide association study of CSF levels of 59 alzheimer's disease candidate proteins: significant associations with proteins involved in amyloid processing and inflammation.

Cerebrospinal fluid (CSF) 42 amino acid species of amyloid beta (Aβ42) and tau levels are strongly correlated with the presence of Alzheimer's disease (AD) neuropathology including amyloid plaques and neurodegeneration and have been successfully used as endophenotypes for genetic studies of AD. Additional CSF analytes may also serve as useful endophenotypes that capture other aspects of AD pathophysiology. Here we have conducted a genome-wide association study of CSF levels of 59 AD-related analytes. All analytes were measured using the Rules Based Medicine Human DiscoveryMAP Panel, which includes analytes relevant to several disease-related processes. Data from two independently collected and measured datasets, the Knight Alzheimer's Disease Research Center (ADRC) and Alzheimer's Disease Neuroimaging Initiative (ADNI), were analyzed separately, and combined results were obtained using meta-analysis. We identified genetic associations with CSF levels of 5 proteins (Angiotensin-converting enzyme (ACE), Chemokine (C-C motif) ligand 2 (CCL2), Chemokine (C-C motif) ligand 4 (CCL4), Interleukin 6 receptor (IL6R) and Matrix metalloproteinase-3 (MMP3)) with study-wide significant p-values (p<1.46×10−10) and significant, consistent evidence for association in both the Knight ADRC and the ADNI samples. These proteins are involved in amyloid processing and pro-inflammatory signaling. SNPs associated with ACE, IL6R and MMP3 protein levels are located within the coding regions of the corresponding structural gene. The SNPs associated with CSF levels of CCL4 and CCL2 are located in known chemokine binding proteins. The genetic associations reported here are novel and suggest mechanisms for genetic control of CSF and plasma levels of these disease-related proteins. Significant SNPs in ACE and MMP3 also showed association with AD risk. Our findings suggest that these proteins/pathways may be valuable therapeutic targets for AD. Robust associations in cognitively normal individuals suggest that these SNPs also influence regulation of these proteins more generally and may therefore be relevant to other diseases.

Treatment of rheumatoid arthritis with traditional chinese medicine.

Rheumatoid arthritis (RA) is a chronic inflammatory disease that will affect quality of life and, working efficiency, and produce negative thoughts for patients. Current therapy of RA is treated with disease-modifying antirheumatic drugs (DMARDs). Although most of these treatment methods are effective, most patients still have a pleasant experience either due to poor efficacy or side effects or both. Interleukin-6 receptor (IL6R) is important in the pathogenesis of RA. In this study, we would like to detect the potential candidates which inhibit IL6R against RA from traditional Chinese medicine (TCM). We use TCM compounds from the TCM Database@Taiwan for virtually screening the potential IL6R inhibitors. The TCM candidate compound, calycosin, has potent binding affinity with IL6R protein. The molecular dynamics simulation was employed to validate the stability of interaction in the protein complex with calycosin. The analysis indicates that protein complex with calycosin is more stable. In addition, calycosin is known to be one of the components of Angelica sinensis, which has been indicated to have an important role in the treatment of rheumatoid arthritis. Therefore, calycosin is a potential candidate as lead compounds for further study in drug development process with IL6R protein against rheumatoid arthritis.

Upregulation of skeletal muscle inflammatory genes links inflammation with insulin resistance in women with the metabolic syndrome

The metabolic syndrome, a combination of interrelated metabolic risk factors, is associated with insulin resistance and promotes the development of cardiovascular diseases and type 2 diabetes mellitus. There is a close link between inflammation and metabolic disease, but the responsible mechanisms remain elusive. The aim of this study was to identify differentially expressed genes in insulin-resistant skeletal muscle tissue of women with the metabolic syndrome compared with healthy control women. Women with the metabolic syndrome (n = 19) and healthy control women (n = 20) were extensively phenotyped, insulin sensitivity was measured using a hyperinsulinaemic euglycaemic clamp, and a skeletal muscle biopsy was obtained. Gene expression levels were compared between the two groups by microarrays. The upregulated genes in skeletal muscle of the women with the metabolic syndrome were primarily enriched for inflammatory response-associated genes. The three most significantly upregulated of this group, interleukin 6 receptor (IL6R), histone deacetylase 9 (HDAC9) and CD97 molecule (CD97), were significantly correlated with insulin resistance. Taken together, these findings suggest an important role for a number of inflammatory-related genes in the development of skeletal muscle insulin resistance.

Species Specificity of ADAM10 and ADAM17 Proteins in Interleukin-6 (IL-6) Trans-signaling and Novel Role of ADAM10 in Inducible IL-6 Receptor Shedding

Hypomorphic ADAM17(ex/ex) mice showed defects in mucosal regeneration due to inefficient enhanced GFR shedding. ADAM17 is the main sheddase of interleukin-6 receptor (IL-6R) to induce IL-6 trans-signaling. However, serum levels of soluble murine IL-6R were not reduced in ADAM17(ex/ex) mice, and murine ADAM17 was not the major sheddase of murine IL-6R. Shedding of murine IL-6R by murine ADAM17 was rescued in chimeric murine IL-6R proteins containing any extracellular domain but not the transmembrane and intracellular domain of human IL-6R. Apoptosis is a physiological stimulus of ADAM17-mediated shedding of human IL-6R. Even though apoptosis induced IL-6R shedding in mice, the responsible protease was identified as ADAM10. ADAM10 also was identified as protease responsible for ionomycin-induced shedding of murine and human IL-6R. However, in ADAM10-deficient murine embryonic fibroblasts, compensatory shedding of human IL-6R was mediated by ADAM17, but loss of ADAM10-mediated shedding of murine IL-6R was compensated by an as-yet-unidentified protease. Finally, we identified physiological purinergic P2X7 receptor stimulation as a novel inducer of murine and human IL-6R shedding solely mediated by ADAM10. In conclusion, we describe an unexpected species specificity of ADAM10 and ADAM17 and identified ADAM10 as novel inducible sheddase of IL-6R in mice and humans, which might have consequences for the interpretation of phenotypes from ADAM17- and ADAM10-deficient mice.

Abstract 1337: Soluble IL-6Rα regulates endothelial survival and vascular integrity and promotes ovarian cancer progression

Abstract The immunoregulatory cytokine interleukin 6 (IL-6) has been recognized as an important modulator of ovarian cancer progression. However, whether IL-6 plays a direct role in the process remains controversial. On target cells, IL-6 binds to the IL-6 receptor (IL-6Rα) and signals via complex formation with the ubiquitously expressed gp130 receptor. Soluble IL-6 receptor protein (sIL-6Rα) binds IL-6 and can activate target cells that are deficient in or lack IL-6Rα, such as endothelial cells. This process is named trans signaling and serves as the major proinflammatory paradigm of IL-6 signaling under various pathologic conditions, including cancers. In the present report, we showed that sIL-6Rα is significantly elevated in malignant ascites from ovarian cancer patients and is associated with poor prognosis in advanced ovarian cancer patients. We identified that IL-6 trans signaling on endothelial cells prevents chemotherapy-induced apoptosis, induces endothelial hyperpermeability, and increases transendothelial migration of ovarian cancer cells. Selectively blocking the MAPK pathway with the ERK inhibitor PD98059 substantially reduced the IL-6/sIL-6Rα complex-mediated endothelial hyperpermeability. The activation of ERK by the IL-6/sIL-6Rα complex modulated endothelial integrity by initiating the activation of Src kinase activities and Y685 phosphorylation of VE-cadherin. Collectively, these data indicate that sIL-6Rα in malignant ascites of ovarian cancer may drive IL-6 signaling on endothelial cells, which, importantly, is involved in ovarian cancer progression. Specifically, blockage of sIL-6α-mediated signaling may present a promising therapeutic strategy for the management of ovarian cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1337.

ADAM17-mediated shedding of the IL6R induces cleavage of the membrane stub by γ-secretase

Interleukin-6 (IL6) signals are mediated by classic and trans-signaling. In classic signaling, IL6 first binds to the membrane bound Interleukin-6 Receptor (IL6R) whereas in trans-signaling, IL6 acts via a soluble form of the IL6R. Trans-signaling via the soluble IL6R (sIL6R) was linked to chronic inflammation and cancer. The release of the IL6R is mediated by the disintegrin and metalloproteinases ADAM10 and ADAM17. To analyze the fate of the C-terminal cleavage fragment after ectodomain shedding we fused the IL6R C-terminally to two Z-domains of Protein-A (2Z-tag) or to GFP. A specific C-terminal fragment of the IL6R protein could be detected after ADAM17-induced shedding. Using γ-secretase inhibitors and gene-deficient cells, we demonstrate that after ADAM17 mediated cleavage, the IL6R C-terminal fragment was cleaved by the γ-secretase at the plasma membrane. We were, however, not able to detect an IL6R intracellular domain. After γ-secretase cleavage IL6R cell surface expression was lost and γ-secretase cleavage product(s) of the IL6R were endocytosed. No GFP-fluorescence of a γ-secretase-cleaved IL6R-GFP fusion protein was observed in the nucleus. We therefore hypothesize that a potential IL6R intracellular domain fragment is not involved in nuclear signaling but rapidly degraded.

Changes in Expression of Interleukin-6 Receptors in Granulosa Cells During Follicular Atresia in Pig Ovaries

More than 99% of follicles undergo a degenerative process known as "atresia" in mammalian ovaries, and only a few follicles ovulate during follicular growth and development. Follicular selection predominantly depends on granulosa cell apoptosis. To reveal the molecular mechanisms of selective follicular atresia, we examined the changes in the levels of interleukin-6 (IL-6) receptors expressed in the granulosa cells of pig ovaries. The levels of IL-6 receptor (IL-6R)-alpha mRNA and protein in granulosa cells were quantified by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting, respectively. IL-6R alpha mRNA and protein were highly expressed in the granulosa cells of progressed atretic follicles. Enzyme-linked immunosorbent assay showed that the expression of IL-6 soluble receptor (IL-6sR) protein in follicular fluid decreased during atresia. Moreover, we isolated porcine cDNA encoding an IL-6 signal transducer, gp130. Porcine gp130 (2,754 bp and 917 amino acids) was identified from a cDNA library prepared using follicular granulosa cells of pig ovaries. Porcine gp130 was highly homologous with human and murine gp130. RT-PCR analysis revealed that the level of gp130 mRNA also decreased during atresia. We presume that IL-6sR and gp130, but not IL-6R alpha, play important roles in regulation of granulosa cell survival.

Increased interleukin-6 receptor expression in the paraventricular nucleus of rats with heart failure

Activation of the hypothalamic-pituitary-adrenal (HPA) axis and augmented plasma and tissue levels of IL-6 are hallmarks of heart failure (HF). Within the forebrain, cardiovascular homeostasis is mediated in part by the paraventricular nucleus (PVN) of the hypothalamus. IL-6, via binding to the IL-6 receptor (IL-6R)/glycoprotein 130 (gp130) complex influences cellular and physiological responses. Thus, in the current study, we hypothesized that PVN IL-6R protein and gene expression are upregulated in HF vs. sham-operated rats, whereas gp130 levels in the same tissues remain stable. Six weeks after coronary ligation surgery, hemodynamic measurements were obtained, and HF rats were divided into moderate noncongestive and severe chronic congestive groups based on cardiac indices. Plasma IL-6 levels were determined and changes in gene and protein expression of IL-6R and gp130 between sham-operated and HF rats were determined via real-time PCR and Western blot analyses, respectively. Plasma levels of IL-6 were elevated in rats with severe, but not moderate, HF compared with sham-operated controls. In both moderate and severe HF rats, protein but not gene expression of IL-6R was significantly increased in PVN tissue but not in non-PVN tissue, compared with sham-operated controls. Gene and protein levels of the gp130 subunit were not altered by HF in either tissue analyzed. Collectively, these data suggest that within the brain of HF rats, IL-6R expression is not a global change. Rather the increased IL-6 levels characteristic of HF may alter PVN-mediated physiological responses via enhanced expression of the IL-6R.

Interleukin‐6 receptor expression in contracting human skeletal muscle: regulating role of IL‐6

Contracting muscle fibers produce and release IL-6, and plasma levels of this cytokine are markedly elevated in response to physical exercise. We recently showed autocrine regulation of IL-6 in human skeletal muscle in vivo and hypothesized that this may involve up-regulation of the IL-6 receptor. Therefore, we investigated IL-6 receptor regulation in response to exercise and IL-6 infusion in humans. Furthermore, using IL-6-deficient mice, we investigated the role of IL-6 in the IL-6 receptor response to exercise. Human skeletal muscle biopsies were obtained in relation to: 3 h of bicycle exercise and rest (n=6+5), or recombinant human IL-6 infusion (rhIL-6) or saline infusion (n=6+6). We further obtained skeletal muscle samples from IL-6 knockout (KO) mice and wild-type C57/BL-6 mice in response to a 1-h bout of exercise. In exercising human skeletal muscle, IL-6 receptor mRNA increased sixfold with a peak at 6 h postexercise. Detection of the IL-6 receptor protein by immunohistochemistry revealed a pronounced staining following exercise that was primarily located at the cell membrane of the muscle fibers, whereas muscle gp130 expression and plasma levels of soluble IL-6 receptor were unaffected. Infusion of rhIL-6 to humans had no effect on the mRNA level of the IL-6 receptor, whereas there was an increase at the protein level. IL-6 receptor mRNA increased similarly in muscle of both IL-6 KO mice and wild-type mice in response to exercise. In conclusion, exercise increases IL-6 receptor production in human skeletal muscle. This effect is most prominent 6 h after the end of the exercise bout, suggesting a postexercise-sensitizing mechanism to IL-6 when plasma IL-6 is concomitantly low. Exercise-induced increases in IL-6 receptor mRNA most likely occurs via an IL-6 independent mechanism as shown in IL-6 KO mice and the human rhIL-6 infusion study, whereas IL-6 receptor protein levels are responsive to elevated plasma IL-6 levels.

Prostaglandin E2 mediates growth arrest in NFS-60 cells by down-regulating interleukin-6 receptor expression.

Interleukin-6 (IL-6), a potent myeloid mitogen, and the immunosuppressive prostanoid prostaglandin E2 (PGE2) are elevated following thermal injury and sepsis. We have previously demonstrated that bone marrow myeloid commitment shifts toward monocytopoiesis and away from granulocytopoiesis during thermal injury and sepsis and that PGE2 plays a central role in this alteration. Here we investigated whether PGE2 can modulate IL-6-stimulated growth in the promyelocytic cell line, NFS-60, by down-regulating IL-6 receptor (IL-6r) expression. Exposure of NFS-60 cells to PGE2 suppressed IL-6-stimulated proliferation as well as IL-6r expression. Receptor down-regulation is functionally significant since IL-6-induced signal transduction through activators of transcription (STAT)-3 is also decreased. Down-regulation of IL-6r correlated with the ability of PGE2 to arrest cells in the G0/G1 phase of the cell cycle. PGE2 appears to signal through EP2 receptors. Butaprost (EP2 agonist) but not sulprostone (EP3 agonist) inhibited IL-6-stimulated proliferation. In addition, an EP2 antagonist (AH6809) alleviated the anti-proliferative effects of PGE2. NFS-60 cells express predominantly EP2 and EP4 receptors. While PGE2 down-regulated both the IL-6r protein and mRNA expression, it had no influence on EP2 or EP4 mRNA expression. The present study demonstrates that PGE2 is a potent down-regulator of IL-6r expression and thus may provide a mechanistic explanation for the granulocytopenia seen in thermal injury and sepsis.

The interleukin-6/interleukin-6-receptorsystem is activated in donor hearts

ObjectivesTo assess the potential of the donor heart to respond to interleukin-6 (IL6), the present study investigated the expression of IL6 receptor components in the myocardium of donor hearts before transplantation. BackgroundDonor heart dysfunction early after transplantation has been associated with the cytokine storm after donor brain death. Proinflammatory cytokines are thought to play a central role in this process. Interleukin-6 is of specific interest because it has been associated with cardiac allograft dysfunction and is related to an impaired prognosis. Its action requires expression of the specific IL6 receptor (IL6R), and the common signal transducer of the IL6 family glycoprotein 130 (gp130) in the donor heart. MethodsThe activation of IL6, IL6R and gp130 messenger ribonucleic acid (mRNA) and protein was studied via reverse transcription-polymerase chain reaction (RT-PCR) and immunohistology in donor hearts (n = 6) and compared with patients undergoing evaluation of ventricular arrhythmias (control, n = 9) or with advanced heart failure (n = 20). ResultsMessenger RNA of IL6, IL6R and gp130 was strongly expressed in all chambers of donor hearts, whereas right ventricles of control patients did not show any expression (donor vs. control: p < 0.005). Right ventricles of failing hearts showed IL6, IL6R and gp130 mRNA levels comparable with those found in donor hearts. Immunohistochemistry paralleled the RT-PCR data on the protein level. While IL6 was mainly expressed by myocytes, both receptor components were preferentially found mainly on interstitial cells. ConclusionsThe expression of the IL6 receptor components in the donor heart before transplantation establishes the condition sine qua non for the response of the donor heart to circulating IL6. This mechanism may explain the close association of elevated IL6 serum levels to acute cardiac allograft dysfunction in the early perioperative period.