Expression Analysis of Suppressor of Cytokine Signaling (SOCS) Genes in Blood of Autistic Patients

The chemokine CXCL12 induces prolonged focal adhesion kinase (FAK) phosphorylation and sustained proadhesive responses in progenitor bone-marrow (BM) B cells, but not in mature peripheral B cells. Here we demonstrate that suppressor of cytokine signaling 3 (SOCS3) regulated CXCL12-induced FAK phosphorylation through the ubiquitin-proteasome pathway. CXCL12 triggered increased FAK ubiquitination in mature B cells, but not in progenitor B cells. Accordingly, SOCS3 expression was low in progenitor B cells, increased in immature B cells, and highest in mature B cells. SOCS3 overexpression in pro-B cells impaired CXCL12-induced FAK phosphorylation and proadhesive responses. Conversely, SOCS3-deficient mature B cells from Cre(MMTV)Socs3(fl/fl) mice exhibited prolonged FAK phosphorylation and adhesion to VCAM-1. In contrast to wild-type mice, Cre(MMTV)Socs3(fl/fl) mice had a 2-fold increase in immature B cells, which were evenly distributed in endosteal and perisinusoidal BM compartments. We propose that the developmental regulation of CXCR4-FAK signaling by SOCS3 is an important mechanism to control the lodgement of B cell precursors in the BM microenvironment.

Enhanced Induction of HIV-specific Cytotoxic T Lymphocytes by Dendritic Cell-targeted Delivery of SOCS-1 siRNA

Myoblast differentiation is characterized by a sequence of events that includes an increase in insulin-like growth factor (IGF)-I and contractile gene expression. The increase in IGF-I expression activates cell signaling mechanisms that participate in the differentiation process. One potential contributor is the SOCS-3 (suppressor of cytokine signaling-3) gene, which regulates signaling mechanisms and may be sensitive to changes in IGF-I concentrations. For the first time, the role of SOCS-3 is investigated in myoblast differentiation. SOCS-3 mRNA levels and SOCS-3 transcriptional activity increase during myoblast differentiation. SOCS-3 gene expression is induced, at least in part, by activation of the IGF-I receptor during myoblast differentiation. Overexpression of SOCS-3 cDNA significantly increased transcriptional activation of the 2.0-kb skeletal alpha-actin promoter in differentiating C2C12 myoblasts. In addition, overexpression of SOCS-3 specifically increased serum response factor-driven transcriptional activity but had no effect on nuclear-factor of activated T cell-driven transcriptional activity. SOCS-3 overexpression induced skeletal alpha-actin transcription in a myoblast cell line that cannot respond to endogenous IGF-I, indicating that SOCS-3 can contribute to the myoblast differentiation process in the absence of IGF-I. These data suggest that IGF-I induces myoblast differentiation, in part, by increasing SOCS-3 expression.

Neuroinflammation is being recognized as a hallmark of different neurodegenerative disorders, including Alzheimer's disease. Suppressor of cytokine signaling 3 (SOCS3) is an anti-inflammatory molecule, which is known to inhibit cytokine signaling and inflammatory gene expression in different cells. However, the pathways by which SOCS3 could be up-regulated in brain cells are poorly understood. Aspirin is a widely available pain reliever that is showing promise beyond its known pain-relieving capacity. This study underlines the importance of aspirin in upregulating SOCS3 in astrocytes and microglia. Aspirin increased the expression of Socs3 mRNA and protein in mouse astrocytes and BV-2 microglial cells in both a time- and dose-dependent manner. While investigating the mechanism, we found that Socs3 gene promoter harbors peroxisome proliferator response element and that aspirin up-regulated SOCS3 in astrocytes isolated from PPARβ (-/-), but not PPARα (-/-), mice. Accordingly, aspirin increased SOCS3 in vivo in the cortex of wild type and PPARβ (-/-), but not PPARα (-/-), mice. Similarly, aspirin treatment increased astroglial and microglial SOCS3 in the cortex of FAD5X, but not FAD5X/PPARα (-/-), mice. Finally, recruitment of PPARα by aspirin to the proximal, but not distal, peroxisome proliferator response element of the Socs3 promoter suggests that aspirin increases the transcription of Socs3 gene via PPARα. This study describes a novel property of aspirin in elevating SOCS3 in glial cells via PPARα and suggests that aspirin may be further considered for therapeutic application in neuroinflammatory and neurodegenerative disorders.

Suppressor of Cytokine Signaling 1/JAB and Suppressor of Cytokine Signaling 3/Cytokine-Inducible SH2 Containing Protein 3 Negatively Regulate the Signal Transducers and Activators of Transcription Signaling Pathway in Normal Human Epidermal Keratinocytes

Suppressor of cytokine signaling (SOCS) proteins constitute a class of negative regulators for Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathways. These intracellular proteins are induced by cytokine signaling, but they can also be induced by stimulation of Toll-like receptors (TLR). It has even been suggested that SOCS proteins are important negative regulators of TLR signaling. Here we have elucidated the nature of the regulatory role of SOCS in TLR signaling. Induction of SOCS-3 and cytokine-inducible Src homology 2-containing protein (CIS) by TLR stimulation was strictly dependent on MyD88 but showed differing needs in case of SOCS-1. However, induction of SOCS proteins by TLR ligands was independent of type I interferon. In macrophages overexpressing SOCS, we were not able to observe an inhibitory effect of SOCS-1, SOCS-2, SOCS-3, or CIS on prototypical TLR target genes such as tumor necrosis factor-alpha. However, we found that TLR-2, TLR-3, TLR-4, and TLR-9 stimulation induced interferon-beta (IFN-beta), which is able to exert auto- and paracrine signaling, leading to the activation of secondary genes like IP-10. SOCS-1 and, to a lesser extent, SOCS-3 and CIS were able to inhibit this indirect signaling pathway following TLR stimulation, whereas neither MAP kinase nor NF kappa B signaling were affected. However, STAT-1 tyrosine phosphorylation following TLR triggering was severely impaired by SOCS-1 overexpression. Thus, our data suggest that SOCS proteins induced by TLR stimulation limit the extent of TLR signaling by inhibiting type I IFN signaling but not the main NF kappa B pathway.

Neuroinflammation plays an important role in the pathogenesis of various neurodegenerative diseases including Alzheimer's disease (AD). Suppressor of cytokine signaling 3 (SOCS3) is an anti-inflammatory molecule that suppresses cytokine signaling and inflammatory gene expression in different cells including microglia. The pathways through which SOCS3 could be upregulated are poorly described. Cinnamic acid is a metabolite of cinnamon, a natural compound that is being widely used all over the world as a spice or flavoring agent. Here, we examined if cinnamic acid could upregulate SOCS3 in microglia. Microglia and astroglia isolated from mouse brain as well as BV-2 microglial cells were treated with cinnamic acid followed by monitoring the level of SOCS3 and different proinflammatory molecules by RT-PCR and real-time PCR. To nail down the mechanism, we also performed ChIP analysis to monitore the recruitment of cAMP response element binding (CREB) to the socs3 gene promoter and carried out siRNA knockdown of CREB. Cinnamic acid upregulated the expression of SOCS3 mRNA and protein in mouse BV-2 microglial cells in dose- and time-dependent manner. Accordingly, cinnamic acid also increased the level of SOCS3 and suppressed the expression of inducible nitric oxide synthase and proinflammatory cytokines (TNFα, IL-1β and IL-6) in LPSstimulated BV-2 microglial cells. Similar to BV-2 microglial cells, cinnamic acid also increased the expression of SOCS3 in primary mouse microglia and astrocytes. We have seen that cAMP response element is present in the promoter of socs3 gene, that cinnamic acid induces the activation of CREB, that siRNA knockdown of CREB abrogates cinnamic acid-mediated upregulation of SOCS3, and that cinnamic acid treatment leads to the recruitment of CREB to the socs3 gene. These studies suggest that cinnamic acid upregulates the expression of SOCS3 in glial cells via CREB pathway, which may be of importance in neuroinflammatory and neurodegenerative disorders.

Rationale: Inadequate expression of suppressor of cytokine signaling 3 (SOCS3) with subsequent activation of its target, the transcription factor STAT3, has been implicated in tumorigenesis and cancer progression in the lung and other organs. Our lab has recently reported the novel capability of alveolar macrophages (AMs) to secrete SOCS3 within microvesicles (MVs). While AM delivery of MV-encapsulated SOCS3 was shown to suppress inflammatory signaling in recipient lung epithelial cells, the potential significance of this process in restraining the development of lung cancer has not been studied. Methods: A KRAS G12D mutant mouse model was utilized to determine dysfunction of AM SOCS3 secretion in lung cancer. Mice were administered adenoviral Cre recombinase via intratracheal instillation, resulting in formation of lung tumors after 16 weeks. Bronchoalveolar lavage (BAL) fluid and AMs were isolated from the lungs of KRAS and wild-type (WT) mice, and analysis of SOCS3 secretion in BAL or AM cell culture medium was done via ELISA after sonication to disrupt vesicles. In vitro experiments utilized human adenocarcinoma cells (A549) or KRAS mutant rat lung epithelial cells (RLE-G12V). Proliferation, apoptosis and transformation were assessed by Cyquant assay, Annexin V staining, and soft agar assay, respectively. For SOCS3 provision studies, natural AM-derived MVs (isolated by ultracentrifugation) or synthetic liposomes containing recombinant SOCS3 were utilized. Results: Levels of secreted SOCS3 were ~50% lower in KRAS mice BAL than in WT BAL fluid. Additionally, although AMs isolated from KRAS mice contained similar amounts of intracellular SOCS3 and released similar numbers of MVs as those from WT mice, their ex vivo capacity for SOCS3 secretion was significantly lower than that of WT AMs. To determine whether provision of exogenous SOCS3 could inhibit tumorigenesis, synthetic SOCS3 liposomes were administered to RLE-G12V cells prior to chemical transformation with N-Methyl-N′-nitro-N-nitrosoguanidine (MNNG). Addition of exogenous SOCS3 had the capacity to significantly inhibit colony formation in soft agar. To investigate effects of exogenous SOCS3 on established tumor cell function, A549 cells were exposed to both natural AM-derived MVs and SOCS3-containing liposomes and effects on proliferation and apoptosis were measured. Both liposomes and natural MVs significantly induced apoptosis and inhibited proliferation. Finally, the reduction in secreted SOCS3 observed in the mouse model was confirmed in BAL samples of a cohort of NSCLC lung cancer patients compared to healthy volunteers. Conclusion: We report a novel dysregulation of immune surveillance in the form of decreased SOCS3 secretion by AMs that is elicited by the tumor microenvironment, and that may promote tumorigenesis via sustained STAT3 activation. Future studies will focus on the mechanism underlying this defect and whether rescuing SOCS3 secretion can inhibit cancer progression in vivo. Citation Format: Jennifer M. Speth, Loka R. Penke, Joseph Bazzill, Daniel J. Schneider, Douglas A. Arenberg, James J. Moon, Venkateshwar G. Keshamouni, Marc Peters-Golden. Vesicular secretion of suppressor of cytokine signaling 3 by alveolar macrophages is dysregulated in NSCLC and its provision inhibits tumor cell function [abstract]. In: Proceedings of the Fifth AACR-IASLC International Joint Conference: Lung Cancer Translational Science from the Bench to the Clinic; Jan 8-11, 2018; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(17_Suppl):Abstract nr A35.

309-LB: Cell-Permeable Truncated SOCS3 Competitively Inhibits the Interaction Between Endogenous SOCS3 and Leptin Receptor (ObR) to Overcome Leptin Resistance on JAK/STAT Signaling

Axl, a tyrosine kinase receptor, was recently identified as an essential component regulating innate immune response. Suppressor of cytokine signaling 1 and suppressor of cytokine signaling 3 are potent Axl-inducible negative inflammatory regulators. This study investigated the role of Axl signaling pathway in immune restoration in an autologous blood-injection mouse model of intracerebral hemorrhage. Recombinant growth arrest-specific 6 (Gas6) and R428 were administrated as specific agonist and antagonist. In vivo knockdown of Axl or suppressor of cytokine signaling 1 and suppressor of cytokine signaling 3 by siRNA was applied. After intracerebral hemorrhage, the expression of endogenous Axl, soluble Axl, and Gas6 was increased, whereas the expression of suppressor of cytokine signaling 1 and suppressor of cytokine signaling 3 was inhibited. Recombinant growth arrest-specific 6 administration alleviated brain edema and improved neurobehavioral performances. Moreover, enhanced Axl phosphorylation with cleavage of soluble Axl (sAxl), and an upregulation of suppressor of cytokine signaling 1 and suppressor of cytokine signaling 3 were observed. In vivo knockdown of Axl and R428 administration both abolished the effect of recombinant growth arrest-specific 6 on brain edema and also decreased the expression suppressor of cytokine signaling 1 and suppressor of cytokine signaling 3. In vivo knockdown of suppressor of cytokine signaling 1 and suppressor of cytokine signaling 3 aggravated cytokine releasing despite of recombinant growth arrest-specific 6. In conclusion, Axl plays essential role in immune restoration after intracerebral hemorrhage. And recombinant growth arrest-specific 6 attenuated brain injury after intracerebral hemorrhage, probably by enhancing Axl phosphorylation and production of suppressor of cytokine signaling 1 and suppressor of cytokine signaling 3.

Expression and Suppressive Effects of Interleukin-19 on Vascular Smooth Muscle Cell Pathophysiology and Development of Intimal Hyperplasia

Suppressor of cytokine signaling 1 (SOCS1) is a negative regulator of multiple cellular pathways. Experimental studies assigned a tumour suppressor function and various mechanisms can result in the functional inactivation of SOCS1. Genomic mutations, for example, result in abnormal stabilization and dysregulation of the JAK/STAT signalling pathway and occur in several neoplasms, including c. 16% of patients with diffuse-large B-cell lymphoma (DLBCL) (Mottok et al, 2009; Capello et al, 2013; Schif et al, 2013). Functionally equivalent to a mutation is DNA methylation of promoter-associated CpG islands resulting in transcriptional silencing. Numerous studies described SOCS1 methylation in a variety of malignancies including lymphomas; however, DLBCL has not been studied. Here, we explored methylation of CpG islands in the SOCS1 gene locus in DLBCL. The CpG density in the SOCS1 locus (Fig 1A) shows two maxima: one extending over the promoter, the other spanning coding exon 2. Mapping of the examined regions from >30 publications (Supplementary Table 1) shows considerable variation – especially when viewed in relation to the functionally characterized promoter region (Fig 1A). Hypermethylation in the coding region (exon 2) has yielded controversial results and is even regarded as functionally questionable (Melzner & Moller, 2003; Chim & Kwong, 2004). With these uncertainties in mind, we chose a more comprehensive approach and used a MassArray assay (Sequenom, San Diego, CA) for locus-spanning, quantitative methylation measurements (referred to as ‘global’; Fig 1B) in combination with pyrosequencing as an orthogonal method (referred to as ‘local’; Fig 1C). As a study cohort, we used 154 comprehensively characterized DLBCL patients (Schif et al, 2013) from the network project ‘Molecular Mechanisms in Malignant Lymphoma’ (MMML; see Supplementary Appendix) (Hummel et al, 2006). Global methylation evaluation on nine fragments (sites MA1–MA9, shown in Supplementary Fig 1A, Supplementary Table 2) covered 9471 CpG-sites (287 CpG-sites in 33 cases; Fig 1B). With an analytical sensitivity of 86% (n = 1375 technical failures), and using three different methylation fraction cut-off points (0·1/0·3/0·5), we found 4·5/0·7/0·2% methylated CpG-sites. Differentially methylated regions (DMRs) were defined as regions containing at least five (or ten) differentially methylated CpGs (DMCs) whose total methylation difference was more than 10% (or 5%). In the absence of multiple measurements from independent samples, the use of ≥5 DMCs can overcome some statistical limitations of individual tests based on any individual sample. We failed to detect any DMRs with either cut-off. When compared to regions from prior studies (Fig 1A), no DLBCL cases showed differential methylation in any of these regions (Fig 1B). Correlation of overall methylation scores by case (and/or fragment) with SOCS1 expression data (Fig 1B) showed no inverse correlation (R = 0·02–0·13; P = 0·04–0·48). Cases with higher methylation (cut-off at 0·1) showed higher SOCS1 expression levels (R = 0·13; P = 0·04); given the overall low methylation scores this result was regarded as a statistical artifact caused by the large number of datapoints with very small confidence intervals. Local assessment using pyrosequencing on four fragments (S1–S4; location shown in Supplementary Fig 1B, Supplementary Table 2) covered 6314 CpG-sites (41 CpG-sites in 154 cases). The analytical sensitivity was 89%, and using three different methylation fraction cut-offs (0·1/0·3/0·5), we found 3·9/3·1/2·5% methylated CpG-sites and again, we failed to detect any DMRs (Supplementary Fig 2A–E). Correlation of methylation status and SOCS1 expression showed no inverse correlation (R: <0·001–0·003; P = 0·53–0·76). We additionally checked 88/154 cases in a separate focused MassArray experiment (using fragment MA5) and found no methylation or DMRs. In addition, we inspected 16 lymphoma cell lines (DOHH-2, HDML-2, Karpas-707, Karpas-1106, KHM-2, L428, L540, Namalwa, REH, SudHL-5, Karpas-422, Nalm6, WSU-NHL, Daudi, Ramos, Raji) by pyrosequencing and found no hypermethylation or DMRs in the promoter region (not shown). Investigation of the methylation status in the four control melanoma cases (Supplementary Fig 2E), three peripheral blood samples (negative), the SOCS1-deleted cell line Karpas-1106 (biological negative control) and seven technical standard methylation controls (Fig 1C) confirmed technical and analytical validity of the applied assays. SOCS1 is not hypermethylated in DLBCL. The negligible fraction of methylated CpG-sites shows no correlation with SOCS1 expression data or mutation status. These data indicate that SOCS1 methylation does not occur in DLBCL. Given that DLBCL is the most common non-Hodgkin lymphoma, it is surprising that SOCS1 methylation has not been comprehensively examined in DLBCL. Some abstracts have mentioned SOCS1 methylation in DLBCL (Cerri et al, 2007; Dou et al, 2010), and one study described SOCS1 methylation ranging from 5% to 50% in various types of immunodeficiency-related non-Hodgkin lymphomas(Capello et al, 2013); however, in these reports the evaluated regions are not well documented. In fact, given the acknowledged controversy of the methylation sites (Melzner & Moller, 2003; Chim & Kwong, 2004), we argue that global examination and the use of orthogonal methods is essential to reliably assess the SOCS1 methylation status. In general terms, our data indicate that the role of SOCS1 methylation in lymphomagenesis must be reconsidered, particularly in relation to the evaluated sites (i.e., exon 2) (Chim & Kwong, 2004). The physiological role of SOCS1 as a silencer of cytokine signalling is to serve as a ‘brake’ of fluctuating cytokine effects in developing B-cells (Corfe et al, 2011). Dysregulation of the tightly controlled SOCS1 signalling network is regarded as pro-lymphomagenic. Although we did not exclude all SOCS1 silencing mechanisms, our data establish that SOCS1 is not hypermethylated in DLBCL and there is experimental evidence that both mutant, as well as wild-type SOCS1 alleles are co-expressed (Mottok et al, 2009; Lennerz et al, 2015). Thus, we revisited the variability in SOCS1 expression levels in DLBCL. Importantly, the depicted expression gradients (Fig 1B, C) are based solely on the relative expression levels within the DLBCL group. Therefore, we compared RNA-expression levels of SOCS1 across 37 different cancer types from the cancer genome atlas (cancergenome.nih.gov). Notably, with the exception of thymoma samples, DLBCL has the overall highest expression with very little variation (Supplementary Fig 3). Thus, the consistently high expression levels of SOCS1 in conjunction with the coexpression of wild-type and mutant alleles suggests that future studies should explore whether DLBCL may, in fact, hitchhike the ‘rheostat’ function of SOCS1. We thank Karola Dorsch, Julia Melzner, Ywona Nerbas, Michaela Buck, Elena Moser and Elena Kelsch for expert technical help. Karin Hoffmann, and Carina Birkhofer for help with experiments. We acknowledge Birgit Schif who helped with logistics, initial experiments and validations as part of her doctoral thesis. We furthermore thank Christian Kohler, Birgt Schif, Olga Ritz and Thomas Barth for thoughtful discussions. The Deutsche Krebshilfe supports the Network Project Molecular Mechanisms in Malignant Lymphoma (Bonn, Germany, grant 70-3173-Tr3). The Else-Kröner Fresenius foundation supported JKL. SEW is supported by the Dres. Bayer foundation (Ravensburg, Germany) and The Hertha Nathorff Program (University Ulm, Ulm, Germany). The Deutsche Forschungsgemeinschaft supports RM (grant: MA2367/6-1) and PM (grant: MO384/6-1). UG is owner and CEO of Varionostic, GmbH, Ulm, Germany. The remaining authors declare no conflicts of interest. Lennerz JK: Designed research, performed research, collected data, analysed and interpreted data, performed statistical analysis and wrote the manuscript; Weissinger SE: Performed research, collected data and critically revised the manuscript. Gerstenmaier U: designed research, performed research, collected data, analysed data and critically revised the manuscript. Marienfeld R: Performed research, collected data, analysed data and critically revised the manuscript. Moeller P: Designed research, interpreted data and critically revised the manuscript. All authors approved the revised and submitted version. 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The interleukin (IL)-6-type cytokines play major roles in a variety of biological processes by signaling through a common receptor subunit, glycoprotein (gp) 130. We performed yeast two-hybrid screening to identify new binding partners of the activated gp130 and the associated Janus kinases. LMO4, a LIM domain-containing protein that belongs to a family of oncogenes, was identified in this assay. Further studies show that LMO4 associates with gp130 and Janus kinase1 in several mammalian cell types. It also interacts with protein-tyrosine phosphatase 2 (SHP2) and suppressor of cytokine signaling 3 (SOCS3). The binding domains involved in these interactions were mapped, and the interactions were shown to be in a direct manner by in vitro binding assays. It is likely that LMO4 exists in the gp130 complex. The cellular localization of LMO4 was detected primarily in the nucleus with a substantial amount also detected in the cytoplasm in several cell types. The effect of LMO4 in IL-6 signaling was subsequently examined. Overexpression of LMO4 enhanced the transcriptional activity and target gene expression of Stat 3 (signal transducers and activators of transcription 3). Consistent with this, silencing LMO4 expression in stable cell lines expressing the small interfering RNA of LMO4 decreased Stat3 activity. Furthermore, the half-life of gp130 was shortened, and the production of acute phase proteins induced by IL-6 was reduced. Together, our data reveal a positive regulatory role of LMO4 in IL-6 signaling, possibly by acting as a scaffold for stabilization of the gp130 complex. These studies may open up a link between the oncogenic effect of LMO proteins and their regulatory role in cytokine signaling in general.

Suppressor of cytokine signaling 1 (SOCS1) is a negative regulator of c-Kit and interleukin-3 (IL-3) receptor signaling. We examined the role of SOCS1 in regulating IL-3-induced cell growth of primary bone marrow-derived mast cells (BMMCs) from SOCS1-/- mice. Instead of showing increased proliferation, SOCS1-deficient BMMCs responded poorly to IL-3 and stem cell factor. SOCS1-/- BMMCs showed increased apoptosis and defective cell cycle entry. We show that the growth retardation of SOCS1-/- BMMCs was due to a cell intrinsic defect. Protein tyrosine phosphorylation following IL-3 stimulation was markedly diminished in SOCS1-/- BMMCs. Intriguingly, JAK2 and STAT5 proteins were selectively diminished in SOCS1-/- BMMCs, which also showed lower molecular mass products of p85 and Vav suggesting proteolytic degradation. Incubation of the SOCS1-/- BMMC lysate with STAT5, p85, and Vav immunoprecipitated from SOCS1+/+ cells directly demonstrated the dysregulated proteolytic activity in SOCS1-/- BMMCs. The proteolytic activity in SOCS1-/- BMMCs was selectively inhibited by phenylmethylsulfonyl fluoride and soybean trypsin inhibitor, suggesting that the protease regulated by SOCS1 is a tryptase. The dysregulated tryptase in SOCS1-/- BMMCs is unlikely to be mMCP6 or mMCP7, because the enzyme activity was not inhibited by Polybrene but was inhibited by normal mouse plasma. SOCS1+/+ BMMC lysate inhibited the proteolytic activity present in SOCS1-/- BMMC lysate, indicating that SOCS1-/- BMMCs lack an endogenous protease inhibitor. These results show that SOCS1 is required for the expression and/or stability of an endogenous protease inhibitor, which protects mast cells from their own proteolytic enzymes.

Molecular characterization, expression pattern and evolution of nine suppressors of cytokine signaling (SOCS) gene in the swamp eel (Monopterus albus)