Suppressor Of Cytokine Signaling 3 Induction Research Articles

Botulinum neurotoxin serotype A inhibited ocular angiogenesis through modulating glial activation via SOCS3.

Pathological angiogenesis causes significant vision loss in neovascular age-related macular degeneration and other retinopathies with neovascularization (NV). Neuronal/glial-vascular interactions influence the release of angiogenic and neurotrophic factors. We hypothesized that botulinum neurotoxin serotype A (BoNT/A) modulates pathological endothelial cell proliferation through glial cell activation and growth factor release. A laser-induced choroidal NV (CNV) was employed to investigate the anti-angiogenic effects of BoNT/A. Fundus fluorescence angiography, immunohistochemistry, and real-time PCR were used to assess BoNT/A efficacy in inhibiting CNV and the molecular mechanisms underlying this inhibition. Neuronal and glial suppressor of cytokine signaling 3 (SOCS3) deficient mice were used to investigate the molecular mechanisms of BoNT/A in inhibiting CNV via SOCS3. In laser-induced CNV mice with intravitreal BoNT/A treatment, CNV lesions decreased > 30%; vascular leakage and retinal glial activation were suppressed; and Socs3 mRNA expression was induced while vascular endothelial growth factor A (Vegfa) mRNA expression was suppressed. The protective effects of BoNT/A on CNV development were diminished in mice lacking neuronal/glial SOCS3. BoNT/A suppressed laser-induced CNV and glial cell activation, in part through SOCS3 induction in neuronal/glial cells. BoNT/A treatment led to a decrease of pro-angiogenic factors, including VEGFA, highlighting the potential of BoNT/A as a therapeutic intervention for pathological angiogenesis in retinopathies.

SOCS3 deficiency in cardiomyocytes elevates sensitivity of ischemic preconditioning that synergistically ameliorates myocardial ischemia reperfusion injury.

Ischemic preconditioning (IPC) is the most powerful endogenous cardioprotective form of cellular adaptation. However, the inhibitory or augmenting mechanism underlying cardioprotection via IPC remains largely unknown. Suppressor of cytokine signaling-3 (SOCS3) is a cytokine-inducible potent negative feedback regulator of the signal transducer and activator of transcription-3 (STAT3) signaling pathway. Here, we aimed to determine whether cardiac SOCS3 deficiency and IPC would synergistically reduce infarct size after myocardial ischemia reperfusion injury. We evaluated STAT3 activation and SOCS3 induction after ischemic conditioning (IC) using western blot analysis and real-time PCR, and found that myocardial IC alone transiently activated myocardial STAT3 and correspondingly induced SOCS3 expression in wild-type mice. Compared with wild-type mice, cardiac-specific SOCS3 knockout (SOCS3-CKO) mice showed significantly greater and more sustained IC-induced STAT3 activation. Following ischemia reperfusion, IPC substantially reduced myocardial infarct size and significantly enhanced STAT3 phosphorylation in SOCS3-CKO mice compared to in wild-type mice. Real-time PCR array analysis revealed that SOCS3-CKO mice after IC exhibited significantly increased expressions of several anti-apoptotic genes and SAFE pathway-related genes. Moreover, real-time PCR analysis revealed that myocardial IC alone rapidly induced expression of the STAT3-activating cytokine erythropoietin in the kidney at 1 h post-IC. We also found that the circulating erythropoietin level was promptly increased at 1 h after myocardial IC. Myocardial SOCS3 deficiency and IPC exert synergistic effects in the prevention of myocardial injury after ischemia reperfusion. Our present results suggest that myocardial SOCS3 is a potent inhibitor of IPC-induced cardioprotection, and that myocardial SOCS3 inhibition augment IPC-mediated cardioprotection during ischemia reperfusion injury.

Genome-Wide Mapping Defines a Role for C/EBPβ and c-Jun in Non-Canonical Cyclic AMP Signalling.

The novel exchange protein activated by cyclic AMP (EPAC1) activator, I942, induces expression of the suppressor of cytokine signalling 3 (SOCS3) gene, thereby inhibiting interleukin 6 (IL6) inflammatory processes in human umbilical vein endothelial cells (HUVECs). Here we use RNA-SEQ and ChIP-SEQ to determine global gene responses to I942, in comparison with cyclic AMP production promoted by forskolin and rolipram (F/R). We found that I942 promoted significant changes in the RNA expression of 1413 genes, largely associated with microtubule stability and cell cycle progression, whereas F/R regulated 197 genes linked to endothelial cell function, including chemokine production and platelet aggregation. A further 108 genes were regulated by both treatments, including endothelial regulatory genes involved in purinergic signalling and cell junction organization. ChIP-SEQ demonstrated that F/R induced genome-wide recruitment of C/EBPβ and c-Jun transcription factors, whereas I942 promoted recruitment of c-Jun to genes associated with IL6 signalling, with little effect on C/EBPβ activation. Despite this, certain key inflammatory genes, including IL6, VEGF, CCL2/MCP1, VCAM1, SELE and ICAM1 were regulated by I942 without significant c-Jun recruitment, suggesting an additional, indirect mode of action for I942. In this regard, SOCS3 induction by I942 was found to require c-Jun and was associated with suppression of IL6-promoted ERK MAP kinase and AKT activity and induction of ICAM1. Pharmacological inhibition of ERK and AKT also potentiated ICAM1 induction by I942. We therefore propose that c-Jun activation by I942 regulates endothelial gene expression in HUVECs through direct mechanisms, involving recruitment of c-Jun or, as for ICAM1, through indirect regulation of tertiary regulators, including SOCS3.

Asaronic Acid Attenuates Macrophage Activation toward M1 Phenotype through Inhibition of NF-κB Pathway and JAK-STAT Signaling in Glucose-Loaded Murine Macrophages.

Macrophage polarization has been implicated in the pathogenesis of obesity and type 2 diabetes, which are recognized as chronic proinflammatory diseases. This study investigated that high level of glucose, similar to lipopolysaccharide (LPS), activated macrophages toward M1 phenotypes and 1-20 μM asaronic acid (AA) counteracted diabetic macrophage activation. AA reduced the LPS-promoted secretion of proinflammatory interleukin (IL)-6 and monocyte chemoattractant protein-1. The LPS markedly elevated the macrophage induction of the M1 markers of Toll-like receptor 4 (TLR4), CD36, and CD68, which was attenuated by AA. Also, the LPS significantly enhanced the nuclear factor (NF)-κB transactivation, signal transducers, and activators of transcription 1 (STAT1)/STAT3 activation and suppressor of cytokine signaling 3 (SOCS3) induction in macrophages. However, AA highly suppressed the aforementioned effects of LPS. Glucose-stimulated macrophages expressed advanced glycation end products (AGEs) and receptor for AGE (RAGE). Administration of 20 μM AA to macrophages partly but significantly attenuated such effects (1.65 ± 0.12 vs 0.95 ± 0.25 times glucose control for AGE; 2.33 ± 0.31 vs 1.40 ± 0.22 times glucose control for RAGE). Furthermore, glucose enhanced the macrophage induction of TLR4 and inducible nitric oxide synthase and IL-6 production, while it demoted the production of anti-inflammatory arginase-1 and IL-10. In contrast, AA reversed the induction of these markers in glucose-loaded macrophages. AA dose-dependently and significantly encumbered NF-κB transactivation, Janus kinase 2 (JAK2) and STAT1/STAT3 activation, and SOCS3 induction upregulated in glucose-supplemented macrophages. These results demonstrated for the first time that AA may limit diabetic macrophage activation toward the M1 phenotype through the inhibition of TLR4-/IL-6-mediated NF-κB/JAK2-STAT signaling entailing AGE-RAGE interaction.

Influenza Virus-Induced Robust Expression of SOCS3 Contributes to Excessive Production of IL-6.

Influenza A virus (IAV) remains a major public health threat in the world, as indicated by the severe pneumonia caused by its infection annually. Interleukin-6 (IL-6) involved excessive inflammatory response to IAV infection profoundly contributes to the virus pathogenesis. However, the precise mechanisms underlying such a response are poorly understood. Here we found from both in vivo and in vitro studies that IAV not only induced a surge of IL-6 release, but also greatly upregulated expression of suppressor of cytokine signaling-3 (SOCS3), the potent suppressor of IL-6-associated signal transducer and activator of transcription 3 (STAT3) signaling. Interestingly, there existed a cytokine-independent mechanism of the robust induction of SOCS3 by IAV at early stages of the infection. Furthermore, we employed SOCS3-knockdown transgenic mice (TG), and surprisingly observed from virus challenge experiments using these mice that disruption of SOCS3 expression provided significant protection against IAV infection, as evidenced by attenuated acute lung injury, a higher survival rate of infected animals and lower viral load in infected tissues as compared with those of wild-type littermates under the same condition. The activity of nuclear factor-kappa B (NFκB) and the expression of its target gene IL-6 were suppressed in SOCS3-knockdown A549 cells and the TG mice after infection with IAV. Moreover, we defined that enhanced STAT3 activity caused by SOCS3 silencing was important for the regulation of NFκB and IL-6. These findings establish a critical role for IL-6-STAT3-SOCS3 axis in the pathogenesis of IAV and suggest that influenza virus may have evolved a strategy to circumvent IL-6/STAT3-mediated immune response through upregulating SOCS3.

The novel exchange protein activated by cyclic AMP 1 (EPAC1) agonist, I942, regulates inflammatory gene expression in human umbilical vascular endothelial cells (HUVECs)

Exchange protein activated by cyclic AMP (EPAC1) suppresses multiple inflammatory actions in vascular endothelial cells (VECs), partly due to its ability to induce expression of the suppressor of cytokine signalling 3 (SOCS3) gene, the protein product of which inhibits interleukin 6 (IL6) signalling through the JAK/STAT3 pathway. Here, for the first time, we use the non-cyclic nucleotide EPAC1 agonist, I942, to determine its actions on cellular EPAC1 activity and cyclic AMP-regulated gene expression in VECs. We demonstrate that I942 promotes EPAC1 and Rap1 activation in HEK293T cells and induces SOCS3 expression and suppresses IL6-stimulated JAK/STAT3 signalling in HUVECs. SOCS3 induction by I942 in HUVECs was blocked by the EPAC1 antagonist, ESI-09, and EPAC1 siRNA, but not by the broad-spectrum protein kinase A (PKA) inhibitor, H89, indicating that I942 regulates SOCS3 gene expression through EPAC1. RNA sequencing was carried out to further identify I942-regulated genes in HUVECs. This identified 425 I942-regulated genes that were also regulated by the EPAC1-selective cyclic AMP analogue, 007, and the cyclic AMP-elevating agents, forskolin and rolipram (F/R). The majority of genes identified were suppressed by I942, 007 and F/R treatment and many were involved in the control of key vascular functions, including the gene for the cell adhesion molecule, VCAM1. I942 and 007 also inhibited IL6-induced expression of VCAM1 at the protein level and blocked VCAM1-dependent monocyte adhesion to HUVECs. Overall, I942 represents the first non-cyclic nucleotide EPAC1 agonist in cells with the ability to suppress IL6 signalling and inflammatory gene expression in VECs.

Induction of SOCS3 by liver X receptor suppresses the proliferation of hepatocellular carcinoma cells.

Liver X receptor (LXR), a member of nuclear receptor superfamily, is involved in the regulation of glucose, lipid and cholesterol metabolism. Recently, it has been reported that LXR suppress different kinds of cancers including hepatocellular carcinoma (HCC). However, the corresponding mechanism is still not well elucidated. In the present study, we found that activation of LXR downregulated cyclin D1 while upregulated p21 and p27 by elevating the level of suppressor of cytokine signaling 3 (SOCS3), leading to the cell cycle arrest at G1/S phase and growth inhibition of HCC cells. Moreover, we demonstrated that LXRα (not LXRβ) mediated the induction of SOCS3 in HCC cells. Subsequently, we showed that LXR activation enhanced the mRNA stability of SOCS3, but had no significant influence on the transcriptional activity of SOCS3 gene promoter. The experiments in nude mice revealed that LXR agonist inhibited the growth of xenograft tumors and enhanced SOCS3 expression in vivo. These results indicate that “LXRα-SOCS3-cyclin D1/p21/p27” is a novel pathway by which LXR exerts its anti-HCC effects, suggesting that the pathway may be a new potential therapeutic target for HCC treatment.

Small acid, big hopes: a rosy outlook for formic acid attracts chemical companies

We aimed to investigate regulation of signal transducer and activator of transcription 3 (STAT3) and suppressor of cytokine signaling 3 (SOCS3) by interferon alpha (IFN-α) and to analyze the relationship between STAT3 and SOCS3 during hepatitis C virus (HCV) infection. Changes in STAT3 and SOCS3 were analyzed at both mRNA and protein levels in human hepatoma cells infected with HCV (J6/JFH1). At 72 h of HCV infection, STAT3 expression was decreased with sustained phosphorylation, and IFN-α increased such decrease and phosphorylation. HCV increased SOCS3 expression, while IFN-α impaired such increase, indicating different regulation of STAT3 and SOCS3 by IFN-α. IFN-α-induced expression and phosphorylation of upstream kinases of the JAK/STAT pathway, Tyk2 and Jak1, were suppressed by HCV. Moreover, knockdown of STAT3 by RNA interference led to decreases in HCV RNA replication and viral protein expression, without affecting either the expression of Tyk2 and Jak1 or the SOCS3 induction in response to IFN-α. These results show that IFN-α antagonizes STAT3 and SOCS3 signaling triggered by HCV and that STAT3 regulation correlates inversely with SOCS3 induction by IFN-α, which may be important in better understanding the complex interplay between IFN-α and signal molecules during HCV infection.

The role of Pdia3-dependent 1α,25-dihydroxy vitamin D3 signaling on IgE-mediated mast cell activation

Abstract Active 1,25(OH)2 Vitamin D3 (1,25D3) is most commonly known for its role in the regulation of bone metabolism. However, research has now indicated it is a potent immune regulator beyond its calcemic effects. Vitamin D deficiency has been implicated in the pathogenesis of allergic diseases including asthma and atopic dermatitis. 1,25D3 is known to signal through the canonical Vitamin D receptor (VDR) facilitating its anti-inflammatory properties and regulating over 100 genes. However, protein disulfide isomerase family A, member 3 (Pdia3), was recently shown to mediate rapid-membrane responses to 1,25D3. In this study, we show that upon IgE-mediated activation, VDR knockout mast cells display enhanced cytokine production. Additionally, 1,25D3 suppresses cytokine production independently of VDR supporting the possibility of an alternative receptor facilitating the suppressive properties of Vitamin D on mast cells. Pdia3 protein and mRNA levels were upregulated in response to 1,25D3 in both WT and VDR KO mast cells. Upon blocking Pdia3 signaling, VDR KO mast cells lost their suppressive response to Vitamin D. Prostaglandin E2, which is released during Pdia3-dependent 1,25D3 signaling, mimicked Vitamin D effects on WT and VDR KO mast cells. Suppressor of Cytokine Signaling 3 (SOCS3), which is known to be upregulated by PGE2, was induced by 1,25D3 in both WT and VDR KO mast cells. Interestingly, VDR KO mast cells showed an enhanced induction of SOCS3 in comparison to WT. Collectively, our data depict a novel signaling mechanism utilized by mast cells to respond to 1,25D3 that could be explored as a potential therapeutic target for the treatment of allergic and inflammatory diseases.

Interferon alpha antagonizes STAT3 and SOCS3 signaling triggered by hepatitis C virus

We aimed to investigate regulation of signal transducer and activator of transcription 3 (STAT3) and suppressor of cytokine signaling 3 (SOCS3) by interferon alpha (IFN-α) and to analyze the relationship between STAT3 and SOCS3 during hepatitis C virus (HCV) infection. Changes in STAT3 and SOCS3 were analyzed at both mRNA and protein levels in human hepatoma cells infected with HCV (J6/JFH1). At 72h of HCV infection, STAT3 expression was decreased with sustained phosphorylation, and IFN-α increased such decrease and phosphorylation. HCV increased SOCS3 expression, while IFN-α impaired such increase, indicating different regulation of STAT3 and SOCS3 by IFN-α. IFN-α-induced expression and phosphorylation of upstream kinases of the JAK/STAT pathway, Tyk2 and Jak1, were suppressed by HCV. Moreover, knockdown of STAT3 by RNA interference led to decreases in HCV RNA replication and viral protein expression, without affecting either the expression of Tyk2 and Jak1 or the SOCS3 induction in response to IFN-α. These results show that IFN-α antagonizes STAT3 and SOCS3 signaling triggered by HCV and that STAT3 regulation correlates inversely with SOCS3 induction by IFN-α, which may be important in better understanding the complex interplay between IFN-α and signal molecules during HCV infection.

Regulation of Anti-Inflammatory Gene Expression in Vascular Endothelial Cells by EPAC1

Suppressor of cytokine signalling 3 (SOCS3) is a potent inhibitor of pro-inflammatory pathways involved in atherogenesis and the development of neo-intimal hyperplasia (NIH), which contributes to the in-stent re-stenosis responsible for the failure of percutaneous coronary intervention (PCI) procedures. We have shown that cyclic AMP sensor EPAC1 triggers induction of the SOCS3 gene in vascular endothelial cells (VECs), thereby attenuating interleukin 6 (IL-6)-mediated pro-inflammatory signalling. We propose that EPAC1 localisation to the nuclear pore controls cyclic AMP-mediated activation of a C/EBPβ/c-Jun transcriptional complex, leading to SOCS3 induction and suppression of pro-inflammatory signalling. Future work in this area will involve an integrated approach to determine the wider significance of the EPAC1-C/EBPβ/c-Jun pathway in controlling human VEC function and identify new therapeutic targets for management of chronic inflammation in vascular settings.

FXR induces SOCS3 and suppresses hepatocellular carcinoma.

Suppressor of cytokine signaling 3 (SOCS3) is regarded as a vital repressor in the liver carcinogenesis mainly by inhibiting signal transducer and activator of transcription 3 (STAT3) activity. Farnesoid X Receptor (FXR), highly expressed in liver, has an important role in protecting against hepatocellular carcinoma (HCC). However, it is unclear whether the tumor suppressive activity of FXR involves the regulation of SOCS3. In the present study, we found that activation of FXR by its specific agonist GW4064 in HCC cells inhibited cell growth, induced cell cycle arrest at G1 phase, elevated p21 expression and repressed STAT3 activity. The above anti-tumor effects of FXR were dramatically alleviated by knockdown of SOCS3 with siRNA. Reporter assay revealed that FXR activation enhanced the transcriptional activity of SOCS3 promoter. Electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) assay displayed that FXR directly bound to IR9 DNA motif within SOCS3 promoter region. The in vivo study in nude mice showed that treatment with FXR ligand GW4064 could decelerate the growth of HCC xenografts, up-regulate SOCS3 and p21 expression and inhibit STAT3 phosphorylation in the xenografts. These results suggest that induction of SOCS3 may be a novel mechanism by which FXR exerts its anti-HCC effects, and the FXR-SOCS3 signaling may serve as a new potential target for the prevention/treatment of HCC.

Suppressor of Cytokine Signaling 3 Is an Inducible Host Factor That Regulates Virus Egress during Ebola Virus Infection.

Ebola virus (EBOV) initially targets monocytes and macrophages, which can lead to the release of proinflammatory cytokines and chemokines. These inflammatory cytokines are thought to contribute to the development of circulatory shock seen in fatal EBOV infections. The VP40 matrix protein is a key viral structural protein that is critical for virion egress. Physical and functional interactions between VP40 and host proteins such as Tsg101 and Nedd4 facilitate efficient release of VP40-driven virus-like particles (VLPs) and infectious virus. Here, we show that host suppressor of cytokine signaling 3 (SOCS3) can also bind to EBOV VP40, leading to enhanced ubiquitinylation and egress of VP40. Indeed, titers of infectious EBOV derived from SOCS3 knockout mouse embryonic fibroblasts (MEFs) were significantly reduced compared to those from wild-type (WT) MEFs at 24 and 48 h postinfection. Importantly, this reduced virus yield could be rescued back to WT levels by exogenously expressing SOCS3. Lastly, we show that SOCS3 expression is induced by EBOV glycoprotein (GP) expression and that VLPs containing EBOV VP40 and GP induced production of proinflammatory cytokines, which induced SOCS3 for negative-feedback regulation. These data indicate that host innate immune protein SOCS3 may play an important role in budding and pathogenesis of EBOV. The VP40 matrix protein is a key structural protein critical for Ebola virus budding. Physical and functional interactions between VP40 and host proteins such as Tsg101 and Nedd4 facilitate efficient release of VLPs and infectious virus. We reported that host TLR4 is a sensor for Ebola GP on VLPs and that the resultant TLR4 signaling pathways lead to the production of proinflammatory cytokines. Host SOCS3 regulates the innate immune response by controlling and limiting the proinflammatory response through negative-feedback inhibition of cytokine receptors. We present evidence that Ebola virus VLPs stimulate induction of SOCS3 as well as proinflammatory cytokines, and that expression of human SOCS3 enhances budding of Ebola VLPs and infectious virus via a mechanism linked to the host ubiquitinylation machinery.

Key role of suppressor of cytokine signaling 3 in regulating gp130 cytokine-induced signaling and limiting chondrocyte responses during murine inflammatory arthritis.

To examine the impact of the gp130 cytokine family on murine articular cartilage and to explore a potential regulatory role of suppressor of cytokine signaling 3 (SOCS-3) in murine chondrocytes. In wild-type (WT) mouse chondrocytes, baseline receptor expression levels and gp130 cytokine-induced JAK/STAT signaling were determined by flow cytometry, and expression of SOCS-3 was assessed by quantitative polymerase chain reaction. The role of endogenous SOCS-3 was examined in cartilage explants and chondrocytes from mice with conditional deletion of Socs3 driven by the Col2a1 promoter in vitro (Socs3(Δ/Δcol2) ) and from mice during CD4+ T cell-dependent inflammatory monarthritis. Bone erosions in the murine joints were analyzed by micro-computed tomography. On chondrocytes from WT mice, gp130 and the oncostatin M (OSM) receptor were strongly expressed, whereas the transmembrane interleukin-6 (IL-6) receptor was expressed at much lower levels. Compared to other gp130 cytokines, OSM was the most potent activator of the JAK/STAT pathway and of SOCS-3 induction. Treatment of Socs3(Δ/Δcol2) mouse cartilage explants and chondrocytes with gp130 cytokines prolonged JAK/STAT signaling, enhanced cartilage degradation, increased the expression of Adamts4, Adamts5, and RANKL, and elevated the production of IL-6, granulocyte colony-stimulating factor, CXCL1, and CCL2. Socs3(Δ/Δcol2) mice developed exacerbated inflammation and joint damage in response to gp130 cytokine injections, and these histopathologic features were also observed in mice with inflammatory monarthritis. The results of this study highlight a key role for SOCS-3 in regulating chondrocyte responses during inflammatory arthritis. Within the gp130 cytokine family, OSM is a potent stimulus of chondrocyte responses, while IL-6 probably signals via trans-signaling. The gp130 cytokine-driven production of RANKL in chondrocytes may link chondrocyte activation and bone remodeling during inflammatory arthritis. Thus, these findings suggest that the inhibition of OSM might reduce the development and severity of structural joint damage during inflammatory arthritis.

Characterisation of the cytokine milieu associated with the up‐regulation of IL‐6 and suppressor of cytokine 3 in chronic hepatitis C treatment non‐responders

In chronic hepatitis C virus infection (CHC), expression of suppressor of cytokine signalling-3 (SOCS3) has been shown to be associated with obesity and non-response to antiviral therapy. In this study, we aimed to determine the effect of SOCS3 induction on the cytokine response in patients receiving Pegylated interferon (PegIFN) and ribavirin (RBV) therapy. Peripheral blood mononuclear cells (PBMC) collected at baseline and at 12 weeks from CHC patients receiving PegIFN/RBV therapy were examined for mRNA and protein SOCS3 expression. Immunological assays were employed to examine cytokine production. There was increased expression of SOCS3 in PBMC of non-responders at week 12 of therapy, when compared to treatment responders (P = 0.0001). The expression of SOCS3 correlated with body mass index (BMI) (r = 0.54; P = 0.01). Patients with low SOCS3 expression at week 12 of therapy had lower HCV-specific IFN-γ production in enzyme-linked immunosorbent spot (ELISpot) assays (P = 0.01), and reduced ex-vivo production of the anti-HCV effector cytokines interleukin (IL)-2 and tumour necrosis factor (TNF)-α(P = 0.01 and P = 0.04 respectively). Analysis of serum cytokine levels revealed higher levels of IL-6 at week 12 in the high SOCS3 expression group (P = 0.02) while IL-6 levels correlated with SOCS3 expression in the entire cohort (P = 0.04). Ex-vivo studies confirmed that IL-6 induced SOCS3, and neutralisation of IL-6 reduced levels of SOCS3. In subjects with increased BMI and non-response to antiviral therapy, the IL-6/SOCS3 axis appears to play a crucial role in altering the anti-HCV-cytokine response associated with antiviral therapy.

Flavanoids induce expression of the suppressor of cytokine signalling 3 (SOCS3) gene and suppress IL-6-activated signal transducer and activator of transcription 3 (STAT3) activation in vascular endothelial cells

The atherogenic cytokine IL-6 (interleukin-6) induces pro-inflammatory gene expression in VECs (vascular endothelial cells) by activating the JAK (Janus kinase)/STAT3 (signal transducer and activator of transcription 3) signalling pathway, which is normally down-regulated by the STAT3-dependent induction of the E3 ubiquitin ligase component SOCS3 (suppressor of cytokine signalling 3). Novel treatments based on the regulation of SOCS3 protein levels could therefore have value in the treatment of diseases with an inflammatory component, such as atherosclerosis. To this end we carried out a screen of 1031 existing medicinal compounds to identify inducers of SOCS3 gene expression and identified the flavanoids naringenin and flavone as effective inducers of SOCS3 protein, mRNA and promoter activity. This was in contrast with the action of traditional JAK/STAT3 inhibitors and the polyphenol resveratrol, which effectively suppress SOCS3 gene expression. Both naringenin and flavone also effectively suppressed IL-6-stimulated phosphorylation of STAT3 (Tyr705) which led to suppression of IL-6-induction of the atherogenic STAT3 target gene MCP1 (monocyte chemotactic protein-1), suggesting that their ability to induce SOCS3 gene expression is STAT3-independent. Supporting this idea was the observation that the general kinase inhibitor compound C inhibits flavone- and cAMP-dependent, but not JAK-dependent, SOCS3 induction in VECs. Indeed, the ability of flavanoids to induce SOCS3 expression requires activation of the ERK (extracellular-signal-regulated kinase)-dependent transcription factor SP3, and not STAT3. In the present paper we therefore describe novel molecular actions of flavanoids, which control SOCS3 gene induction and suppression of STAT3 signalling in VECs. These mechanisms could potentially be exploited to develop novel anti-atherogenic therapies.

Pien Tze Huang suppresses IL-6-inducible STAT3 activation in human colon carcinoma cells through induction of SOCS3

IL-6/STAT3 is one of the most critical cellular signal transduction pathways known to malfunction in colorectal cancer (CRC). As a target gene of signal transducer and activator of transcription 3 (STAT3) signaling, suppressor of cytokine signaling 3 (SOCS3) can be quickly induced by interleukin-6 (IL-6) stimulation but it then strongly inhibits IL-6-mediated STAT3 activation, functioning as a negative feedback regulator of the IL-6/STAT3 pathway. Aberrant activation of STAT3 and/or reduced expression of SOCS are strongly correlated with carcinogenesis, which therefore becomes a promising target for the development of novel anticancer chemotherapies. Pien Tze Huang (PZH) is a well-known traditional Chinese formula that was first prescribed by a royal physician 450 years ago in the Ming Dynasty. It has been used in China and Southeast Asia for centuries as a folk remedy for various types of cancer including CRC. However, the precise mechanism of its antitumor activity remains largely unclear. In the present study, we found that PZH could significantly and dose-dependently inhibit IL-6-mediated increase of STAT3 phosphorylation levels and transcriptional activity in the human colon carcinoma HT-29 cells, resulting in the suppression of cell proliferation and the induction of apoptosis. In addition, PZH treatment profoundly inhibited IL-6-induced upregulation of cyclin D1 and Bcl-2, two key target genes of the STAT3 pathway. Moreover, PZH treatment increased the expression of SOCS3. These results suggest that PZH could effectively inhibit proliferation and promote apoptosis of human colon carcinoma cells via modulation of the IL-6/STAT3 signaling pathway and its target genes.

STATI, STAT6 and Adenosine 3′,5′-Cyclic Monophosphate (cAMP) Signaling Drive SOCS3 Expression in Inactive Ulcerative Colitis

Ulcerative colitis (UC) is a chronic disease associated with long periods of quiescent disease followed by fulminant exacerbation. Imminent relapse in UC is associated with high mucosal expression of suppressor of cytokine signaling 3 (SOCS3); hence, knowledge of the mechanisms driving mucosal SOCS3 expression may provide important clues as to rational therapy. Thus, here we aim to characterize the molecular forces driving SOCS3 expression in the mucosal compartment, focusing on druggable pathways. The colon epithelial cell line Caco-2 was stimulated with interferon (IFN)-γ, interleukin (IL)-4 or prostaglandin E(2) (PGE(2)) to allow correlations between SOCS3 expression with signal transducer and activator of transcription 1 (STAT1), STAT6 and adenosine 3',5'-cyclic monophosphate (cAMP) signaling, respectively. The physiological relevance of the findings obtained was assessed by immunohistochemical staining for the activated forms of STAT1, STAT6, protein kinase A (PKA)-Cγ and cAMP response element-binding protein (CREB) in biopsies from inactive UC patients and controls. Stimulation with IFN-γ, IL-4 or PGE(2) induced activation of STAT1, STAT6 and cAMP, respectively, in colonic cells, without any signs of concomitant STAT3 activation. Forced activation of all these signaling pathways was sufficient for SOCS3 expression. Biopsies from patients with inactive UC showed significant increase of phosphorylated STAT1 (p-STAT1) (p < 0.0001), p-STAT6 (p = 0.0001), p-PKA-Cγ (p = 0.0003) and p-CREB (p = 0.0025) expression compared with controls. STAT3-independent SOCS3 induction in inactive UC involves multiple proinflammatory signaling pathways and contradicts the usefulness of pathway-specific antiinflammatory drugs for preventing relapse. Our findings suggest that broad-spectrum antiinflammatory drugs are essential to counteract increases in SOCS3 expression and exacerbation of disease. Our results highlight the multifactorial nature of the factors that cause exacerbation in UC.

G-CSF-treated donor CD4+ T cells attenuate acute GVHD through a reduction in Th17 cell differentiation

The immunoregulatory effects of granulocyte colony-stimulating factor (G-CSF) on allogeneic peripheral blood cell transplantation (PBCT) have been demonstrated to reduce acute graft-versus-host disease (GVHD). However, the underlying mechanism is still not clear. In this study, we focused on the direct effects of G-CSF on donor CD4+ T cell responses after transplantation. We observed that lethally irradiated B6D2F1 recipient mice that are transplanted with CD4+ T cells from G-CSF-treated B6 donors showed mild attenuations in severity and mortality compared with recipients transplanted with PBS-treated CD4+ T cells. Notably, skin GVHD was significantly reduced, but no such reduction was observed in other organs. Although there was no difference with respect to alloreactive expansion or Foxp3+ Treg induction, the use of G-CSF-treated CD4+ T cells significantly reduced the numbers of IL-17-producing and RORγt-expressing cells in the secondary lymphoid organs of allogeneic recipients after transplantation compared with the use of the control cells. Finally, we found that the suppressor of cytokine signaling-3 (SOCS3) expression in G-CSF-treated donor CD4+ T cells was much higher than that in control CD4+ T cells. Our results demonstrate that the inhibition of Th17 cell differentiation by SOCS3 induction is associated with the immunoregulatory role of G-CSF in CD4+ T cell-mediated acute GVHD.

FXR ligands protect against hepatocellular inflammation via SOCS3 induction

Because of the anti-inflammatory actions of farnesoid X receptor (FXR) agonists, FXR has received much attention as a potential therapeutic target. However, the molecular mechanisms of actions have not yet been elucidated. In the present study, we reported that in the animal model of LPS-induced liver injury, administration of the FXR natural ligand CDCA could attenuate hepatocyte inflammatory damage, reduce transaminase activities, suppress inflammation mediators (IL-6, TNF-α and ICAM-1) expression and inhibit STAT3 phosphorylation. These protective effects of FXR were accompanied by an increased expression of suppressor of cytokine signaling 3 (SOCS3), which is a negative feedback regulator of cytokine–STAT3 signaling. We then demonstrated that the beneficial effects of FXR agonist in STAT3 activation were weakened by small interfering RNA-mediated SOCS3 knockdown in hepacytes. Moreover we observed both natural ligand CDCA and synthetic ligand GW4064 could upregulate SOCS 3 expression by enhancing the promoter activity in hepatocytes. These results suggest modulation of SOCS3 expression may represent a novel mechanism through which FXR activation could selectively affect cytokine bioactivity in inflammation response. FXR ligands may be potentially therapeutic in the treatment of liver inflammatory diseases via SOCS3 induction.