Isoproterenol is a positive regulator of the suppressor of cytokine signaling-3 gene expression in 3T3-L1 adipocytes

SOCS (suppressor of cytokine signaling)-3 has recently been shown to be an insulin- and tumor necrosis factor (TNF)-alpha-induced negative regulator of insulin signaling. To further clarify a potential involvement of SOCS-3 in the development of insulin resistance, we measured differentiation-dependent SOCS-3 mRNA expression in 3T3-L1 adipocytes and studied its regulation by various hormones known to impair insulin signaling using quantitative real-time RT-PCR. There was a differentiation-dependent downregulation of SOCS-3 mRNA by 50% over the 9 day adipocyte differentiation course. Interestingly, besides insulin and TNF-alpha, chronic treatment of differentiated 3T3-L1 cells with 10 microM isoproterenol for 16 h stimulated SOCS-3 gene expression by about 3.5-fold. Furthermore, isoproterenol stimulated SOCS-3 mRNA expression in a dose-dependent manner with significant activation detectable at concentrations as low as 10 nM isoproterenol. Moreover, a strong 27- and 47-fold activation of SOCS-3 mRNA expression could be seen after 1 h of isoproterenol and GH treatment respectively. The stimulatory effect of isoproterenol could be almost completely reversed by pretreatment of 3T3-L1 cells with the beta-adrenergic antagonist propranolol. Finally, isoproterenol's action could be mimicked by stimulation of G(S)-proteins with cholera toxin and of adenylyl cyclase with forskolin and dibutyryl cAMP. Taken together, our results demonstrate a differentiation-dependent downregulation of SOCS-3 in adipocytes and suggest that SOCS-3 gene expression is stimulated by beta-adrenergic agents via activation of a G(S)-protein-adenylyl cyclase-dependent pathway. As SOCS-3 is a novel inhibitor of insulin signaling, the data support a possible role of this protein as a selectively regulated mediator of catecholamine-induced insulin resistance.     

Methylation status of suppressor of cytokine signaling-1 gene in hepatocellular carcinoma

Background Silencing of the suppressor of cytokine signaling (SOCS-1) by aberrant methylation at the CpG island in the coding region gene has been reported in hepatocellular carcinoma (HCC). However, principally, it is methylation in the 5-noncoding region but not that in the coding region which determines the regulation of gene expression.Methods Methylation-specific PCR was performed for the analysis of methylation status both in the 5-noncoding region and the CpG island of SOCS-1 from 22 HCC tissue samples with adjacent non-HCC tissue samples and from two cell lines.Results Using primers in the CpG island, 9 of 22 HCC samples exhibited aberrant methylation of SOCS-1, while only 1 of 22 adjacent non-HCC samples did so. The unmethylation pattern was detected in 1 of 22 HCC and in 5 of 22 non-HCC samples. Thus, aberrant methylation of SOCS-1 was significantly associated with HCC (P = 0.0076 by Fishers exact test). Using primers in the 5-noncoding region, aberrant methylation was observed in 12 of 22 HCC and in 2 non-HCC samples. The unmethylated pattern was observed in 5 of 22 HCC and in 10 of 22 non-HCC samples (P = 0.0042). There was no significant correlation between the methylation status of SOCS-1 and clinicopathological findings, such as the presence or absence of cirrhosis or the histological grade of HCC.Conclusions Aberrant methylation of the SOCS-1 had a significant correlation with HCC. The rate of aberrant methylation was similar in the 5-noncoding region and in the CpG island. Aberrant methylation of SOCS-1 may be associated with hepatocarcinogenesis, although further studies are necessary.     

Endothelin-1 regulates adiponectin gene expression and secretion in 3T3-L1 adipocytes via distinct signaling pathways

Adiponectin, which is specifically and highly expressed in adipose tissue, has pleiotropic insulin-sensitizing effects. Endothelin-1 (ET-1) is a potent vasoconstrictive peptide mainly produced by endothelial cells. We previously showed that ET-1 can induce insulin resistance in vitro and in vivo and proposed that it might regulate adiponectin expression and secretion, thus affecting the homeostasis of whole-body energy metabolism. In the present study, we explored the regulatory effects of ET-1 on adiponectin expression and secretion and the underlying mechanisms in 3T3-L1 adipocytes using Northern blotting and ELISA. ET-1 was found to cause a significant time- and dose-dependent decrease in adiponectin expression, and this effect was inhibited by the ET type A receptor (ETAR) antagonist BQ-610 but not by the ETBR antagonist BQ-788. To explore the underlying mechanism, we examined the involvement of the cAMP-dependent protein kinase A-, phospholipase A2-, protein kinase C-, and MAPK-mediated pathways using inhibitors and found that only PD98059 and U0126, inhibitors that blocked MAPK/ERK kinase's ability to activate the ERKs, prevented ET-1-induced down-regulation of adiponectin. Furthermore, acute ET-1 treatment significantly stimulated adiponectin secretion by 3T3-L1 adipocytes, and this effect was inhibited by the ETAR antagonist BQ-610, the inositol-1,4,5-triphosphate receptor blocker 2-APB, and phospholipase C inhibitor U73122, showing that the release of adiponectin stimulated by ET-1 was mediated through the ETAR and the inositol-1,4,5-triphosphate pathway. In conclusion, ET-1 regulates adiponectin expression and secretion by two different signaling pathways in 3T3-L1 adipocytes. These findings suggested that the cardiovascular system affects adipocyte physiology by regulating the expression of adipocytokines and, consequently, energy homeostasis via vasoactive factors, such as ET-1.     

The SOCS box of suppressor of cytokine signaling-1 is important for inhibition of cytokine action in vivo

Suppressor of Cytokine Signaling-1 (SOCS-1) is an essential physiological inhibitor of IFN-gamma signaling. Mice lacking this gene die in the early postnatal period from a disease characterized by hyperresponsiveness to endogenous IFN-gamma. The SOCS box is a C-terminal domain shared with over 30 other proteins that links SOCS proteins to an E3 ubiquitin ligase activity and the proteasome, but whether it contributes to inhibition of cytokine signaling is currently disputed. We have deleted only the SOCS box of the SOCS-1 gene in mice and show that such mice have an increased responsiveness to IFN-gamma and slowly develop a fatal inflammatory disease. These results demonstrate that deletion of the SOCS box leads to a partial loss of function of SOCS-1.     

慢性乙型肝炎患者外周血单个核细胞中细胞信号转导抑制因子-1mRNA的表达

目的检测慢性乙型肝炎（CHB）患者外周血单个核细胞（PBMCs）中,细胞信号转导抑制因子-1（SOCS-1）mRNA的表达水平,分析其表达与肝脏不同炎症活动程度之间的关系。方法 CHB患者分为轻、中、重度组各15例,乙型肝炎病毒（HBV）携带者15例为携带组,15例健康体检者作为对照组。采集外周静脉血,密度梯度离心法分离PBMCs。RT-PCR检测SOCS-1 mRNA的表达。结果重度组SOCS-1 mRNA的表达（1.15±0.12）明显高于对照组（0.86±0.10）、携带组（0.89±0.18）和轻度组（0.93±0.12）（q=4.183～5.472,P〈0.01）。CHB患者SOCS-1 mRNA的表达水平与肝脏不同炎症活动程度呈正相关（rs=0.664,P〈0.01）。结论 CHB患者外周血PMBCs中SOCS-1 mRNA的表达与肝脏炎症活动程度有关。     

The SOCS box of suppressor of cytokine signaling-3 contributes to the control of G-CSF responsiveness in vivo

Suppressor of cytokine signaling 3 (SOCS3) is a negative regulator of granulocyte-colony stimulating factor (G-CSF) signaling in vivo. SOCS proteins regulate cytokine signaling by binding, via their SH2 domains, to activated cytokine receptors or their associated Janus kinases. In addition, they bind to the elongin B/C ubiquitin ligase complex via the SOCS box. To ascertain the contribution of the SOCS box of SOCS3 to in vivo regulation of G-CSF signaling, we generated mice expressing a truncated SOCS3 protein lacking the C-terminal SOCS box (SOCS3(Delta SB/Delta SB)). SOCS3(Delta SB/Delta SB) mice were viable, had normal steady-state hematopoiesis, and did not develop inflammatory disease. Despite the mild phenotype, STAT3 activation in response to G-CSF signaling was prolonged in SOCS3(Delta SB/Delta SB) bone marrow. SOCS3(Delta SB/Delta SB) bone marrow contained increased numbers of colony-forming cells responsive to G-CSF and IL-6. Treatment of the mice with pharmacologic doses of G-CSF, which mimics emergency granulopoiesis and therapeutic use of G-CSF, revealed that SOCS3(Delta SB/Delta SB) mice were hyperresponsive to G-CSF. Compared with wild-type mice, SOCS3(Delta SB/Delta SB) mice developed a more florid arthritis when tested using an acute disease model. Overall, the results establish a role for the SOCS box of SOCS3 in the in vivo regulation of G-CSF signaling and the response to inflammatory stimuli.     

Overexpression of suppressor of cytokine signaling-1 impairs pre-T-cell receptor-induced proliferation but not differentiation of immature thymocytes

Cytokines play an essential role during early T-cell development. However, the mechanisms controlling cytokine signaling in developing thymocytes have not been elucidated. Cytokine receptor signaling can be modulated by suppressor of cytokine signaling-1 (SOCS-1), which acts as a negative regulator of Janus kinases. SOCS-1 is normally expressed throughout thymocyte development; however, retroviral-mediated overexpression of SOCS-1 in fetal liver-derived hematopoietic progenitors prevented their progression beyond the earliest stage of T-cell development. Further analysis revealed that SOCS-1 expression is transiently suppressed following pre-T-cell receptor (TCR) signaling. Moreover, constitutive expression of SOCS-1 abrogated pre-TCR- mediated expansion of immature thymocytes but did not interfere with differentiation. These findings reveal that SOCS-1 serves to regulate cytokine signaling at critical checkpoints during early T-cell development.     

细胞因子信号转导抑制因子1与肺部疾病

细胞因子信号转导抑制因子1(suppressor of cytokine signaling-1,SOCS1)能反馈性抑制干扰素、白介素4等多种细胞因子的信号转导,在转录、翻译、蛋白各水平受到精密调控.适当的SOCS1表达可抑制哮喘的炎症反应,但不恰当的SOCS1表达可能增加发生哮喘的几率.在肺部感染中,SOCS1能防...     

Endothelin-1 stimulates arterial VCAM-1 expression via NADPH oxidase-derived superoxide in mineralocorticoid hypertension

Although hypertension is a major risk factor for atherosclerosis, its underlying mechanisms remain to be delineated. We have recently reported that both endothelin-1 (ET-1) and vascular cellular adhesion molecule-1 (VCAM-1) levels, key early markers of atherosclerosis, are significantly elevated in carotid arteries of deoxycorticosterone acetate (DOCA)-salt hypertensive rats, a model known for its suppressed plasma renin levels. This study tested the hypothesis that ET-1 augments arterial VCAM-1 expression through NADPH oxidase-derived superoxide (O2-). Carotid arteries of DOCA-salt or sham-operated rats were transduced ex vivo with extracellular superoxide dismutase (EC-SOD), dominant negative HA-tagged N17Rac1 that inhibits Rac1, the small GTPase component of NADPH oxidase, or beta-galactosidase (beta-gal) reporter gene (5x10(10) plaque formation units [pfu]/mL), and the effect of transgene expression on O2- and VCAM-1 levels was assayed 24 hours afterward. The arterial activity of NADPH oxidase but not xanthine oxidase was significantly higher in DOCA-salt than in sham rats, which was abolished by the selective ETA receptor antagonist ABT-627 (3x10(-8) mol/L), NADPH oxidase inhibitor apocynin (10(-4) mol/L), or dominant negative Rac1 gene transfer. The levels of O2- and VCAM-1 were significantly increased in arteries of DOCA-salt rats, an effect that was ameliorated after EC-SOD or dominant negative Rac1 but not beta-gal reporter gene transfer. ABT-627 and apocynin also significantly reduced elevated VCAM-1 levels in ET-1-treated arteries of normal rats and arteries of DOCA-salt rats. The results of this study indicate that ET-1 stimulates arterial VCAM-1 expression by producing O2- from an ETA receptor/NADPH oxidase pathway in low-renin mineralocorticoid hypertension.     

L02细胞乙型肝炎病毒X蛋白和SOCS-1基因水平研究*

目的 探讨乙型肝炎病毒X蛋白(HBx)影响细胞因子信号传导抑制因子-1(SOCS-1)基因水平的可能机制。方法 收集22例乙型肝炎相关肝细胞癌(HCC)手术后癌组织和癌旁肝组织,采用RT-PCR法检测组织HBx、DNMT3A、DNMT3B和SOCS-1 mRNA水平。通过脂质体法构建表达HBx的L02细胞和空质粒L02细胞,采用CCK8法检测5-氮杂-2′-脱氧胞苷(5-Aza-c)对L02细胞存活率的影响,采用RT-PCR法和Western blot法分别检测细胞HBx、DNA甲基转移酶(DNMT)3A、DNMT3B和SOCS-1 mRNA水平及其蛋白表达。结果 肝癌组织HBx和DNMT3A mRNA水平分别为(65.2±3.5)和(77.2 ± 3.8),显著高于癌旁肝组织【分别为(22.5±4.0)和(42.1± 2.9),P<0. 05】,而SOCS-1 mRNA水平为(33.1±3.0),显著低于癌旁肝组织【(75.6 ±2.6),P<0. 05】;与对照细胞比,表达HBx的L02细胞活力随5-Aza-c作用浓度增高而下降(P<0. 05);过表达HBx的L02细胞DNMT3A mRNA水平及其蛋白表达量显著高于空载质粒组(P<0.05),而SOCS-1 mRNA水平及其蛋白表达量显著低于空载质粒组(P<0. 05);在5-Aza-c干预表达HBx的L02细胞,DNMT3A mRNA水平及其蛋白表达量显著低于对照组(P<0. 05),而SOCS-1 mRNA水平及其蛋白表达量显著高于对照(P<0. 05)。结论 HBx通过上调DNMT3A表达使SOCS-1基因表达下调,表明HBx可能通过调控DNMT3A影响抑癌基因SOCS-1表达从而促进肝癌的发生。     

Amylin stimulates fatty acid esterification in 3T3-L1 adipocytes

Amylin is co-localized and co-secreted with insulin, however its direct effects on adipocytes are unexplored. In 3T3-L1 preadipocytes, amylin increased thymidine incorporation (174%; p < 0.05) and Myc mRNA expression (378%; p < 0.01). Amylin supplementation during differentiation enhanced triglyceride accumulation (272%; p < 0.001). In 3T3-L1 adipocytes, amylin increased fatty acid uptake (238%; p < 0.01) and further potentiated the effects of insulin (insulin 158%; p < 0.01, amylin + insulin 335%; p < 0.001 vs CTL, p < 0.001 vs insulin). By contrast, amylin inhibited glycerol release in 3T3-L1 adipocytes (−50%; p < 0.05) and primary adipocytes (−34%; p < 0.05). Amylin stimulated cytokine secretion (monocyte chemotactic protein-1 + 166%, keratinocyte-derived chemokine + 174%; both p < 0.05) and mRNA expression of PPARγ (163%; p < 0.01), C/EBPβ (121%, p < 0.05), DGAT1 (157%; p < 0.01), FABP4 (122%; p < 0.01), and CD36 (122%; p < 0.05). In human adipose tissue, mRNA expression of amylin receptor genes (CALCR and RAMP3) correlated with numerous lipid and insulin signaling genes, plasma glucose and HOMA. Altogether amylin directly stimulates fat cells, potentiates the effects of insulin and may influence insulin resistance.     

Endothelin-1 stimulates small artery VCAM-1 expression through p38MAPK-dependent neutral sphingomyelinase

Endothelin-1 (ET-1) stimulates vascular cell adhesion molecule (VCAM-1) expression, a process associated with arterial remodelling. However, the pathways activated by ET-1 that lead to VCAM-1 expression are not fully understood. It is reported that sphingomyelinases are necessary for VCAM-1 expression in response to cytokines. Our aim was to investigate the role of sphingomyelinases in ET-1-induced VCAM-1 expression. Acid and neutral sphingomyelinase activities were measured in extracts from rat mesenteric small arteries (RMSA). ET-1 (1-100 nmol/l) stimulated neutral but not acid sphingomyelinase. The activation was rapid, peaking within 5 min and transient, returning towards baseline by 10 min and inhibited by BQ-788, GW4869 and SB203580, which are inhibitors of ET(B) receptor, neutral sphingomyelinase and p38MAPK, respectively. Both GW4869 and SB203580 are reported to inhibit activation of neutral sphingomyelinase 2 implicating it in the response to ET-1. Accordingly we investigated the expression of this isoform and found it was present in RMSA, predominantly in endothelial cells. Treatment of RMSA with ET-1 (1-100 nmol/l) for 16 h increased VCAM-1 expression, which was inhibited by GW4869 and SB203580. These results indicate that ET-1 stimulates arterial VCAM-1 expression through p38MAPK-dependent activation of neutral sphingomyelinases. This suggests a role for sphingolipids in ET-1-induced vascular inflammation in cardiovascular disease.     

Insulin stimulates IGFBP-2 expression in 3T3-L1 adipocytes through the PI3K/mTOR pathway

Insulin-like growth factor binding protein 2 (IGFBP-2) has been implicated in the etiology of several diseases, including the metabolic syndrome. Although IGFBP-2 derives mostly from the liver, recent evidence in mice and humans indicate that aging and obesity are associated with altered IGFBP-2 levels in white adipocytes. The present study was aimed at determining the mechanisms that control IGFBP-2 expression in mature adipocytes. IGFBP-2 mRNA and protein expression in serum-deprived 3T3-L1 adipocytes were twofold increased by acute insulin treatment. Co-treatments with the phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin or the mammalian target of rapamycin (mTOR) inhibitor rapamycin blunted the effects of insulin. Coherently, IGFBP-2 mRNA levels were robustly increased in adipocytes lacking either TSC2 or 4E-BP1. Insulin triggered the recruitment of CAAT/enhancer binding protein α (C/EBPα) to the IGFBP-2 proximal promoter. These findings suggest that insulin upregulates IGFBP-2 expression through a PI3K/mTOR/C/EBPα pathway in white adipocytes.     

C反应蛋白对3T3-L1脂肪细胞脂联素表达和分泌的影响

目的 观察C反应蛋白(CRP)对313-L1脂肪细胞脂联素的表达和分泌的影响,并探讨其致胰岛素抵抗的作用机制.方法 分别用Northem印迹、Western印迹等方法观察CRP对脂联素表达及分泌的影响.结果 (1)Northern印迹显示25和50μg/ml CRP作用24 h分别使脂联素mRNA表达下降约31%和52%(均P<0.01),呈剂量依赖趋势;50 μg/ml CRP干预12和24 h分别使脂联素的表达下降约42%和52%(均P<0.01),呈时间依赖趋势.(2)Western印迹显示25和50μg/ml CRP作用24 h分别使脂联素的分泌下降约19%和41%(均P<0.01),呈剂量依赖趋势;50μs/ml CRP干预12和24 h分别使脂联素的分泌下降约29%和41%(均P<0.01).(3)用10 μmol/L磷脂酰肌醇3激酶(PDK)抑制剂LY294002与50μg/ml CRP干预313-L1细胞24 h,可部分逆转CRP对脂联素mRNA表达的抑制作用,使脂联素的表达恢复到对照组的77%.结论 CRP通过PDK途径抑制脂肪细胞脂联索的表达和分泌,可能是其导致胰岛素抵抗机制之一.     

C-reactive protein inhibits adiponectin gene expression and secretion in 3T3-L1 adipocytes

C-reactive protein (CRP) is considered as one of the most sensitive markers of inflammation. The aim of the present study is to investigate the effects of CRP on the production of adiponectin in 3T3-L1 adipocytes. Northern and western blot analysis revealed that CRP treatment inhibited adiponectin mRNA expression and secretion in a dose- and time-dependent manner. Co-incubation of adipocytes with rosiglitazone and CRP decreased induction of adiponectin gene expression by rosiglitazone. However, luciferase reporter assays did not show that CRP affected the activity of approximately 2.1 kb adiponectin gene promoter, which was increased by rosiglitazone alone. Pharmacological inhibition of phosphatidylinositol (PI)-3 kinase by LY294002 partially reversed inhibition of adiponectin gene expression by CRP. These results collectively suggest that CRP suppresses adiponectin gene expression partially through the PI-3 kinase pathway, and that decreased production of adiponectin might represent a mechanism by which CRP regulates insulin sensitivity.     

Signal transducer and activator of transcription-3/suppressor of cytokine signaling-3 (STAT3/SOCS3) axis in myeloid cells regulates neuroinflammation

Suppressor of cytokine signaling (SOCS) proteins are feedback inhibitors of the JAK/STAT pathway. SOCS3 has a crucial role in inhibiting STAT3 activation, cytokine signaling, and inflammatory gene expression in macrophages/microglia. To determine the role of SOCS3 in myeloid cells in neuroinflammation, mice with conditional SOCS3 deletion in myeloid cells (LysMCre-SOCS3(fl/fl)) were tested for experimental autoimmune encephalomyelitis (EAE). The myeloid-specific SOCS3-deficient mice are vulnerable to myelin oligodendrocyte glycoprotein (MOG)-induced EAE, with a severe, nonresolving atypical form of disease. In vivo, enhanced infiltration of inflammatory cells and demyelination is prominent in the cerebellum of myeloid-specific SOCS3-deficient mice, as is enhanced STAT3 signaling and expression of inflammatory cytokines/chemokines and an immune response dominated by Th1 and Th17 cells. In vitro, SOCS3-deficient macrophages exhibit heightened STAT3 activation and are polarized toward the classical M1 phenotype. SOCS3-deficient M1 macrophages provide the microenvironment to polarize Th1 and Th17 cells and induce neuronal death. Furthermore, adoptive transfer of M2 macrophages into myeloid SOCS3-deficient mice leads to delayed onset and reduced severity of atypical EAE by decreasing STAT3 activation, Th1/Th17 cells, and proinflammatory mediators in the cerebellum. These findings indicate that myeloid cell SOCS3 provides protection from EAE through deactivation of neuroinflammatory responses.