SOCS3 mRNA Expression Research Articles

Differential Early Secreted Antigen Target (ESAT) 6 kDa–induced IFN-γ and SOCS1 expression distinguishes latent and active tuberculosis

Expression of Suppressor of cytokine signaling (SOCS)-1 molecules is increased in patients with tuberculosis (TB). Early Secreted Antigen Target (ESAT)-6 kDa - induced IFN-γ responses indicate Mycobacterium tuberculosis infection. The effect of ESAT6- stimulation on SOCS1 in the host is not known. Healthy asymptomatic controls had a negative (n = 16) or a positive ( n = 13) tuberculin skin test (TST). ESAT6-induced IFN-γ responses classified these controls as positive (EC ESAT6 IFN-γ (+), n = 5) or negative (EC ESAT6 IFN-γ (-), n = 24) responders. Patients had pulmonary (n = 21) or extra-pulmonary (n = 30) tuberculosis. Peripheral blood cells were stimulated with ESAT6 and mRNA expression of IFN-γ and SOCS1 was determined. ESAT6-induced IFN-γ expression was raised in EC ESAT6 IFN-γ (+) as compared with EC ESAT6 IFN-γ (-), p = 0.019. ESAT6-induced SOCS1 mRNA expression was increased in both pulmonary TB and extra-pulmonary TB patients as compared with both EC groups. ESAT6-induced IFN-γ/SOCS1 mRNA expression ratio was decreased in TB patients as compared with both EC groups. M. tuberculosis infection induces increased ESAT6-induced IFN-γ responses in both latent and active TB. Our data shows down-regulation of IFN-γ / SOCS1 expression to be induced only in active TB cases, distinguishing them from healthy individuals likely to have latent TB. A decreasing IFN-γ /SOCS1 ratio may leads to reduced Th1 immunity which contributes to inability of the host to control clinical disease.

Respiratory Syncytial Virus NS1 Protein Degrades STAT2 by Inducing SOCS1 Expression

Objectives: Respiratory syncytial virus (RSV) nonstructural protein NS1 (NS1) has been shown to block interferon (IFN)-inducible antiviral signaling. The suppressor of cytokine signaling (SOCS) gene family could utilize a feedback loop to block the activation of the JAK/STAT signaling pathway, further inhibiting the activation of host type I IFN. We evaluated the role of the SOCS1 and SOCS3 genes in this antiviral mechanism. Material and Methods: A humanized stable NS1 (rich in GC)-expressing plasmid was constructed, and A549 cells were transfected with it. Expression of the SOCS1, SOCS3, RIG-I, and TLR3 mRNAs was measured with real-time PCR. STAT2 and pSTAT2 expression was determined with Western blotting. Results: RSV NS1 upregulated SOCS1 mRNA expression 30-fold increase compared with the baseline level in very early phase (p < 0.01), and silence of RIG-I or TLR3 mRNA did not affect NS1-induced SOCS1 expression. NS1 inhibited IFN-α-induced STAT2 phosphorylation and degraded STAT2 in a time-dependent manner compared with the empty-vector control (p < 0.05). Conclusion: RSV NS1 upregulates SOCS1 expression in a RIG-I- and TLR3-independent pathway, to inhibit STAT2 phosphorylation.

Cytokine signaling-1 suppressor is inducible by IL-1beta and inhibits the catabolic effects of IL-1beta in chondrocytes: its implication in the paradoxical joint-protective role of IL-1beta

IntroductionAlthough IL-1β is believed to be crucial in the pathogenesis of osteoarthritis (OA), the IL-1β blockade brings no therapeutic benefit in human OA and results in OA aggravation in several animal models. We explored the role of a cytokine signaling 1 (SOCS1) suppressor as a regulatory modulator of IL-1β signaling in chondrocytes.MethodsCartilage samples were obtained from patients with knee OA and those without OA who underwent surgery for femur-neck fracture. SOCS1 expression in cartilage was assessed with immunohistochemistry. IL-1β-induced SOCS1 expression in chondrocytes was analyzed with quantitative polymerase chain reaction and immunoblot. The effect of SOCS1 on IL-1β signaling pathways and the synthesis of matrix metalloproteinases (MMPs) and aggrecanase-1 was investigated in SOCS1-overexpressing or -knockdown chondrocytes.ResultsSOCS1 expression was significantly increased in OA cartilage, especially in areas of severe damage (P < 0.01). IL-1β stimulated SOCS1 mRNA expression in a dose-dependent pattern (P < 0.01). The IL-1β-induced production of MMP-1, MMP-3, MMP-13, and ADAMTS-4 (aggrecanase-1, a disintegrin and metalloproteinase with thrombospondin motifs 4) was affected by SOCS1 overexpression or knockdown in both SW1353 cells and primary human articular chondrocytes (all P values < 0.05). The inhibitory effects of SOCS1 were mediated by blocking p38, c-Jun N-terminal kinase (JNK), and nuclear factor κB (NF-κB) activation, and by downregulating transforming growth factor-β-activated kinase 1 (TAK1) expression.ConclusionsOur results show that SOCS1 is induced by IL1-β in OA chondrocytes and suppresses the IL-1β-induced synthesis of matrix-degrading enzymes by inhibiting IL-1β signaling at multiple levels. It suggests that the IL-1β-inducible SOCS1 acts as a negative regulator of the IL-1β response in OA cartilage.

The Interferon Score Towards Interferon Alpha Tailored Therapy In Essential Thrombocythemia

IntroductionInterferon-alpha 2 (IFN) is able to induce hematological response in about 70-80% of ET patients but some of them could be defined as bad responders.IFN binding its receptor results in tyrosine cross-phosphorylation and auto-phosphorylation of the JAKs proteins (Tyk2 and Jak1). These phosthyrosines recruit and activate STAT family member such as STAT1 and STAT3. These proteins induce the transcription of SOCSs, whose role is to extinguish cytokine signaling by inhibition of JAK kinase-activity directly through the KIR-domain, and indirectly promoting the proteasomal degradation of Jak2, by SOCS-box-motif.In summary, IFN induces the expression of SOCSs, which inhibit TPO mediated signaling through Jak2 double inhibition. This allows IFN-α and TPO pathway to cross-talks by means of the JAK-STAT-SOCS cascade. AimsTo identify molecular markers that identify those patients who respond to IFN, we analyzed bone marrow cells transcript levels of specific genes involved in the IFN receptor pathway, whose signal cross-talks with the TPO dependent JAK-STAT pathway. In particular we investigated the mRNA expression of JAK1, TYK2, STAT1, STAT3, SOCS1 and SOCS3. MethodsWe analyzed 60 ET patients treated with 3 million units of IFN-α-2b 5 times a week as induction (3 months), and 3 times a week as maintenance. Responses were classified as follow: Good-Responders(R) (n=44), those who achieved complete response according to European Leukemia Net criteria, and Bad-Responders(NR) (n=17) who didn't reach the criteria.The mRNA expression of genes of interest was measured in bone marrow samples from ET patients by RTq-PCR and tested for their predictive value using receiver operating characteristics (ROC) curves.Data were normalized as following: [mRNA normalized copy number (NCN)=mRNA target gene/mRNA GUSB].An IFN score was calculated as an average in log2 of mRNA levels of genes differently expressed between Good-R and Bad-R. ResultsMain clinical characteristics were similar between the two groups of response. JAK2 V617F mutation was detected in 56,8% of Good-R and 58,8% of Bad-R (p=0,81) and no difference was found in JAK2V617F allele burden (p=0,17) and mRNA expression (p=0,2). Patients showed a median spleen volume of 500 ml in Good-R and 250 ml in Bad-R group (p=0.01).Bad-R compared with Good-R showed higher mRNA expression of JAK1 (13.4 vs 4.7; p<0.00001), STAT3 (2.7 vs 2.4; p=0.0002) and SOCS3 (1.1 vs 2; p=0,002). The AUC, using the normalized gene expression values, was 0.88 for JAK1, 0.81 for STAT3 and 0.7 for SOCS3.Average expression in log2 of these three genes was calculated and used as IFN score. The ROC curve AUC analysis for IFN-score revealed an AUC of 0.9 (95% CI:0.8-1.0). The score value with highest combined sensitivity (94.1%, 95% CI: 71.3-99.8) and specificity (88.6%, 95% CI: 75.4-96.2) was 4.74, with a likelihood ratio of 8.28. In our cohort, all Bad-R patients but one, showed IFN-score higher than the established cut off, and this further support the accuracy of our IFN-responsive score for ET patients. ConclusionsWe identified a set of three genes whose expression could be compounded into IFN score that showed a significant correlation with response in ET patients.The IFN score could represent a predictive biomarker for responsiveness to IFN and may likely become a substantial aid to the physician. Disclosures:No relevant conflicts of interest to declare.

Production of interferon α and β, pro-inflammatory cytokines and the expression of suppressor of cytokine signaling (SOCS) in obese subjects infected with influenza A/H1N1

Obesity was recognized as an independent risk factor for morbidity and mortality during last influenza A/H1N1 pandemic. Mechanisms involved in the high mortality risk from obesity during influenza A virus include reduced type I interferon production and delayed pro-inflammatory response, which lead to a higher rate of morbidity and mortality in murine models. In this study, we evaluated the production of type I interferons, pro-inflammatory and anti-inflammatory cytokines in peripheral blood mononuclear cells (PBMCs) from obese and lean subjects with and without confirmed infection of influenza A/H1N1. The expression levels of the suppressor of cytokine signaling-1 (SOCS1), SOCS3 and nuclear factor-kB were also evaluated. Cytokines were measured by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) and/or by ELISA in PBMCs stimulated with toll like receptor-3 (TLR-3) and TLR-7 ligands. The mRNA expression of SOCS1 and SOCS3 were evaluated by qRT-PCR. The obese volunteers infected with influenza A/H1N1 showed a diminished ability to produce type I interferon in response to TLR-3 ligand. Interestingly, the pro-inflammatory response was also affected in TLR-3 stimulated PBMCs. Obese influenza-free volunteers showed an increased basal expression of SOCS3, but not SOCS1. During influenza infection, SOCS1 and SOCS3 expression was higher in the lean infected volunteers in contrast to those who were obese infected. These data suggest that obesity is related to TLR-3 impairment and explain, at least in part, the inadequate immune response of obese individuals during infection with influenza A/H1N1 virus.

Transcriptional down-regulation of suppressor of cytokine signaling (SOCS)-3 in chronic obstructive pulmonary disease.

BackgroundTobacco is a leading environmental factor in the initiation of respiratory diseases and causes chronic obstructive pulmonary disease (COPD). Suppressor of cytokine signaling (SOCS) family members are involved in the pathogenesis of many inflammatory diseases and SOCS-3 has been shown to play an important role in the regulation, onset and maintenance of airway allergic inflammation indicating that SOCS-3 displays a potential therapeutic target for anti-inflammatory respiratory drugs development. Since chronic obstructive pulmonary disease (COPD) is also characterized by inflammatory changes and airflow limitation, the present study assessed the transcriptional expression of SOCS-3 in COPD.MethodsReal-time PCR was performed to assess quantitative changes in bronchial biopsies of COPD patients in comparison to unaffected controls.ResultsSOCS-3 was significantly down-regulated in COPD at the transcriptional level while SOCS-4 and SOCS-5 displayed no change.ConclusionsIt can be concluded that the presently observed inhibition of SOCS-3 mRNA expression may be related to the dysbalance of cytokine signaling observed in COPD.

Long-term fructose feeding changes the expression of leptin receptors and autophagy genes in the adipose tissue and liver of male rats: a possible link to elevated triglycerides

Long-term fructose consumption has been shown to evoke leptin resistance, to elevate triglyceride levels and to induce insulin resistance and hepatic steatosis. Autophagy has been suggested to function in processes such as lipid storage in adipose tissue and inflammation in liver. Autophagy and the leptin system have also been suggested to regulate each other. This study aimed to identify the changes caused by fetal undernourishment and postnatal fructose diet in the gene expression of leptin, its receptors (LEPR-a, LEPR-b, LEPR-c, LEPR-e and LEPR-f) and autophagy genes in the white adipose tissue (WAT) and liver of adult male rats in order to clarify the mechanism behind the metabolic alterations. The data clearly revealed that the long-term postnatal fructose diet decreased leptin levels (p<0.001), LEPR (p<0.001), especially LEPR-b (p=0.011) and LEPR-f (p=0.005), as well as SOCS3 (p<0.001), ACC (p=0.006), ATG7 (p<0.001), MAP1LC3β (p<0.001) and LAMP2 (p=0.004) mRNA expression in WAT. Furthermore, LEPR (p<0.001), especially LEPR-b (p=0.001) and LEPR-f (p<0.001), ACC (p=0.010), ATG7 (p=0.024), MAP1LC3β (p=0.003) and LAMP2 (p<0.001) mRNA expression in the liver was increased in fructose-fed rats. In addition, the LEPR expression in liver and MAP1LC3β expression in WAT together explained 55.7% of the variation in the plasma triglyceride levels of the rats (R adj. (2) =0.557, p<0.001). These results, together with increased p62 levels in WAT (p<0.001), could indicate decreased adipose tissue lipid storing capacity as well as alterations in liver metabolism which may represent a plausible mechanism through which fructose consumption could disturb lipid metabolism and result in elevated triglyceride levels.

IL-4 confers resistance to IL-27–mediated suppression on CD4+ T cells by impairing signal transducer and activator of transcription 1 signaling

TH2 cells play a critical role in the pathogenesis of allergic asthma. Established TH2 cells have been shown to resist reprogramming into TH1 cells. The inherent stability of TH2 cells poses a significant barrier to treating allergic diseases. We sought to understand the mechanisms by which CD4(+) T cells from asthmatic patients resist the IL-27-mediated inhibition. We isolated and cultured CD4(+) T cells from both healthy subjects and allergic asthmatic patients to test whether IL-27 can inhibit IL-4 production by the cultured CD4(+) T cells using ELISA. Culturing conditions that resulted in resistance to IL-27 were determined by using both murine and human CD4(+) T-cell culture systems. Signal transducer and activator of transcription (STAT) 1 phosphorylation was analyzed by means of Western blotting and flow cytometry. Suppressor of cytokine signaling (Socs) mRNA expression was measured by using quantitative PCR. The small interfering RNA method was used to knockdown the expression of Socs3 mRNA. We demonstrated that CD4(+) T cells from asthmatic patients resisted the suppression of IL-4 production mediated by IL-27. We observed that repeated exposure to TH2-inducing conditions rendered healthy human CD4(+) T cells resistant to IL-27-mediated inhibition. Using an invitro murine culture system, we further demonstrated that repeated or higher doses of IL-4 stimulation, but not IL-2 stimulation, upregulated Socs3 mRNA expression and impaired IL-27-induced STAT1 phosphorylation. The knockdown of Socs3 mRNA expression restored IL-27-induced STAT1 phosphorylation and IL-27-mediated inhibition of IL-4 production. Our findings demonstrate that differentiated TH2 cells can resist IL-27-induced reprogramming toward TH1 cells by downregulating STAT1 phosphorylation and likely explain why the CD4(+) T cells of asthmatic patients are resistant to IL-27-mediated inhibition.

An integrated analysis of SOCS1 down‐regulation in HBV infection‐related hepatocellular carcinoma

Persistent inflammation together with genetic/epigenetic aberrations is strongly associated with chronic Hepatitis B virus (HBV) infection-related hepatocarcinogenesis. Here, we investigated the alterations of the suppressor of cytokine signalling (SOCS) family genes in HBV-related hepatocellular carcinoma (HCC). A total of 116 patients with HCC were enrolled in this study. The methylation statuses of SOCS1-7 and CISH genes were quantitatively measured and clinicopathological significance of SOCS1 methylation was statistically analysed. The gene copy number variation was assayed by aCGH. Luciferase reporter assay and Western blot were used to detect the involvement of SOCS1 in p53 signalling. We found high frequencies of SOCS1 gene hypermethylation in both tumour (56.03%) and adjacent nontumour tissues (54.31%), but tumour tissues exhibited increased methylation intensity (24.01% vs 13.11%, P < 0.0001), particularly in patients with larger tumour size or cirrhosis background (P < 0.0001). In addition, the frequency and intensity of SOCS1 hypermethylation in tumour tissues were both significantly higher than those in nontumour tissues in male gender patients and in patients ≥45 years old (P = 0.0214 and P < 0.0001, P = 0.0232 and P < 0.0001, respectively). SOCS1 gene deletion was found in 8 of 25 aCGH assayed tumour specimens, which was associated with lower SOCS1 mRNA expression (P = 0.0448). Furthermore, ectopic SOCS1 overexpression could activate the p53 signalling pathway in HCC cell lines. Hypermethylation of SOCS2-7 and CISH genes was seldom found in HCC. Our results suggested that the gene loss and epigenetic silencing of SOCS1 were strongly associated with HBV-related HCC.

Interleukin‐6‐induced satellite cell proliferation is regulated by induction of theJAK2/STAT3 signalling pathway through cyclin D1 targeting

To determine whether interleukin-6 (IL-6) stimulates rat muscle satellite cell proliferation in culture, and if so, to clarify the signalling mechanisms. Primary satellite cells were isolated from thirty male F344 rats, 11weeks of age. IL-6 at concentrations of 0.01, 0.1, 1, 10 or 100ng/ml was added to culture media. IL-6 at 0.01-1ng/ml induced dose-dependent increase in cell proliferation. After treatment with 1ng/ml IL-6, cell proliferation increased by 31%, and p-STAT3(+) /MyoD(+) cells increased in number compared to those in control media (P<0.05). Inhibitors of JAK2 (AG 490) and STAT3 (STAT3 peptide) blocked the increase in BrdUrd(+) cell numbers at 6h post stimulation with 1ng/ml IL-6 (P<0.05). Furthermore, cyclin D1 mRNA expression and cyclin D1(+) /MyoD(+) cell numbers significantly increased in cultures treated with 1ng/ml IL-6 compared to those in control media (P<0.05). In contrast, treatment with 10 and 100ng/ml IL-6 did not stimulate cell proliferation. Treatment with 10ng/ml IL-6 induced greater SOCS3 mRNA expression than with 1ng/ml IL-6 and control media. Moreover, co-localization of SOCS3 and myogenin was observed after treatment with 10ng/ml IL-6. IL-6 induced dose-dependent increase in satellite cell proliferation by activating the JAK2/STAT3/cyclin D1 pathway.

Effects of fibroblast growth factor-21 gene knockdown on the expression of hepatic gluconeogenesis-related genes and the JAK2/STAT3 signaling pathway in ApoE-/-mice

Objective To investigate the effects of fibroblast growth factor-21 (FGF-21) gene knockdown on the expression of hepatic glyconeogenesis-related genes in ApoE-/-mice and its mechanism.Methods Twenty-four 8-week-old male ApoE-/-mice were randomly divided into chow diet fed group(NC,n =6),chow diet+pAd-shFGF-21group(NF,n=6),high-fat diet fed group(HC,n=6),and high-fat diet+pAd-shFGF-21 group(HF,n=6).Mice were fed for 16 weeks.NC and HC groups injected with pAd-shGFP,however NF and HF groups were injected with pAd-shFGF-21 through the tail vein at the end of the 15th week.At the end of the 16th week,blood samples were collected for measurement of plasma glucose,insulin and lipid levels.Hepatic tissues were collected for measurement of mRNA and protein levels of glyconeogenesis-related genes and JAK2/STAT3 signaling pathway via real-time PCR and Western-blot,respectively.Results The fasting plasma glucose (FBG),fasting plasma insulin (PIns),triglycerides,total cholesterol,and low-density lipoprotein-cholesterol (LDL-C) were significantly higher,while the high-density lipoprotein-cholesterol(HDL-C)was lower in FGF-21-knockdown groups compared with control groups,respectively(P＜0.05 or P＜0.01).When fed with normal and high-fat diet,hepatic FGF-21 mRNA levels were decreased by 76.6％ (P＜0.05) and 40.9％ (P＜0.05) compared with those in control groups,while hepatic FGF-21protein levels were decreased by 67.8％ (P＜0.05) and 49.6％ (P＜0.05),respectively.The hepatic glucose-6-phosphatase(G6pase) and phosphoenolpyruvate carboxykinase (PEPCK)mRNA and protein expression in FGF-21-knockdown groups were significantly increased compared with those in control groups(P＜0.05).At the same time we observed decreased expression of hepatic SOCS3 mRNA and decreased expression of phosphorylated STAT3 and SOCS3 protein in FGF-21 knockdown groups.Conclusions FGF-21 can repress the expression of liver glyconeogenesis-related genes and its mechanism might involve the JAK2/STAT3 signaling pathway. Key words: Fibroblast growth factor-21; Gluconeogenesis; JAK2/STAT3 signaling pathway; Insulin resistance

CpG ODN-induced suppressor of cytokine signaling-3 expression is required for the activation of MAP kinase and NF-κB via toll-like receptors-9. (P6303)

Abstract Toll-like receptors (TLRs) recognize pathogen-derived structures which can activate the innate immune system and play an important role in the recognition of various microbial-derived molecules; lipopolysaccharide (LPS), lipoteichoic acid (LTA), peptidoglycan (PGN), and bacterial DNA/synthetic CpG oligodeoxynucleotides (CpG ODN). In this study, we demonstrated that unmethylated CpG ODNs induces suppressor of cytokine signaling (SOCS-3) expression from murine macrophage RAW 264.7 cells. CpG ODN-mediated SOCS-3 expression is regulated at transcriptional level and requires de novo protein synthesis. Expression of SOCS-3 mRNA and protein was blocked by inhibitors of TLR-9, MAP kinases, and NF-κB, indicating that TLR-9 dependent MAP kinases and the subsequent NF-κB activation is required for the CpG ODN-induced SOCS-3 expression.

Maternal pre‐gravid body mass index and adiposity influence umbilical cord gene expression at term in AGA infants

While maternal obesity is associated with unfavorable maternal and fetal outcomes, the influence of maternal obesity on fetal gene expression is less clear. Umbilical cords (UC) from 12 lean (pre‐gravid BMI &lt; 25) and 10 overweight/obese (OB, pre‐gravid BMI ≥25) women without gestational diabetes were collected at term and utilized for gene expression analysis using Human Primeview microarrays (Affymetrix). Although offspring birth weight and infant adiposity (at 2‐wk) did not differ between groups, expression of 232 transcripts was affected in UC from OB compared to lean mothers. GSEA analysis revealed an up‐regulation of genes related to metabolism, stimulus and defense response and inhibitory to insulin signaling in the OB group. We confirmed that Egr‐1, periostin, FosB, and NEDD9 mRNA expression was induced in UCs from OB moms, while endothelin receptor B, Klf10, Peg3 and Egln3 expression was decreased. mRNA expression of Egr‐1, FosB, MEST and SOCS1 was positively associated (p&lt;0.05) with mother's 1st trimester body fat mass (%). Metabolic and hormonal assessments were also assayed in mother's serum at 30 weeks of gestation and in UC plasma. Our data indicate that maternal obesity per se causes changes in UC gene expression favoring inflammation and insulin resistance, possibly predisposing infants of overweight moms to become overweight later on in life. Funding USDA ARS‐CRIS 6251–51000‐007–04S.

Multiple roles of SOCS proteins: Differential expression of SOCS1 and SOCS3 in atherosclerosis

Pro-inflammatory cytokines play a key pathogenic role in atherosclerosis, which are induced by the Janus kinase/signal transducer and activator of transduction (JAK/STAT) pathway. Furthermore, the JAK/STAT pathway is negatively regulated by the suppressor of cytokine signaling (SOCS) proteins. However, the change in SOCS expression levels and the correlation between SOCS expression and cholesterol levels in atherosclerosis is not yet well understood. To this end, a mouse model of atherosclerosis was established using apolipoprotein-deficient (ApoE(-/-)) mice. The mice were fed either a chow or high-fat diet. The mRNA and protein expression of SOCS1 and SOCS3 in plaque and vessels were determined at different time points. Furthermore, SOCS1 and SOCS3 mRNA expression was detected in the peripheral blood mononuclear cells (PBMCs) obtained from 18 male subjects with no coronary heart disease (non-CHD) population. The expression of SOCS1 in the ApoE(-/-) mice first increased and then decreased and the high-fat diet accelerated the appearance of the peak; the expression of SOCS3 increased with the increased feeding duration, and this trend was more pronounced in the mice fed the high-fat diet. SOCS1/CD68 and SOCS3/CD68 showed opposite trends in expression with the increased duration of the high-fat diet. Interleukin-6 (IL-6) expression in the main aorta of the ApoE(-/-) mice fed the high-fat diet also increased with the increased feeding duration. In the non-CHD population, the total serum cholesterol levels positively correlated with SOCS3 mRNA expression in the PBMCs (r=0.433, P=0.012). These results demonstrate the differential expression of SOCS1 and SOCS3 in atherosclerosis and suggest that SOCS3, together with IL-6 may promote the formation and development of atherosclerosis.

166 COMBINATION THERAPY WITH HUMANIZED ANTIHUMAN IL-6R ANTIBODY AND INTERFERON-α SUCCESSFULLY PREVENTS THE GROWTH OF 786-O RENAL CELL CARCINOMA CELLS

You have accessJournal of UrologyKidney Cancer: Basic Research (I)1 Apr 2013166 COMBINATION THERAPY WITH HUMANIZED ANTIHUMAN IL-6R ANTIBODY AND INTERFERON-α SUCCESSFULLY PREVENTS THE GROWTH OF 786-O RENAL CELL CARCINOMA CELLS Kei Ishibashi, Toshiki Oguro, Shin Kumagai, Norio Takahashi, Nobuhiro Haga, Masanori Nomiya, Tomohiko Yanagida, Ken Aikawa, and Yoshiyuki Kojima Kei IshibashiKei Ishibashi Fukushima, Japan More articles by this author , Toshiki OguroToshiki Oguro Fukushima, Japan More articles by this author , Shin KumagaiShin Kumagai Fukushima, Japan More articles by this author , Norio TakahashiNorio Takahashi Fukushima, Japan More articles by this author , Nobuhiro HagaNobuhiro Haga Fukushima, Japan More articles by this author , Masanori NomiyaMasanori Nomiya Fukushima, Japan More articles by this author , Tomohiko YanagidaTomohiko Yanagida Fukushima, Japan More articles by this author , Ken AikawaKen Aikawa Fukushima, Japan More articles by this author , and Yoshiyuki KojimaYoshiyuki Kojima Fukushima, Japan More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2013.02.1546AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Clinical studies have revealed that increased serum IL-6 concentrations in patients are associated with advanced tumor stages of renal cell carcinoma (RCC) and short survival. We evaluated the effect of combination therapy with humanized antihuman IL-6R antibody tocilizumab and interferon-α in vitro and in vivo against RCC 786-O cells which induce autocrine secretion of IL-6 by interferon-α. METHODS MTT assays were performed for determination of cellular proliferation and viability by tocilizumab and/or interferon-α use in 786-O cells. Real-time quantitative RT-PCR and western blotting analysis were performed to detect SOCS3 mRNA expression and phosphorylation of STAT proteins and ERK. Xenograft assays in nude mice were used for analysis in vivo effects of combination therapy with tocilizumab and interferon-α. RESULTS Use of tocilizumab or interferon-α alone could not suppress the growth of 786-O cells. A significant interferon-α-induced growth inhibitory effect was observed when both tocilizumab and interferon-α were added to 786-O cells. Real-time quantitative PCR revealed that the absolute level of SOCS-3 mRNA was significantly increased upon interferon-α treatment. Tocilizumab significantly decreased the SOCS3 expression level after IFN stimulation in 786-O cells (p=0.023). Western blot analysis revealed that the addition of tocilizumab enhanced the interferon-induced phosphorylation of STAT1 and inhibited SOCS3 expression and the phosphorylation of both STAT3 and ERK. The growth of tumors in athymic mice receiving combination therapy with tocilizumab and interferon-α was retarded in comparison with those in the tocilizumab, interferon-α and untreated groups. The tumor volume was significantly lower in the combination therapy group than in the other groups at the end of the study (p<0.05). Morphological observation of the tumors revealed apoptosis, invasion of inflammatory cells and fibrosis. A remarkable reduction in interferon-induced SOCS3 protein expression by tocilizumab was confirmed. CONCLUSIONS Inhibition of SOCS3 by tocilizumab enhanced the anti-tumor activity of interferon-α, both in vitro and in vivo. Our findings suggest that combination therapy using an antihuman IL-6R antibody with interferon may represent a novel therapeutic approach for the treatment of RCC. © 2013 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 189Issue 4SApril 2013Page: e68-e69 Advertisement Copyright & Permissions© 2013 by American Urological Association Education and Research, Inc.MetricsAuthor Information Kei Ishibashi Fukushima, Japan More articles by this author Toshiki Oguro Fukushima, Japan More articles by this author Shin Kumagai Fukushima, Japan More articles by this author Norio Takahashi Fukushima, Japan More articles by this author Nobuhiro Haga Fukushima, Japan More articles by this author Masanori Nomiya Fukushima, Japan More articles by this author Tomohiko Yanagida Fukushima, Japan More articles by this author Ken Aikawa Fukushima, Japan More articles by this author Yoshiyuki Kojima Fukushima, Japan More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Expression of M. tuberculosis-induced suppressor of cytokine signaling (SOCS) 1, SOCS3, FoxP3 and secretion of IL-6 associates with differing clinical severity of tuberculosis

BackgroundAppropriate immune activation of T cells and macrophages is central for the control of Mycobacterium tuberculosis infections. IFN-γ stimulated responses are lowered in tuberculosis (TB), while expression of Suppressor of Cytokine Signaling (SOCS) molecules – 1 and 3 and CD4+CD25+FoxP3+T regulatory cells is increased. Here we investigated the association of these molecules in regard to clinical severity of TB.MethodsPeripheral blood mononuclear cells (PBMCs) were isolated from patients with pulmonary TB (PTB, n = 33), extra-pulmonary TB (ETB, n = 33) and healthy endemic controls (EC, n = 15). Cases were classified as moderately advanced or far advanced PTB, and less severe or severe disseminated ETB. M. tuberculosis -stimulated IFN-γ, SOCS1, SOCS3 and FoxP3 gene expression and secretion of Th1 and Th2 cytokines was measured. Statistical analysis was performed using Mann–Whitney U, Wilcoxon Rank and Kruskal Wallis non-parametric tests.ResultsIn un-stimulated PBMCs, IL-6 (p = 0.018) and IL-10 (p = 0.013) secretion levels were increased in PTB while IL-10 was also increased in ETB (p = 0.003), all in comparison with EC. M. tuberculosis-stimulated IL-6 (p = 0.003) was lowered in ETB as compared with EC. SOCS1 mRNA expression in M. tuberculosis stimulated PBMCs levels in moderately advanced PTB (p = 0.022), far advanced (p = 0.014) PTB, and severe ETB (p = 0.009) were raised as compared with EC. On the other hand, SOCS1 mRNA titers were reduced in less severe ETB, in comparison with severe ETB (p = 0.027) and far advanced PTB (p = 0.016). SOCS3 mRNA accumulation was reduced in far advanced PTB (p = 0.007) and FoxP3 mRNA expression was increased in less severe ETB as compared with EC (p = 0.017).ConclusionsThe lowered SOCS1 mRNA levels in patients with less severe extra-pulmonary TB as compared to those with more severe ETB and PTB may lead to elevated IFN-γ pathway gene expression in the latter group. As localized ETB has shown to be associated with more effective Th1 immunity and adaptive responses, this suggests a role for SOCS1 in determining disease outcome in extra-pulmonary TB.

Impact and long-term effect of three prescriptions regulating and tonifying lung and kidney on JAK/STAT signaling in rats

To evaluate the impact and long-term effect of three prescriptions regulating and tonifying lung and kidney (prescription tonifying lung and spleen, prescription tonifying lung and kidney, and prescription tonifying Qi and kidney) on JAK/STAT signaling of COPD rats. Rats were randomly divided into the control group, the model group, the Bufeijianpi group, the Bufeiyishen group, the Yiqizishen group and the aminophyline group. The COPD rat model was established by smoke inhalations and bacterial infections. In the 9th week, the control group and the model group were administered with normal saline, while the remaining groups are orally given corresponding medicines. In the 20th and 32nd week, the rats were sacrificed in batches to observe the pathology in their lung tissues, protein expressions of JAK2, STAT1, STAT3, STAT5, and expressions of JAK2 and SOCS3 mRNA. In the 20th and 32nd week, protein expressions of JAK2 mRNA and phosphorylation-JAK2, STAT1, STAT3 and STAT5 in the model group were higher than the control group (P < 0.01), whereas the three traditional Chinese medicine (TCM) (Bufeijianpi, Bufeiyishen and Yiqizishen) groups and the aminophyline group were significantly lower (P < 0.05, P < 0.01). The expression of SOCS3 mRNA in the model group was higher than the control group (P < 0.01), whereas the level was notably higher in the three TCM groups and the aminophylline group (P < 0.01). The three TCM groups were remarkably higher than the aminophylline group (P < 0.05, P < 0.01). Compared with the figures in the 20th week, JAK2 mRNA and phosphorylation-JAK2, STAT3 and STAT5 were significantly lower in the Bufeijianpi group in the 32nd week (P < 0.05, P < 0.01), and so did phosphorylation-STAT3 in Bufeiyishen group (P < 0.01) and phosphorylation-STAT3 and STAT5 in the Yiqizishen group (P < 0.05, P < 0.01). However, the aminophylline group showed no significant difference in above indicators. The three medicines regulating and tonifying lung and kidney can effectively relieve injury of lung tissues, and have long-term effect, which may be related to the regulation of JAK/ STAT signaling. Specifically, prescription tonifying lung and spleen shows good effect in reducing JAK2, STAT3 and STAT5, prescription tonifying lung and kidney shows good effect in reducing p-STAT3, and prescription tonifying Qi and kidney shows good effect in reducing p-STAT3 and p-STAT5.

Cycloheximide stimulates suppressor of cytokine signaling-3 gene expression in 3T3-L1 adipocytes via the extracellular signal-regulated kinase pathway

Suppressor of cytokine signaling (SOCS)-3 can act as a regulator of energy metabolism and cytokine signaling in fat cells. It is regulated by hormones and toxicological factors. However, the action of cycloheximide on expression of adipocyte SOCS-3 gene is unknown. Using 3T3-L1 adipocytes, we found that cycloheximide up-regulated SOCS-3 mRNA expression in dose- and time-dependent manners. Treatment with actinomycin D prevented cycloheximide-stimulated SOCS-3 mRNA expression, suggesting that the effect of cycloheximide requires new mRNA synthesis. While cycloheximide was shown to increase activities of MEK1 and JNK, signaling was demonstrated to be inhibited by pretreatment with either MEK1 inhibitors U0126 and PD98059, or with the JNK inhibitor SP600125. U0126 and PD98059, respectively, reduced cycloheximide-stimulated SOCS-3 mRNA expression, but SP600125 did not antagonize cycloheximide effect. Moreover, cycloheximide was observed to up-regulate expression of other SOCS family members, such as SOCS-1, -2, -4, -5, -6, -7, and cytokine-inducible SH2-containing protein (CIS)-1 mRNAs. Such effects varied with the dosage and duration of cycloheximide treatment. These results imply the functional MEK1/ERK-mediated pathway is necessary for the cycloheximide stimulation of SOCS-3 gene expression.

Effects of SOCS 1/3 gene silencing on the expression of C/EBPα and PPARγ during differentiation and maturation of rat preadipocytes

Suppressor of cytokine signaling-1 and -3 (SOCS-1 and SOCS-3) are two important negative regulators in the insulin-signaling pathway, and their overexpression may aggravate insulin resistance. Subjects with insulin resistance are often obese and have increased expressions of SOCS-1 and SOCS-3. We speculated that SOCS-1 and SOCS-3 may be involved in abnormal deposition of adipose tissues during insulin resistance. A catch-up growth intrauterine growth retardation (CG-IUGR) rat model with insulin resistance was established; mRNA and protein expression of SOCS-1, SOCS-3, the CCAAT/enhancer binding protein (C/EBPα), and peroxisome proliferator-activated receptor (PPARγ) in adipose tissue were measured by real-time PCR and western blot; plasmids carrying small hairpin RNAs (shRNAs) targeting the SOCS-1 and SOCS-3 genes were constructed and transfected into preadipocytes, which were then induced to mature. At 72 h after differentiation was induced, the expressions of C/EBPα and PPARγ, two important molecules promoting the differentiation of preadipocytes, were detected. Expressions of SOCS-1, SOCS-3, C/EBPα, and PPARγ were markedly increased in adipose tissues of CG-IUGR rats, whereas the expressions of C/EBPα and PPARγ were significantly reduced after gene silencing of SOCS-1 or SOCS-3 in adipocytes. Overexpression of SOCS-1 and SOCS-3 may enhance the expression of C/EBPα and PPARγ, resulting in abnormal deposition of adipose tissues during insulin resistance.

Expression of suppressor of cytokine signaling in peripheral blood monocular cells of patients with sympathetic ophthalmia

Objective To investigate the mRNA and protein expression of suppressor of cytokine signaling (SOCS) in peripheral blood mononuclear cells(PBMC) of patients with sympathetic ophthalmia.Methods Blood samples were taken from 14 sympathetic ophthalmia patients with active uveitis,13 quiescent patients,and 15 healthy individuals.IFN-γ,IL-12 and IL-4 in the serum were measured by ELISA.PBMC were subjected to analysis of SOCS mRNA and protein expression using quantitative RT-PCR and western blot,respectively.Results The level of IFN-γ,IL-12 and IL-4 were all significantly higher in the serum of sympathetic ophthalmia patients with active uveitis than that in controls and quiescent patients(P ＜0.05).There were no significant differences between the quiescent patients and the controls(P ＞ 0.05).Compared to healthy volunteers,the expressions of cytokine inducible SH2 containing protein (CIS),SOCS1,SOCS3 and SOCS5 mRNA and protein were significantly increased.There were no significant differences in expression of CIS,SOCS1,SOCS3 mRNA and protein in quiescent patients,but SOCS5 mRNA and protein were significantly increased.Conclusion Up regulation of SOCS1 and SOCS5 expression may involve in the development of a Th1 mediated immune response in sympathetic ophthalmia patients with active uveitis.CIS and SOCS3 may play important roles in mitigating the pathogenic effects of proinflammatory cytokines.It were kept balance in quiescent patients,and up-regulation of SOCS5 may have protective functions. Key words: Suppressor of cytokine signaling; Sympathetic ophthalmia