Induction Of SOCS3 Research Articles

Sphingosine 1-phosphate induced anti-atherogenic and atheroprotective M2 macrophage polarization through IL-4

Sphingosine 1-phosphate (S1P) has been implicated in anti-atherogenic properties of high-density lipoproteins. However, the roles and signaling of S1P in macrophages, the main contributor to atherosclerosis, have not been well studied. Furthermore, pro-inflammatory M1 and anti-inflammatory M2 macrophage phenotypes may influence the development of atherosclerosis. Therefore, we investigated the effects of S1P on macrophage phenotypes, especially on M2 polarization and its signaling in relation to the anti-atherogenic properties of S1P. It was found that S1P induced anti-inflammatory M2 polarization via IL-4 secretion and its signaling, and induced IL-4Rα and IL-2Rγ. In addition, down-stream signalings, such as, stat-6 phosphorylation, SOCS1 induction, and SOCS3 suppression were also observed in macrophages in response to S1P. Furthermore, S1P-induced ERK activation, and the inhibitions of p38 MAPK and JNK were found to be key signals for IL-4 induction. Moreover, the anti-atherogenic effect of S1P in HDL was confirmed by the observation that oxidized LDL-induced lipid accumulation was attenuated in S1P-treated M2 macrophages. Furthermore, the atheroprotective effect of S1P was demonstrated by its anti-apoptotic effect on S1P-treated macrophages. The present study shows that S1P-induced M2 polarization of macrophages could be mediated via IL-4 signaling, and suggests that M2 polarization by S1P is responsible for the anti-atherogenic and atheroprotective properties of high-density lipoproteins in vivo.

Inhibition of leptin-induced vascular extracellular matrix remodelling by adiponectin.

Vascular extracellular matrix (ECM) remodelling, which is the result of disruption in the balance of ECM synthesis and degradation, induces vessel fibrosis and thereby leads to hypertension. Leptin is known to promote tissue fibrosis, while adiponectin has recently been demonstrated to be anti-fibrogenic in tissue fibrosis. In this study, we aimed to evaluate the leptin-antagonist function of adiponectin and to further elucidate the mechanisms through which adiponectin dampens leptin signalling in vascular smooth muscle cells, thus preventing excess ECM production, in our already established 3D co-culture vessel models. Our 3D co-culture vessel model, which mimics true blood vessels, is composed of vascular endothelial cells, vascular smooth muscle cells and collagen type I. We validated the profibrogenic effects of leptin and analysed matrix metalloproteinase 2 (MMP2), MMP9, tissue inhibitor of metalloproteinase 1 (TIMP1) and collagen types II/IV secretion in 3D vessel models. The protective/inhibitory effects of adiponectin were re-analysed by inhibiting adiponectin receptor 1 (AdipoR) and AdipoR2 expression in endothelial cells using RNAi technology. In the 3D vessel models, adiponectin blocked the leptin-stimulated secretion of collagen types II/IV and TIMP1 while significantly increasing MMP2/9 activity. In endothelial cells, adiponectin induced phosphorylation of AMPK, thereby suppressing leptin-mediated STAT3 phosphorylation through induction of SOCS3 in smooth muscle cells. Our findings indicate that adiponectin disrupted the leptin-induced vascular ECM remodelling via AdipoR1 and enhanced AMPK signalling in endothelial cells, which, in turn, promoted SOCS3 up-regulation in smooth muscle cells to repress leptin-stimulated phosphorylation of STAT3.

Innate immune response to a H3N2 subtype swine influenza virus in newborn porcine trachea cells, alveolar macrophages, and precision-cut lung slices

Viral respiratory diseases remain of major importance in swine breeding units. Swine influenza virus (SIV) is one of the main known contributors to infectious respiratory diseases. The innate immune response to swine influenza viruses has been assessed in many previous studies. However most of these studies were carried out in a single-cell population or directly in the live animal, in all its complexity. In the current study we report the use of a trachea epithelial cell line (newborn pig trachea cells – NPTr) in comparison with alveolar macrophages and lung slices for the characterization of innate immune response to an infection by a European SIV of the H3N2 subtype. The expression pattern of transcripts involved in the recognition of the virus, interferon type I and III responses, and the host-response regulation were assessed by quantitative PCR in response to infection. Some significant differences were observed between the three systems, notably in the expression of type III interferon mRNA. Then, results show a clear induction of JAK/STAT and MAPK signaling pathways in infected NPTr cells. Conversely, PI3K/Akt signaling pathways was not activated. The inhibition of the JAK/STAT pathway clearly reduced interferon type I and III responses and the induction of SOCS1 at the transcript level in infected NPTr cells. Similarly, the inhibition of MAPK pathway reduced viral replication and interferon response. All together, these results contribute to an increased understanding of the innate immune response to H3N2 SIV and may help identify strategies to effectively control SIV infection.

Sphingosine 1‐phosphate induces anti‐inflammatory M2 polarization through IL‐4 signaling in macrophages (662.14)

Phenotypes of macrophages, classic pro‐inflammatory M1 and alternative anti‐inflammatory M2, could influence many disease states like atherosclerosis. Lipopolysaccharide (LPS)‐activated macrophages increase pro‐inflammatory cytokine secretion. Such LPS‐induced‐M1 phenotype expression was suppressed by sphingosine 1‐phosphate (S1P), an anti‐atherogenic lipid mediator in HDL. Previously, suppression of NF‐κB activity through S1P1 was suggested as a key mechanism for the suppression of M1 phenotype (Hughes et al. Circ Res. 2008; 102: 950‐958). In the present study, we addressed how S1P induces M2 macrophage polarization in mouse macrophages and found that S1P induced anti‐inflammatory M2 polarization through IL‐4 secretion and its signaling. S1P induced not only induction and secretion of IL‐4 but also induction of their receptors, IL‐4Rα and IL‐2Rγ. In addition, down‐stream signaling such as phosphorylation of stat‐6, induction of SOCS1, and suppression of SOCS3 expression was also observed in response to S1P. Furthermore, S1P‐induced activation of ERK and inhibition of p38 MAPK and JNK were found to be key signaling for IL‐4 induction. Present data suggest that anti‐atherogenic effect of S1P in HDL may be mediated through IL‐4 secretion and its phenotype switching from M1 to M2 anti‐inflammatory macrophages.Grant Funding Source: This research was supported by Basic Science Research Program through the NRF (2011‐0021158)

The role of c‐Jun in controlling the EPAC1‐dependent induction of the SOCS3 gene in HUVECs

The cyclic AMP sensor, EPAC1, activates AP1-mediated transcription in HUVECs. Correspondingly, induction of the SOCS3 minimal promoter by EPAC1 requires a single AP1 site that constitutively binds phosphorylated (Ser63) c-Jun in DNA-pull-down assays. c-Jun (Ser63) becomes further phosphorylated following cyclic AMP stimulation and specific activation of protein kinase A (PKA), but not through selective activation of EPAC1. Moreover, despite a requirement for c-Jun for SOCS3 induction in fibroblasts, phospho-null c-Jun (Ser63/73Ala) had little effect on SOCS3 induction by cyclic AMP in HUVECs. AP1 activation and SOCS3 induction by EPAC1 in HUVECs therefore occur independently of c-Jun phosphorylation on Ser63.

Jak/Stat signaling stimulates zebrafish optic nerve regeneration and overcomes the inhibitory actions of Socs3 and Sfpq.

The regenerative failure of mammalian optic axons is partly mediated by Socs3-dependent inhibition of Jak/Stat signaling (Smith et al., 2009, 2011). Whether Jak/Stat signaling is part of the normal regenerative response observed in animals that exhibit an intrinsic capacity for optic nerve regeneration, such as zebrafish, remains unknown. Nor is it known whether the repression of regenerative inhibitors, such as Socs3, contributes to the robust regenerative response of zebrafish to optic nerve damage. Here we report that Jak/Stat signaling stimulates optic nerve regeneration in zebrafish. We found that IL-6 family cytokines, acting via Gp130-coupled receptors, stimulate Jak/Stat3 signaling in retinal ganglion cells after optic nerve injury. Among these cytokines, we found that CNTF, IL-11, and Clcf1/Crlf1a can stimulate optic axon regrowth. Surprisingly, optic nerve injury stimulated the expression of Socs3 and Sfpq (splicing factor, proline/glutamine rich) that attenuate optic nerve regeneration. These proteins were induced in a Jak/Stat-dependent manner, stimulated each other's expression and suppressed the expression of regeneration-associated genes. In vivo, the injury-dependent induction of Socs3 and Sfpq inhibits optic nerve regeneration but does not block it. We identified a robust induction of multiple cytokine genes in zebrafish retinal ganglion cells that may contribute to their ability to overcome these inhibitory factors. These studies not only identified mechanisms underlying optic nerve regeneration in fish but also suggest new molecular targets for enhancing optic nerve regeneration in mammals.

“Activated” STAT Proteins: A Paradoxical Consequence of Inhibited JAK-STAT Signaling in Cytomegalovirus-Infected Cells

We have previously characterized mouse CMV (MCMV)-encoded immune-evasive IFN signaling inhibition and identified the viral protein pM27 as inducer of proteasomal degradation of STAT2. Extending our analysis to STAT1 and STAT3, we found that MCMV infection neither destabilizes STAT1 protein nor prevents STAT1 tyrosine Y701 phosphorylation, nuclear translocation, or the capability to bind γ-activated sequence DNA-enhancer elements. Unexpectedly, the analysis of STAT3 revealed an induction of STAT3 Y705 phosphorylation by MCMV. In parallel, we found decreasing STAT3 protein amounts upon MCMV infection, although STAT3 expression normally is positive autoregulative. STAT3 phosphorylation depended on the duration of MCMV infection, the infectious dose, and MCMV gene expression but was independent of IFNAR1, IL-10, IL-6, and JAK2. Although STAT3 phosphorylation did not require MCMV immediate early 1, pM27, and late gene expression, it was restricted to MCMV-infected cells and not transmitted to bystander cells. Despite intact STAT1 Y701 phosphorylation, IFN-γ-induced target gene transcription (e.g., IRF1 and suppressor of cytokine signaling [SOCS] 1) was strongly impaired. Likewise, the induction of STAT3 target genes (e.g., SOCS3) by IL-6 was also abolished, indicating that MCMV antagonizes STAT1 and STAT3 despite the occurrence of tyrosine phosphorylation. Consistent with the lack of SOCS1 induction, STAT1 phosphorylation was prolonged upon IFN-γ treatment. We conclude that the inhibition of canonical STAT1 and STAT3 target gene expression abrogates their intrinsic negative feedback loops, leading to accumulation of phospho-tyrosine-STAT3 and prolonged STAT1 phosphorylation. These findings challenge the generalization of tyrosine-phosphorylated STATs necessarily being transcriptional active and document antagonistic effects of MCMV on STAT1/3-dependent target gene expression.

Pattern-Recognition Receptor Signaling Regulator mRNA Expression in Humans and Mice, and in Transient Inflammation or Progressive Fibrosis

The cell type-, organ-, and species-specific expression of the pattern-recognition receptors (PRRs) are well described but little is known about the respective expression profiles of their negative regulators. We therefore determined the mRNA expression levels of A20, CYLD, DUBA, ST2, CD180, SIGIRR, TANK, SOCS1, SOCS3, SHIP, IRAK-M, DOK1, DOK2, SHP1, SHP2, TOLLIP, IRF4, SIKE, NLRX1, ERBIN, CENTB1, and Clec4a2 in human and mouse solid organs. Humans and mice displayed significant differences between their respective mRNA expression patterns of these factors. Additionally, we characterized their expression profiles in mononuclear blood cells upon bacterial endotoxin, which showed a consistent induction of A20, SOCS3, IRAK-M, and Clec4a2 in human and murine cells. Furthermore, we studied the expression pattern in transient kidney ischemia-reperfusion injury versus post-ischemic atrophy and fibrosis in mice. A20, CD180, ST2, SOCS1, SOCS3, SHIP, IRAK-M, DOK1, DOK2, IRF4, CENTB1, and Clec4a2 were all induced, albeit at different times of injury and repair. Progressive fibrosis was associated with a persistent induction of these factors. Thus, the organ- and species-specific expression patterns need to be considered in the design and interpretation of studies related to PRR-mediated innate immunity, which seems to be involved in tissue injury, tissue regeneration and in progressive tissue scarring.

Vitamin D reduces development and progression of virus-induced demyelinating disease (P5131)

Abstract Multiple sclerosis (MS) is a demyelinating disease of the central nervous system (CNS) that is associated with an inflammatory immune response. Microglia are the CNS resident immune cells which have been shown to be activated during MS. Virus infections have been suggested as a causative agent for MS, and vitamin D insufficiency has been correlated with disease development. Thus, we wanted to determine whether higher vitamin D levels at the time of virus infection could alter development of demyelinating disease. Theiler’s murine encephalomyelitis virus (TMEV) induced demyelinating disease is an animal model of MS. TMEV infection of susceptible mice leads to development of a demyelinating disease associated with an inflammatory immune response in the CNS. Our studies showed that increased levels of vitamin D3 at the time of virus infection reduced development and progression of demyelinating disease. Vitamin D reduced the number of CD4+ T cells expressing IFNγ and increased the number of CD25+CD4+ T cells. Most interestingly, vitamin D reduced the expression of pro-inflammatory cytokines and increased the expression of IL-10 and type I interferons by microglia. The reduction in the inflammatory immune response by microglia was dependent on IL-10 induction of SOCS3. Overall, these results show that vitamin D reduced the inflammatory immune response in the CNS and reduced the development and progression of demyelinating disease following virus infection.

Abstract 4396: Caveolin-1 as an oncopromoter in solidtumors: A role mediated by STAT3 in vitro.

Abstract Introduction Caveolin-1(Cav-1) is a multifunctional scaffolding protein essential structural constituent of caveolae, regulating multiple processes in cancer. A growing body of evidence links elevated expression of Cav-1 to an aggressive phenotype of cancer cells and to a poor prognosis in several tumors. The molecular mechanisms underlying the pro-survival and tumor-promoting functions of Cav-1 and the down-stream mediators of Cav-1 functional role in metastatic cancer have jet to be fully identified. The aim of the present work was to prosecute the investigation on such molecular pathways in solid tumors, based on our recent data obtained in cell lines from SCLC (SCLC-R1) and NSCLC (RAL) and from glioblastoma (A-172) where we demonstrated an oncopromoter activity of Cav-1 through the activation of STAT3 signaling pathway. Here we present the data obtained in a cell line from osteosarcoma (143B), where Cav-1 was highly expressed. Methods Cav-1 was knocked-down by siRNA using the transfection reagent oligofectamine. Cell proliferation was assessed by Trypan Blue exclusion test and by MTT test. Protein expressions were evaluated by Western Blotting. Results The siRNA-mediated down-regulation of Cav-1 caused the arrest of proliferation in the cell line, which remains stable up to 24 h after the removal of siRNA and then gradually returns to normal levels. Interestingly, both Cav-1 expression and proliferation goes down and turn up in the same way and at the same extent. A marked reduction of cyclin D1 and of CDK4 expression was evident in the cells transfected with Cav-1 siRNA and consequently of phosphoRb. The down-stream effector was STAT3 in the 143B osteosarcoma cell line, due to a significant induction of SOCS-1. Conclusions Together, our findings obtained in malignant tumors like SCLC, NSCLC, glioblastoma and osteosarcoma indicate that Cav-1 silencing induces an arrest of proliferation in vitro in each of them by inhibiting the pathway mediated by STAT3, through different upstream molecules depending on tumor: AKT and ERK in metastatic lung cancer, c-Src and p38 alpha in glioblastoma and SOCS-1 in osteosarcoma. These results confirm that Cav-1 exerts a role as a promoter of cancer proliferation and its activity strictly depends on cell/tumor type. They indicate the necessity to study this protein's function in each specific tumor context and provide new insights into the molecular mechanisms underlying the pro-survival and tumor-promoting functions of Cav-1. Grants from MIUR (RFO,PRIN), Roberto Pallotti's Legacy and Cornelia Pallotti's Legacy for Cancer Research, University of Bologna, Italy Citation Format: Anna Gasperi-Campani, Fabia Pancotti, Laura Roncuzzi. Caveolin-1 as an oncopromoter in solidtumors: A role mediated by STAT3 in vitro. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4396. doi:10.1158/1538-7445.AM2013-4396

Suppressor of cytokine signalling protein SOCS1 and UBP43 regulate the expression of type I interferon-stimulated genes in human microvascular endothelial cells infected with Rickettsia conorii

Rickettsia conorii, the causative agent of Mediterranean spotted fever, preferentially infects human microvascular endothelium and activates pro-inflammatory innate immune responses as evidenced by enhanced expression and secretion of cytokines and chemokines. Our recent studies reveal that human microvascular endothelial cells (HMECs) infected with R. conorii also launch 'antiviral' host defence mechanisms typically governed by type I interferons. To summarize, infected HMECs secrete IFN-β to activate STAT1 in an autocrine/paracrine manner and display increased expression of IFN-stimulated genes, for example ISG15, which in turn activate innate responses to interfere with intracellular replication of rickettsiae. We now present evidence that UBP43 and SOCS1, known negative regulators of JAK/STAT signalling, are also induced in R. conorii-infected HMECs, of which UBP43 but not SOCS1 functions to negatively regulate STAT1 activation. Interestingly, UBP43 induction is almost completely abolished in the presence of IFN-β-neutralizing antibody, implicating an important role for UBP43 as a feedback inhibitor for IFN-β-mediated STAT1 activation. In contrast, SOCS1 expression is only partially affected by IFN-β neutralization, implicating potential involvement of as-yet-unidentified IFN-independent mechanism(s) in SOCS1 induction during R. conorii infection. A number of IFN-stimulated genes, including ISG15, OAS1, MX1, IRF1, IRF9 and TAP1 are also induced in an IFN-β-dependent manner, whereas GBP1 remains unaffected by IFN-β neutralization. Increased STAT1 phosphorylation in HMECs subjected to UBP43 knockdown led to transcriptional activation of OAS1, MX1 and GBP1, confirming the negative regulatory role of UBP43. Although IRF1, IRF9 and TAP1 were induced by IFN-β, siRNA-mediated silencing of UBP43 or SOCS1 did not significantly affect their transcriptional activation. Expression of ISG15 was, however, increased in HMECs transfected with siRNA for UBP43 and SOCS1. Thus, unique regulatory patterns of induced expression of UBP43, SOCS1 and IFN-stimulated genes represent pathogen-specific responses underlying IFN-β-mediated host endothelial signalling during the pathogenesis of spotted fever group rickettsiosis.

Lactobacillus rhamnosus (LGG) Regulates IL-10 Signaling in the Developing Murine Colon through Upregulation of the IL-10R2 Receptor Subunit

The intestinal microflora is critical for normal development, with aberrant colonization increasing the risk for necrotizing enterocolitis (NEC). In contrast, probiotic bacteria have been shown to decrease its incidence. Multiple pro- and anti-inflammatory cytokines have been identified as markers of intestinal inflammation, both in human patients with NEC and in models of immature intestine. Specifically, IL-10 signaling attenuates intestinal responses to gut dysbiosis, and disruption of this pathway exacerbates inflammation in murine models of NEC. However, the effects of probiotics on IL-10 and its signaling pathway, remain poorly defined. Real-time PCR profiling revealed developmental regulation of MIP-2, TNF-α, IL-12, IL-10 and the IL-10R2 subunit of the IL-10 receptor in immature murine colon, while the expression of IL-6 and IL-18 was independent of postnatal age. Enteral administration of the probiotic Lactobacillus rhamnosus GG (LGG) down-regulated the expression of TNF-α and MIP-2 and yet failed to alter IL-10 mRNA and protein expression. LGG did however induce mRNA expression of the IL-10R2 subunit of the IL-10 receptor. IL-10 receptor activation has been associated with signal transducer and activator of transcription (STAT) 3-dependent induction of members of the suppressors of cytokine signaling (SOCS) family. In 2 week-old mice, LGG also induced STAT3 phosphorylation, increased colonic expression of SOCS-3, and attenuated colonic production of MIP-2 and TNF-α. These LGG-dependent changes in phosphoSTAT3, SOCS3, MIP-2 and TNF-α were all inhibited by antibody-mediated blockade of the IL-10 receptor. Thus LGG decreased baseline proinflammatory cytokine expression in the developing colon through upregulation of IL-10 receptor-mediated signaling, most likely due to the combined induction of phospho-STAT3 and SOCS3. Furthermore, LGG-dependent increases in IL-10R2 were associated with reductions in TNF-α, MIP-2 and disease severity in a murine model of intestinal injury in the immature colon.

Simvastatin inhibits secretion of Th17‐polarizing cytokines and antigen presentation by DCs in patients with relapsing remitting multiple sclerosis

Statins, widely used cholesterol-lowering agents, have also been demonstrated to have antiinflammatory effects. Here, we characterize the capacity of simvastatin to target DCs and modulate T-cell priming and Th17-cell differentiation, in a cohort of patients with relapsing remitting multiple sclerosis (RRMS). We report that simvastatin inhibits IL-1β, IL-23, TGF-β, IL-21, IL-12p70, and induces IL-27 secretion from DCs in RRMS patients, providing an inhibitory cytokine milieu for Th17 and Th1-cell differentiation. The effect on DCs is mediated via induction of SOCS1, SOCS3, and SOCS7 gene expression, which are associated with the inhibition of STAT1, STAT3, and ERK1/2 phosphorylation. A geranylgeranyl transferase inhibitor replicated simvastatin's effects on DC cytokine secretion, implicating that simvastatin-induced depletion of isoprenoids mediates this effect. Simvastatin inhibited antigen presentation by DCs via suppression of the MHC class I expression, costimulatory molecules CD80 and CD40, and by inducing a dramatic loss of dendritic processes. The changes in DC morphology were also mediated via inhibition of geranylgeranylation. The therapeutic use of geranylgeranylation inhibitors may provide selective inhibition of key pathogenic cytokines that drive the autoimmune response in MS; their use represents a promising therapeutic approach that requires further clinical testing.

Dectin-1 Stimulation Induces Suppressor of Cytokine Signaling 1, Thereby Modulating TLR Signaling and T Cell Responses

Suppressor of cytokine signaling (SOCS) proteins serve as negative regulators of cytokine receptor signaling. However, SOCS proteins are not only induced via the JAK/STAT pathway, but are also transcribed on triggering of pattern recognition receptors such as TLRs. We now show that SOCS1 can also be induced by the non-TLR pattern recognition receptor Dectin-1 in murine bone marrow-derived dendritic cells and macrophages (BMMs). The C-type lectin Dectin-1 binds to yeasts and signals either in an autonomous manner or can be triggered in combination with TLRs. In our study, SOCS1 was expressed independently of any TLR engagement as a direct target gene of the Dectin-1 ligand Zymosan. Induction of SOCS1 was mediated by a novel pathway encompassing the tyrosine kinases Src and Syk that activated the downstream kinase proline-rich tyrosine kinase 2. Proline-rich tyrosine kinase 2, in turn, caused activation of the MAPK ERK, thereby triggering SOCS1 induction. SOCS1 did not modulate Dectin-1 signaling but affected TLR signaling, leading to decreased and abbreviated NF-κB activation in BMMs triggered by TLR9. Furthermore, IL-12 and IL-10 secretion were inhibited by SOCS1. We additionally observed that IL-17-producing Th cells were clearly increased by SOCS1 in BMMs. Our results show that SOCS1 is expressed via a new, NF-κB-independent pathway in Dectin-1-triggered murine BMMs and influences TLR cross talk and T cell priming.

IL‐6 Indirectly Modulates The Induction Of Glyceroneogenic Enzymes In Adipose Tissue During Exercise

Glyceroneogenesis is an important step in the control of fatty acid re‐esterification with PEPCK and PDK4 being key enzymes in this process. It has been suggested that IL‐6 regulates adipose tissue metabolism and gene expression during exercise. The purpose of this investigation was to determine the role of IL‐6 in modulating the effects of exercise on the expression of glyceroneogenic enzymes in mouse adipose tissue. To achieve these goals, we utilized IL‐6 ex vivo treatment in cultured adipose tissue in combination with IL‐6−/−mice model. Treatment of cultured epididymal adipose tissue (eWAT), increased the phosphorylation of AMPK, ACC and STAT3 and induced SOCS3 mRNA levels while decreasing PEPCK mRNA. The activation of AMPK was independent of increases in lipolysis. An acute bout of treadmill running did not result in the induction of SOCS3 or increases in the phosphorylation of STAT3 in eWAT from WT mice. Exercise‐induced increases in PEPCK and PDK4 mRNA expression were attenuated in eWAT from IL‐6−/− mice in parallel with a greater relative increase in AMPK phosphorylation compared to exercised WT mice. These changes occurred independent of alterations in beta‐adrenergic signalling in adipose tissue from IL‐6−/− mice. Our findings question the role of IL‐6 signalling in adipose tissue during exercise and demonstrate an indirect effect of this cytokine in the regulation of adipose tissue gene expression.This work was supported by a Bridge Funding Operating Grant from the Canadian Institute of Health Research, Institute of Nutrition, Metabolism and Diabetes to David C. Wright.

Fluticasone propionate and Salmeterol combination induces SOCS-3 expression in airway epithelial cells

Fluticasone propionate (FP) and Salmeterol (SAL) are commonly used in combination therapy for patients with Chronic obstructive pulmonary disease (COPD). Clinical studies show that FP/SAL used in combination therapy was found to inhibit airway inflammation in COPD patients. However, the mechanisms associated with FP/SAL induced anti-inflammatory effects were not clear. We have evaluated the effect of FP/SAL and tobacco smoke (TS) on SOCS-3 and interleukine-6 expression in bronchial airway epithelial cells (BAEpCs). Human BAEpCs were exposed to TS and subsequently treated with FP or SAL alone or in combinations in the presence and absence of mitogen activated protein kinase (MAPK) inhibitors for either Erk1/ Erk2, or p38 or PI3 kinase. In BAEpCs, TS induced IL-6 expression via ERK1/ERK2 MAPK pathway and FP/SAL inhibited TS mediated IL-6 expression. TS down regulated the SOCS-3 expression via activation of Erk1/ Erk2, and p38 MAPK signaling. When TS exposed BAEpCs were treated with FP/SAL SOCS-3 expression was restored. FP/SAL combinations induced significantly higher expression of SOCS-3 in BAEpCs when compared to individual drug. Pretreatment with Ly294002 a PI3 MAPK inhibitor significantly attenuated FP/SAL induced SOCS-3 expression in BAEpCs. Furthermore, FP/SAL blunted TS induced phosphorylation of Erk1/ Erk2 and p38 MAPK in BAEpCs. Our study suggests that TS inhibits SOCS-3, combination of FP/SAL has a profound synergistic effect on SOCS-3 induction in BAEpCs and it is dependent on PI3 kinase signaling pathway. SOCS-3 may represent a potential biomarker for understanding the efficacy and a novel anti-inflammatory mechanism of FP/SAL combination therapy in the treatment of COPD.

Regulation of Suppressors of Cytokine Signaling as a Therapeutic Approach in Autoimmune Diseases, with an Emphasis on Multiple Sclerosis

Multiple sclerosis (MS) is an inflammatory demyelinating, presumably autoimmune disease of the central nervous system (CNS). Among the available MS therapies, interferon (IFN)β and the recently introduced statins have been reported to exert their immunomodulatory effects through the induction of SOCS1 and SOCS3 in various inflammatory cell subsets. The SOCS proteins negatively regulate cytokine and Toll-like receptors- (TLR-) induced signaling in the inflammatory cells. SOCS1 and SOCS3 have been reported to play an important role in the regulation of Th17-cell differentiation through their effects on the cells of the innate and adaptive immune systems. IFNβ and statins inhibit Th17-cell differentiation directly and indirectly via induction of SOCS1 and SOCS3 expression in monocytes, dendritic cells (DCs), and B-cells. Due to their rapid induction and degradation, and SOCS-mediated regulation of multiple cytokine-signaling pathways, they represent an attractive therapeutic target in the autoimmune diseases, and particularly relapsing remitting (RR) MS.

A novel small molecule deubiquitinase inhibitor blocks Jak2 signaling through Jak2 ubiquitination

AG490 is a tyrosine kinase inhibitor with activity against Jak2 and apoptotic activity in specific leukemias. Due to its weak kinase inhibitory activity and poor pharmacology, we conducted a cell-based screen for derivatives with improved Jak2 inhibition and activity in animals. Two hits emerged from an initial small chemical library screen, and more detailed structure–activity relationship studies led to the development of WP1130 with 50-fold greater activity in suppressing Jak2-dependent cytokine signaling than AG490. However, WP1130 did not directly suppress Jak2 kinase activity, but mediated Jak2 ubiquitination resulting in its trafficking through HDAC6 to perinuclear aggresomes without cytokine stimulation or SOCS-1 induction. Jak2 primarily contained K63-linked ubiquitin polymers, and mutation of this lysine blocked Jak2 ubiquitination and mobilization in WP1130-treated cells. Further analysis demonstrated that WP1130, but not AG490, acts as a deubiquitinating enzyme (DUB) inhibitor, possibly through a Michael addition reaction. We conclude that chemical modification of AG490 resulted in development of a DUB inhibitor with activity against a DUB capable of modulating Jak2 ubiquitination, trafficking and signal transduction.

Toll-like receptor 4 and myeloid differentiation factor 88 provide mechanistic insights into the cause and effects of interleukin-6 activation in mouse liver regeneration

Partial hepatectomy (PH) consistently results in an early increase of circulating interleukin-6 (IL-6), which is thought to play a major role in liver regeneration. Activation of this cytokine after PH requires the adaptor protein, MyD88, but the specific MyD88-related receptors involved remain unidentified. It is also unknown whether the magnitude of IL-6 elevation determines the extent of subsequent hepatocyte proliferation. Here, we uncovered artifacts in the assessment of circulating IL-6 levels when using cardiac puncture in mice after PH. By using retro-orbital bleed sampling, we show that the circulating levels of IL-6 after PH were not directly correlated with the extent of hepatocyte DNA synthesis in individual mice. The IL-6 increase after PH was attenuated in all lipopolysaccharide-hyporesponsive mouse strains studied (e.g., C3H/HeJ, Tlr4 null, Cd14 null, Tlr2,4,9 null, and Tlr2,4-Caspase1 null) and was severely abrogated in Myd88 null mice. Despite attenuated IL-6 levels, Tlr4 null mice showed normal signaling downstream of IL-6 and normal hepatocyte proliferation. In contrast, Myd88 null mice showed severe impairments in signal transducer and activator of transcription 3 phosphorylation and Socs3 induction, but had enhanced and prolonged extracellular signal-related kinase 1 and 2 phosphorylation in the first 6 hours after PH. Unexpectedly, these changes were associated with accelerated initiation of hepatocyte proliferation, as assessed by hepatocyte bromodeoxyuridine incorporation, phospho-histone H3 immunostaining, and cyclin E and A protein expression. TLR-4 signaling contributes to IL-6 activation after PH, but the Tlr4-independent component appears sufficient for ensuring intact signaling downstream of IL-6. The lack of correlation between IL-6 levels and hepatocyte proliferation after PH, and the accelerated start of hepatocyte proliferation in Myd88 null mice despite abrogated cytokine activation, may highlight relevant antiproliferative effects of IL-6 signaling, possibly via Socs3, in the regulation of liver regeneration.

Both type I and II IFN induce insulin resistance by inducing different isoforms of SOCS expression in 3T3-L1 adipocytes

Although elevation of the blood glucose level is a causal adverse effect of treatment with interferon (IFN), the precise underlying molecular mechanism is largely unknown. We examined the effects of type I and type II IFN (IFN-β and IFN-γ) on insulin-induced metabolic signaling leading to glucose uptake in 3T3-L1 adipocytes. IFN-β suppressed insulin-induced tyrosine phosphorylation of IRS-1 without affecting its expression, whereas IFN-γ reduced both the protein level and tyrosine phosphorylation. Although both IFNs stimulated phosphorylation of STAT1 (at Tyr(701)) and STAT3 (at Tyr(705)) after treatment for 30 min, subsequent properties of induction of the SOCS isoform were different. IFN-β preferentially induced SOCS1 rather than SOCS3, whereas IFN-γ strongly induced SOCS3 expression alone. In addition, adenovirus-mediated overexpression of either SOCS1 or SOCS3 inhibited insulin-induced tyrosine phosphorylation of IRS-1, whereas the reduction of IRS-1 protein was observed only in SOCS3-expressed cells. Notably, IFN-β-induced SOCS1 expression and suppression of insulin-induced tyrosine phosphorylation of IRS-1 were attenuated by siRNA-mediated knockdown of STAT1. In contrast, adenovirus-mediated expression of a dominant-negative STAT3 (F-STAT3) attenuated IFN-γ-induced SOCS3 expression, reduction of IRS-1 protein, and suppression of insulin-induced glucose uptake but did not have any effect on the IFN-β-mediated SOCS1 expression and inhibition of insulin-induced glucose uptake. Interestingly, pretreatment of IFN-γ with IL-6 synergistically suppressed insulin signaling, even when IL-6 alone had no significant effect. These results indicate that type I and type II IFN induce insulin resistance by inducing distinct SOCS isoforms, and IL-6 synergistically augments IFN-γ-induced insulin resistance by potentiating STAT3-mediated SOCS3 induction in 3T3-L1 adipocytes.