Suppressor Of Cytokine Signaling 3 Gene Research Articles

The JAK2-Selective Inhibitor NS-018 Preferentially Suppresses CFU-GM Colony Formation By Bone-Marrow Cells From High-Risk Myelodysplastic Syndrome Patients

BackgroundCell signaling mediated by the JAK2-STAT (Janus kinase 2-signal transducer and activator of transcription) pathway plays a critical role in hematopoiesis, and its aberrant activation is associated with the progression of hematological malignancies. For instance, somatic mutations in the JAK2 gene that lead to constitutive activation of STATs are involved in the pathogenesis of myeloproliferative neoplasms (MPN) and refractory anemia with ringed sideroblasts with thrombocytosis, one of the myelodysplastic/myeloproliferative neoplasms, unclassifiable (MDS/MPN, U). In addition, insufficient inactivation of the JAK-STAT pathway due to repression of SOCS-1 (suppressor of cytokine signaling 1) through hypermethylation of the SOCS-1 gene is involved in the disease progression of high-risk myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) (Brakensiek et al.; Br. J. Haematol. 2005;130:209). These observations prompted us to investigate the effect of the JAK2-selective inhibitor NS-018, which is in an early-phase clinical trial for MPN, on the colony formation by bone marrow hematopoietic progenitor cells from high-risk de novo MDS patients and the phosphorylation status of STAT3 in these cells. MethodsBone marrow mononuclear cells (BMMNCs) from six MDS patients and three healthy volunteers were collected with informed consent in accordance with the Declaration of Helsinki and with the approval of the Institutional Review Board. The MDS subtypes of the six patients included two RCMD, three RAEB-1 and one RAEB-2 according to the WHO classification. All MDS patients had complex cytogenetic abnormalities and their prognostic risks were defined to be high (N=1) or very high (N=5) in the IPSS-R. Commercially available normal human CD34+ BM cells were also examined for comparison. Cells were incubated in methylcellulose medium containing cytokines with or without NS-018. Burst forming unit-erythroid (BFU-E) and colony forming unit-granulocyte/macrophage (CFU-GM) were scored on day 14 of culture. Colony-forming cells were then collected and subjected to western blotting analysis. ResultsWe first examined the colony-formation capacity of MDS-derived BMMNCs. CFU-GM colony formation was observed in four of six MDS samples, although the absolute CFU-GM numbers from MDS BMMNCs were less than those from normal BMMNCs and normal CD34+BM cells. MDS-derived BMMNCs produced larger numbers of CFU-GM colonies (78–99% of total colonies) than BFU-E, similar to the findings usually observed in AML, whereas normal BMMNCs and normal CD34+BM cells formed virtually the same numbers of CFU-GM colonies and BFU-E colonies. These results suggest that BMMNCs from four of the MDS patients enrolled in this study had characteristics similar to AML BMMNCs, although they showed less proliferation than AML BMMNCs. We next examined the effect of NS-018 on CFU-GM colony formation. NS-018 treatment decreased the numbers of CFU-GM colonies from MDS-derived BMMNCs in a dose-dependent manner, and this effect was significantly more potent against MDS-derived than against normal cells (57.2% inhibition in MDS cells vs. 12.8% inhibition in normal cells at 0.5 mM NS-018; see Figure). These results indicate that NS-018 is preferentially efficacious in inhibiting CFU-GM formation from BMMNCs from high risk MDS. In addition, the level of phospho-STAT3 in MDS-derived colony-forming cells was twice as high as in colony-forming cells from normal BMMNCs, and 1.0 mM NS-018 completely suppressed the phosphorylation of STAT3 in CFU-GM colony-forming cells from MDS. ConclusionOur results show for the first time that a JAK2 inhibitor, NS-018, potently suppresses the formation of MDS colonies. NS-018 could be a new therapeutic option for high-risk MDS patients. [Display omitted] Disclosures:Kodama:Nippon Shinyaku: Employment. Naito:Nippon Shinyaku: Employment. Nakaya:Nippon Shinyaku: Employment. Taniwaki:Novartis: Honoraria.

SOCS3-Mediated Blockade Reveals Major Contribution of JAK2/STAT5 Signaling Pathway to Lactation and Proliferation of Dairy Cow Mammary Epithelial Cells in Vitro

Suppressor of cytokine signaling 3 (SOCS3) is a cytokine-induced negative feedback-loop regulator of cytokine signaling. More and more evidence has proved it to be an inhibitor of signal transducers and activators of transcription 5 (STAT5). Here, we used dairy cow mammary epithelial cells (DCMECs) to analyze the function of SOCS3 and the interaction between SOCS3 and STAT5a. The expression of SOCS3 was found in cytoplasm and nucleus of DCMECs by fluorescent immunostaining. Overexpression and inhibition of SOCS3 brought a remarkable milk protein synthesis change through the regulation of JAK2/STAT5a pathway activity, and SOCS3 expression also decreased SREBP-1c expression and fatty acid synthesis. Inhibited STAT5a activation correlated with reduced SOCS3 expression, which indicated that SOCS3 gene might be one of the targets of STAT5a activation, DCMECs treated with L-methionine (Met) resulted in a decrease of SOCS3 expression. SOCS3 could also decrease cell proliferation and viability by CASY-TT detection. Together, our findings indicate that SOCS3 acts as an inhibitor of JAK2/STAT5a pathway and disturbs fatty acid synthesis by decreasing SREBP-1c expression, which validates its involvement in both milk protein synthesis and fat synthesis. In aggregate, these results reveal that low SOCS3 expression is required for milk synthesis and proliferation of DCMECs in vitro.

Effect of hepatitis B virus X protein on suppressor of cytokine signaling-1 expression

To investigate the effect of hepatitis B virus-encoded X protein (HBx) on the expression of host-encoded suppressor of cytokine signaling-1 (SOCS-1) and to explore the possibility of an underlying mechanism involving modulation of CpG island methylation in the SOCS-1 gene promoter. The immortalized human derived non-tumor liver cell line QSG7701 was transfected with a recombinant HBx plasmid (pcDNA-X) or an empty vector control plasmid (pcDNA3.0) and stably transfected clones were selected by G418 resistance screening. Untransfected cells served as negative controls. Expression of SOCS-1 mRNA and protein was detected by real-time quantitative PCR and western blotting. The methylation status of SOCS-1 was detected by methylation-specific PCR (MSP). The significance of intergroup differences was analyzed by one-way ANOVA or pairwise comparison with post-hoc LSD test. SOCS-1 mRNA level was significantly lower in the pcDNA-X/QSG7701 cells compared to that in the pcDNA3.0/QSG7701 and untransfected cells (0.3249+/-0.0536 vs. 1.0543+/-0.1937 and 1.00; F = 19.6, P = 0.042). SOCS-1 protein level was similarly lower in the pcDNA-X/QSG7701 cells (0.1496+/-0.0106 vs. 0.1984+/-0.0438 and 0.2152+/-0.0816; F = 19.4, P = 0.048). The SOCS-1 promoter region showed methylation only in the pcDNA-X/QSG7701 cells. HBx-expressing human hepatocytes have down-regulated SOCS-1 expression, both at the mRNA and protein levels, and this effect corresponds to increased methylation in the SOCS-1 promoter region harboring CpG islands.

Inhibitory effects of suppressor of cytokine signaling 3 on inflammatory cytokine expression and migration and proliferation of IL-6/IFN-γ-induced vascular smooth muscle cells

The main pathogenesis of saphenous vein graft neointimal hyperplasia after coronary artery bypass grafting (CABG) is inflammation-caused migration and proliferation of vascular smooth muscle cells (VSMCs). Janus kinase 2/signal transducer and activators of transcription 3 (JAK2/STAT3) pathway is an important signaling pathway through which VSMCs phenotype conversion occurs. Suppressor of cytokine signaling 3 (SOCS3) is the classic negative feedback inhibitor of JAK2/STAT3 pathway. Growing studies show that SOCS3 plays an important anti-inflammatory role in numerous autoimmune diseases, inflammatory diseases and inflammation-related tumors. However, the effect and mechanism of SOCS3 on vein graft disease is unclear. The purpose of this study was to investigate the effects of SOCS3 on the inflammation, migration and proliferation of VSMCs in vitro and the mechanism. The small interference RNA plasmid targeting rat SOCS3 (SiRNA-rSOCS3) and the recombinant adenovirus vector carrying rat SOCS3 gene (pYrAd-rSOCS3) were constructed, and the empty plamid (SiRNA-control) and vector (pYrAd-GFP) only carrying GFP reported gene were constructed as control. The rat VSMCs were cultured. There were two large groups of A (SOCS3 up-regulated): control group, IL-6/IFN-γ group, IL-6/IFN-γ+pYrAd-rSOCS3 group, IL-6/IFN-γ(+)pYrAd-GFP group; and B (SOCS3 down-regulated): control group, IL-6/IFN-γ group, IL-6/IFN-γ+SiRNA-rSOCS3 group and IL-6/ IFN -γ+SiRNA-control group. The pYrAd-rSOCS3 and SiRNA-rSOCS3 were transfected into VSMCs induced by IL-6/IFN-γ. After 24 h, real-time reverse transcription polymerase chain reaction (RT-PCR) and Western blotting were used to detect the mRNA and protein expression of SOCS3, STAT3 (only by Western blotting), P-STAT3 (only by Western blotting), IL-1β, IL-6, TNF-α, MCP-1 and ICAM-1. The MTT, Transwell assay and flow cytometry were used to examine VSMCs proliferation, migration and cell cycle progression, respectively. As compared with control group, the mRNA and protein expression of SOCS3, STAT3, P-STAT3, IL-1β, IL-6, TNF-α, MCP-1 and ICAM-1 was significantly up-regulated in VSMCs stimulated by IL-6/IFN-γ. However, in VSMCs transfected with pYrAd-rSOCS3 before stimulation with IL-6/IFN-γ, the expression of SOCS3 mRNA and protein was further up-regulated, and that of STAT3, P-STAT3, IL-1β, IL-6, TNF-α, MCP-1 and ICAM-1 was significantly down-regulated as compared with IL-6/IFN-γ group and IL-6/IFN-γ+pYrAd-GFP group. The expression of those related-cytokines in IL-6/IFN-γ+SiRNA-rSOCS3 group was markedly increased as compared with IL-6/IFN-γ group and IL-6/IFN-γ+SiRNA-control group. The absorbance (A) values, the number of cells migrating to the lower chamber, and percentage of cells in the G2/M+S phase were increased in VSMCs stimulated by IL-6/IFN-γ. In VSMCs incubated with pYrAd-rSOCS3 or SiRNA-rSOCS3 before IL-6/IFN-γ stimulation, the A values, the number of cells migrating to the lower chamber, and the percentage of cells in the G2/M+S phase were significantly decreased, and increased respectively. These results imply that IL-6/IFN-γ, strong inflammatory stimulators, can promote transformation of VSMCs phenotype form a quiescent contractile state to a synthetic state by activating JAK2/STAT3 pathway. Over-expresssed SOCS3 might inhibit pro-inflammatory effect, migration and growth of VSMCs by blocking STAT3 activation and phosphorylation. These data in vitro confirm that SOCS3 may play a negatively regulatory role in development and progression of vein graft failure. These conclusions can provide a novel strategy for clinical treatment of vein graft diseases and a new theoretic clue for related drug development.

Down-regulation of SOCS3 gene in hypothalamus attenuates diet-induced obesity in young rats

Aims Acquired childhood obesity is becoming increasingly apparent with the changes in children`s life-style and eating environment, which become a severe social and medical problem. Our previous studies have found that the leptin concentrations were high in obese children, supporting that leptin resistance is a main mechanism of childhood obesity. The suppressor of cytokine signaling 3 (SOCS3) is a negative-feedback regulator of leptin signaling involved in leptin resistance, therefore suppression of SOCS3 is a potential therapy for leptin-resistance in obesity. In the studies, we investigate whether hypothalamic silencing of SOCS3 would attenuate diet-induced obesity and leptin resistance in young rats.

Parasite Infections in Multiple Sclerosis Modulate Immune Responses through a Retinoic Acid–Dependent Pathway

We recently demonstrated better outcomes in helminth-infected multiple sclerosis (MS) patients, compared with uninfected ones. The present study evaluates the role of TLR2 and retinoic acid (RA) in parasite-driven protection in MS patients. RA serum levels were significantly higher in helminth-infected MS patients than in uninfected MS subjects or healthy controls. Genes involved in RA biosynthesis and metabolism, such as Adh1 and Raldh2, as well as RA receptors and IL-10, were induced in dendritic cells (DCs) via TLR2-dependent ERK signaling. This programmed DCs to induce FOXP3(+) T regulatory cells and suppressed production of proinflammatory cytokines (IL-6, IL-12, IL-23, and TNF-α) via induction of suppressor of cytokine signaling 3 (SOCS3), an effect mediated by soluble egg Ag (SEA) obtained from Schistosoma mansoni, and by RA. SEA-activated DCs also inhibited IL-17 and IFN-γ production through autoreactive T cells. These inhibitory effects were abrogated when SOCS3 gene expression was silenced, indicating that SEA-mediated signaling inhibited production of these cytokines by T cells, through a SOCS3-dependent pathway. Overall, helminth-related immunomodulation observed in MS patients was mediated by TLR2- and RA-dependent pathways, through two different mechanisms, as follows: 1) induction of IL-10 and FOXP3(+) T regulatory cells, and 2) suppression of proinflammatory cytokine production mediated by SOCS3.

Molecular mechanism underlying the antiproliferative effect of suppressor of cytokine signaling‐1 in non‐small‐cell lung cancer cells

Lung cancer (LC) is the major cause of death by cancer and the number of LC patients is increasing worldwide. This study investigated the therapeutic potential of gene delivery using suppressor of cytokine signaling 1 (SOCS-1), an endogenous inhibitor of intracellular signaling pathways, for the treatment of LC. To examine the antitumor effect of SOCS-1 overexpression on non-small-cell lung cancer (NSCLC) cells, NSCLC cells (A549, LU65, and PC9) were infected with adenovirus-expressing SOCS-1 vector. The cell proliferation assay showed that A549 and LU65, but not PC9, were sensitive to SOCS-1 gene-mediated suppression of cell growth. Although JAK inhibitor I could also inhibit proliferation of A549 and LU65 cells, SOCS-1 gene delivery appeared to be more potent as SOCS-1 could suppress focal adhesion kinase and epidermal growth factor receptor, as well as the JAK/STAT3 signaling pathway. Enhanced phosphorylation of the p53 protein was detected by means of phospho-kinase array in SOCS-1 overexpressed A549 cells compared with control cells, whereas no phosphorylation of p53 was observed when JAK inhibitor I was used. Furthermore, treatment with adenoviral vector AdSOCS-1 in vivo significantly suppressed NSCLC proliferation in a xenograft model. These results suggest that the overexpression of SOCS-1 gene is effective for antitumor therapy by suppressing the JAK/STAT, focal adhesion kinase, and epidermal growth factor receptor signaling pathways and enhancing p53-mediated antitumor activity in NSCLC.

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.

GW24-e2291 Association of SOCS3 genetic polymorphisms with insulin resistance in Xinjiang Uygur population

ObjectivesTo investigate the association between suppressor of cytokine signalling 3(SOCS3) genetic polymorphisms and insulin resistance (IR) in Xinjiang Uygur population.MethodsIn this cross-sectional study on the metabolic diseases (e.g. obesity) among...

Overexpression of suppressor of cytokine signaling 3 in the arcuate nucleus of juvenile Phodopus sungorus alters seasonal body weight changes

The profound seasonal cycle in body weight exhibited by the Djungarian hamster (Phodopus sungorus) is associated with the development of hypothalamic leptin resistance during long day photoperiod (LD, 16:8h light dark cycle), when body weight is elevated relative to short day photoperiod (SD, 8:16h light dark cycle). We previously have shown that this seasonal change in physiology is associated with higher levels of mRNA for the potent inhibitor of leptin signaling, suppressor of cytokine signaling-3 (SOCS3), in the arcuate nucleus (ARC) of LD hamsters relative to hamsters in SD. The alteration in SOCS3 gene expression preceded the body weight change suggesting that SOCS3 might be the molecular switch of seasonal body weight changes. To functionally characterize the role of SOCS3 in seasonal body weight regulation, we injected SOCS3 expressing recombinant adeno-associated virus type-2 (rAAV2-SOCS3) constructs into the ARC of leptin sensitive SD hamsters immediately after weaning. Hamsters that received rAAV2 expressing enhanced green fluorescent protein (rAAV2-EGFP) served as controls. ARC-directed SOCS3 overexpression led to a significant increase in body weight over a period of 12 weeks without fully restoring the LD phenotype. This increase was partially due to elevated brown and white adipose tissue mass. Gene expression of pro-opiomelanocortin was increased while thyroid hormone converting enzyme DIO3 mRNA levels were reduced in SD hamsters with SOCS3 overexpression. In conclusion, our data suggest that ARC-directed SOCS3 overexpression partially overcomes the profound seasonal body weight cycle exhibited by the hamster which is associated with altered pro-opiomelanocortin and DIO3 gene expression.

Immunochemoradiotherapy for Patients with Oral Squamous Cell Carcinoma: Augmentation of OK-432-Induced Helper T Cell 1 Response by 5-FU and X-ray Irradiation

Eighty-one patients with oral squamous cell carcinoma (OSCC) received oral fluoropyrimidine UFT and radiotherapy (RT) with or without an immunotherapeutic agent OK-432. Both overall survival and progression-free survival of patients who received RT + UFT + OK-432 were significantly longer than those of patients who received RT + UFT (P = .0075 and P = .0175, respectively). Clinical response was also more favorable in RT + UFT + OK-432 group than in RT + UFT group (P = .0066). Next, in vitro experiments were conducted to examine the effect of 5-fluorouracil (5-FU) and X-ray irradiation in OK-432-induced immunity. Human peripheral blood mononuclear cells stimulated with OK-432 produced helper T cell 1 (Th1)-type cytokines as well as interleukin-10 (IL-10) and transforming growth factor-β (TGF-β), which are produced by Th2 and regulatory T cells (Tregs), respectively, and are inhibitory in antitumor immunity. OK-432-induced IL-10 and TGF-β but not Th1 cytokines were significantly inhibited by 5-FU and/or X-ray. 5-FU and X-ray also inhibited the expression of mRNAs for GATA-3 and Foxp3, which are transcription factors for Th2 and Tregs, respectively, but not for T-bet, a transcription factor for Th1. In addition, 5-FU and X-ray decreased the expression of mRNAs for suppressor of cytokine signaling 1 (SOCS1) and SOCS3. Antisense oligonucleotides for SOCS1 and SOCS3 markedly reduced OK-432-induced IL-10 and TGF-β. This is the first report clearly demonstrating that OK-432-based immunotherapy significantly enhanced the therapeutic effects of chemoradiotherapy in patients with OSCC as well as elucidating the mechanism of the synergistic effect of immunochemoradiotherapy in which 5-FU and radiation enhanced OK-432-induced Th1 response mediated by the inhibition of SOCS1 and SOCS3 gene expression.

MiR-19a Promotes Cell Growth and Tumorigenesis through Targeting SOCS1 in Gastric Cancer

Accumulating evidence has shown that microRNAs are involved in cancer development and progression. However, it remains unknown about the potential role of miR-19a in the pathogenesis of gastric cancer. Here, we report that suppressor of cytokine signaling 1 (SOCS1) is a novel target of miR-19a in gastric cancer cells and that miR-19a expression is inversely correlated with SOCS1 expression in gastric cancer cells and a subset of gastric cancer tissues. Ectopic expression of miR-19a dramatically promoted proliferation and tumorigenicity of gastric cancer cells both in vitro and in vivo. Moreover, we showed that silencing of SOCS1 promoted cell growth and colony formation resembling that of miR-19a overexpression, whereas re-introduction of SOCS1 (without the 3'-UTR) attenuated the pro-tumorigenic functions. Taken together, our findings suggest that the SOCS1 gene is a direct target of miR-19a, which functions as an oncogenic miRNA in gastric cancer by repressing the expression of tumor suppressor SOCS1.

SOCS3 promotor hypermethylation and STAT3-NF-κB interaction downregulate SOCS3 expression in human coronary artery smooth muscle cells

Suppressor of cytokine signaling-3 (SOCS3) is an intracellular negative regulator of cytokine signaling pathway. We recently found significant reduction in SOCS3 expression in coronary artery smooth muscle cells (CASMCs) of atherosclerotic swine and also in vitro cultured cells. Here, we investigated the underlying mechanisms of SOCS3 downregulation by IGF-1 and TNF-α in human CASMCs(hCASMCs). We propose that hypermethylation of CpG islands in the SOCS3 promoter is responsible for decrease in SOCS3 expression involving STAT3 and NFkB-p65 interaction. Western blot and qPCR data revealed significant upregulation of SOCS3 (6- to 10-fold) in hCASMC when treated individually with TNF-α (100 ng/ml) or IGF-1 (100 ng/ml). However, a significant decrease (5-fold) was observed by the combined treatment with TNF-α and IGF-1 compared with individual stimulation. IGF-1 phosphorylated STAT3 and TNF-α-activated NF-κB in hCASMCs. In the nuclear extract of hCASMCs stimulated with both TNF-α and IGF-1, there was an interaction between NF-κB-p65 and pSTAT3, as determined by co-immunoprecipitation. Knockdown of STAT3 by small interfering RNA abolished SOCS3 expression in response to IGF-1. Methylation-specific PCR confirmed hypermethylation of SOCS3 promoter in hCASMCs stimulated with both TNF-α and IGF-1, and this was positively associated with elevated levels of DNA methyltransferase-I (9- to 10-fold). Knockdown of DNMT1 increased SOCS3 expression in IGF-1+TNF-α-stimulated cells. Downregulation of SOCS3 in the presence of both TNF-α and IGF-1 in hCASMCs is due to SOCS3 promoter hypermethylation involving STAT3-NFkBp65 interaction. Because TNF-α and IGF-1 are released due to mechanical injury during coronary intervention, hypermethylation of SOCS3 gene could be an underlying mechanism of intimal hyperplasia and restenosis.

EFFECTS OF SUPPRESSOR OF CYTOKINE SIGNALLING-1 ON PROLIFERATION INDUCED BY OXIDATIVE LOW-DENSITY LIPOPROTEIN IN VASCULAR SMOOTH MUSCLE CELLS

ObjectivesExcessive proliferation vascular smooth muscle cells(VSMC) can promote the development of atherosclerosis.And, in the development of atherosclerosis, oxidative low-density lipoprotein (oxLDL) enhance excessive proliferation of VSMC.As studies have showed, down-regulation...

ASSOCIATION OF SOCS3 GENE POLYMORPHISMS WITH OVERWEIGHT AND OBESITY IN UYGUR POPULATION IN XINJIANG HETIAN AREA OF CHINA

ObjectivesTo explore the relationship between suppressor of cytokine signalling 3 (SOCS3) genetic polymorphisms and overweight and obesity in Uygur Chinese general population in Xinjiang.Methods1407 Uygur individuals were enrolled (530 males,...

Enhanced suppressor of cytokine signaling 3 in arthritic cartilage dysregulates human chondrocyte function

To determine the expression of suppressor of cytokine signaling 3 (SOCS-3) in human articular chondrocytes and its functional consequences. Chondrocytes were isolated from the cartilage of patients with osteoarthritis (OA), patients with rheumatoid arthritis (RA), and trauma patients and from the healthy cartilage of patients with a femoral neck fracture. The human chondrocyte cell line G6 and primary bovine chondrocytes were used in validation experiments. SOCS-3 messenger RNA (mRNA) expression was measured by quantitative polymerase chain reaction, and SOCS-3 protein levels were determined by Western blotting and immunohistochemical analysis. To ascertain the role of SOCS-3 in the chondrocyte response to interleukin-1β (IL-1β) or lipopolysaccharide (LPS), the expression of SOCS3 was either reduced by small interfering RNA or enhanced by viral transduction. The expression of SOCS-3 mRNA (but not that of SOCS-1 mRNA) was significantly enhanced in chondrocytes obtained from OA cartilage (mean ± SD ΔC(t) 3.4 ± 1.0) and RA cartilage (ΔC(t) 3.4 ± 1.4) compared with cartilage obtained from patients with femoral neck fracture (ΔC(t) 5.3 ± 1.2). The expression of SOCS3 correlated significantly with that of other genes known to be expressed in arthritic chondrocytes, such as MMP13 (r = 0.743), ADAMTS4 (r = 0.779), and ADAMTS5 (r = 0.647), and an inverse relationship was observed with COL2A1 (r = -0.561). Up-regulation of SOCS-3 by IL-1 in G6 chondrocytes and its spontaneous expression in OA chondrocytes were reduced by mithramycin, a specific inhibitor of transcription factor Sp-1. Overexpression of SOCS-3 in bovine chondrocytes reduced IL-1- and LPS-induced nitric oxide production and insulin-like growth factor 1-induced proteoglycan synthesis. Interestingly, a similar impairment of function was observed in OA chondrocytes, which was partially restored by SOCS-3 gene knockdown. This study demonstrated that both SOCS-3 mRNA and SOCS-3 protein are expressed in human arthritic chondrocytes and affect cellular responses involved in cartilage pathology.

Downregulating SOCS3 with siRNA ameliorates insulin signaling and glucose metabolism in hepatocytes of IUGR rats with catch-up growth

Individuals with intrauterine growth retardation (IUGR) who demonstrate a catch-up in body weight are prone to insulin resistance. High expressions of suppressor of cytokine signaling 3 (SOCS3) are thought to aggravate insulin resistance. We hypothesized that downregulating SOCS3 expression via small interfering RNA (siRNA) might have beneficial effects on insulin-resistant hepatocytes of catch-up growth IUGR rats (CG-IUGRs). An IUGR rat model was employed via maternal nutritional restriction. After evaluating metabolic states of CG-IUGR offspring, effective SOCS3-specific siRNA (siSOCS3) was transfected into cultured hepatocytes using liposomes. mRNA levels of SOCS3, insulin receptor substrates (IRSs), phosphatidylinositol 3-kinase (PI3K), and Akt2, key gluconeogenesis genes, were assessed via real-time PCR. Protein expression and phosphorylation changes were evaluated via western blot. CG-IUGR hepatocytes showed increases in SOCS3 and gluconeogenic gene expressions, and decreases in IRS1 and PI3K expressions as compared with controls. After transfecting CG-IUGR hepatocytes with siSOCS3, protein levels of IRS1, PI3K, and phosphorylated Akt2 were higher as compared with those of untransfected CG-IUGR cells. Transcriptional suppression effects of gluconeogenesis genes were more obvious in siSOCS3-treated cells after insulin stimulation. Additional insights were provided to understand mechanisms of insulin resistance in CG-IUGR rats. Downregulating SOCS3 might improve insulin signaling transduction and ameliorate hepatic glucose metabolism in insulin-resistant CG-IUGR rats in vitro.

Silencing of microRNA-122 enhances interferon-α signaling in the liver through regulating SOCS3 promoter methylation

Hepatitis C virus (HCV) infection is a major cause of chronic liver disease worldwide. Although novel drugs against HCV are under development, the current standard therapy consists principally of interferon (IFN). To improve the response to IFN treatment by enhancing interferon-stimulated response element (ISRE)-mediated gene transcription, we screened 75 microRNAs highly expressed in hepatocytes for their ability to modulate ISRE activity. Overexpression of microRNA-122 (miR122) significantly suppressed ISRE activity. Conversely, silencing of miR122 function enhanced IFN-induced ISRE activity, by decreasing expression of suppressor of cytokine signaling 3 (SOCS3). This decrease in SOCS3 level was not mediated by microRNA target gene suppression, but rather by enhanced methylation at SOCS3 gene promoter. Taken together, our data, along with the fact that antisense oligonucleotides of miR122 also directly inhibit HCV replication, suggest that a combination therapy comprising IFN and silencing of miR122 function may be a promising therapeutic option in the near future.

129 ASSOCIATION OF SOCS3 GENE POLYMORPHISMS WITH OVERWEIGHT AND OBESITY IN UYGUR POPULATION IN XINJIANG OF CHINA

Objective: To explore the relationship between suppressor of cytokine signaling 3 (SOCS3) genetic polymorphisms and overweight and obesity in Uygur Chinese general population in Xinjiang. Methods: 1407 Uygur individuals were enrolled (530 males, 876 females), The 3 single nucleotide polymorphisms (rs4949169, rs9914220, rs12953258) in SOCS3 gene were selected, Measure the height, weight, waist and hip circumference, calculate body mass index (BMI). Results: (1) Significant association was found between rs4949168 and the overweight, obesity in males, BMI(GG:AG:AA:(26.44±4.16) kg/m2vs.(27.23±4.34) kg/m2 vs.(27.78±4.48) kg/m2, P=0.042). Rs9914220 was associated with overweight and obesity in Uygur females in Xinjiang HETIAN area. But rs12953258 was not associated with overweight and obesity in Xinjiang HETIAN area. Conclusion: SOCS3 gene was significant associated with overweight and obesity in Uygur population in Xinjiang HETIAN area, and SOCS3 gene associated with overweight and obesity express distinctive in different gender.

Expression of SOCS3 throughout liver regeneration is not regulated by DNA methylation

While suppressor of cytokine signaling 3 (SOCS3) plays a crucial role in suppressing dysplasia and tumorigenesis, it also offers a typical instance of DNA methylation in the regulation of gene transcription, since the promoter region of the SOCS3 gene is rich in CpG islands (CGIs). During liver regeneration initiated by partial hepatectomy, SOCS3 acts as a suppressor to balance the acute-phase response and terminate the regeneration. This study aimed to determine whether the variation of SOCS3 expression throughout liver regeneration is also regulated by its DNA methylation. We established a 70% partial hepatectomy mouse model and the animals were sacrificed at indicated times to assess the SOCS3 expression. We performed bisulfite sequencing PCR and DNA sequencing to investigate the detailed cytosine methylation in the SOCS3 gene. Within the promoter sequence, 58 CGIs were identified and 30 were found variously methylated before or after operation; however, methylation remained at a very low level. No evidence indicated that the total methylation level or the methylation of any CpG site regularly changed throughout liver regeneration. DNA methylation or demethylation seems to be a relatively stable modification of cytosine, but not a dynamic and reversible process to regulate gene transcription in daily and acute pathophysiological events.