Suppressor Of Cytokine Signaling-3 mRNA Levels Research Articles

MiR-522 Modulated the Expression of Proinflammatory Cytokines and Matrix Metalloproteinases Partly via Targeting Suppressor of Cytokine Signaling 3 in Rheumatoid Arthritis Synovial Fibroblasts.

microRNAs have been reported to play important roles in the pathogenesis of rheumatoid arthritis (RA). This study examined the effects of miR-522 on the biological behaviors of RA synovial fibroblasts. The expression levels of miR-522 and relevant genes were measured by quantitative real-time PCR. The protein levels of cytokines were determined by ELISA assay. The protein levels of matrix metalloproteinases (MMPs) and suppressor of cytokine signaling 3 (SOCS3) were determined by western blot assay. Luciferase reporter assay was used to confirm the potential target of miR-522. Our results showed that miR-522 was upregulated in synovial fibroblasts from RA patients, and miR-522 expression level was significantly associated with the RA-associated clinical parameters. miR-522 overexpression increased the mRNA and protein expression levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and MMPs (MMP-1, MMP-3, and MMP-13) in RA synovial fibroblasts. Lipopolysaccharide induced the upregulation of TNF-α, IL-1β, and MMPs in RA synovial fibroblasts, which was reversed by miR-522 knockdown. Bioinformatics analysis identified SOCS3 as a potential target of miR-522, and this target of miR-522 was confirmed by luciferase reporter assay, and miR-522 overexpression suppressed the mRNA and protein expression levels of SOCS3. The enforced expression of SOCS3 attenuated the enhanced effects of miR-522 on mRNA expression levels of TNF-α, IL-1β, and MMPs. Collectively, our results suggested that miR-522 regulated the expression of proinflammatory cytokines and MMPs partly via targeting SOCS3 in RA synovial fibroblasts, which may contribute to pathogenesis of RA.

MiRNA-3473b contributes to neuroinflammation following cerebral ischemia

MicroRNAs play an essential role in stroke pathology. Here, we investigated the role of a newly identified microRNA, miR-3473b, in stroke pathology. The expression of miR-3473b was upregulated in the cortex and striatum in mice following transient middle cerebral artery occlusion (MCAO). Intracerebroventricular injection of the miR-3473b antagomir prior to MCAO remarkably attenuated ischemia-induced expression of miR-3473b and pro-inflammatory factors in the ischemic brain and decreased infarct volumes in mice following MCAO. Using in vitro approaches, we showed that the miR-3473b antagomir reduced the mRNA and protein levels of pro-inflammatory factors (iNOS, COX-2, TNF-α, and IL-6) in BV2 microglial cells subjected to LPS stimulation. The miR-3473b antagomir also decreased the expression of pro-inflammatory factors in BV2 cells activated with conditioned medium collected from oxygen-glucose deprivation (OGD)-treated neurons. Suppressor of cytokine signaling 3 (SOCS3), a physiological regulator of innate and adaptive immunity, was predicted to be a potential target of miR-3473b. We verified that the miR-3473b mimic decreased SOCS3 expression in BV2 cells. Meanwhile, the miR-3473b antagomir significantly increased both SOCS3 mRNA and protein levels in the BV2 cells treated with LPS as well as in the ischemic brain. By using the dual luciferase assay, we further showed that the 3′-untranslational region of SOCS3 was directly targeted by miR-3473b. In conclusion, induction of miR-3473b, which is likely targeted to SOCS3, contributes to stroke pathogenesis by enhancing post-stroke neuroinflammation injury.

Momordica charantia extracts ameliorate insulin resistance by regulating the expression of SOCS-3 and JNK in type 2 diabetes mellitus rats

Context: Momordica charantia L. (Cucurbitaceae) has long been widely used as a traditional remedy for diabetes mellitus in some countries. However, detailed antidiabetic mechanisms are largely unknown. Objectives: This study clarified the ameliorating effects of M. charantia ethanol extracts (MCE) on the insulin resistance in type 2 diabetes mellitus (T2DM) rats. Materials and methods: T2DM rat model was established by high-fat diet and streptozotocin (STZ) injection. Diabetic rats were randomized into five groups: the model control group (n = 8) (common diet), the high-fat diet metformin (50 mg/kg/d), and the three-dose MCE (100, 200, and 400 mg/kg/d) groups (n = 8 each). After 8 weeks, the fasting serum glucose, insulin, TNF-α, and IL-6 were measured, and the relevant factors of glucose and insulin were monitored by glycogen dyeing, RT-PCR, and western blot, respectively. Results: The 8-week treatment of 400 mg/kg MCE significantly lowered body weight (330.1 versus 365.9 g), serum glucose (7.41 versus 16.63 mmol/L), insulin (12.06 versus 15.89 mIU/L), TNF-α (52.72 versus 81.83 ng/L), and IL-6 (104.81 versus 135.74 ng/L) in comparison with those of the diabetic control group (p < 0.05). It was the same for skeletal muscle glucose transporter 4 (GLUT-4) protein, and glycogen level, suppressor of cytokine signaling-3 (SOCS-3), c-Jun N-terminal kinase (JNK), and Akt expression at both protein and mRNA levels in liver (p < 0.05). Conclusions: MCE can ameliorate insulin resistance in T2DM rats. This effect may be related to the regulation of mRNA and protein levels of SOCS-3 and JNK.

Expression of the anti-inflammatory suppressor of cytokine signaling 3 (SOCS3) in human clear cell renal cell carcinoma.

The oncogenic transcription factor signal transducer and activator of transcription 3 (STAT3) is a cytokine-activated transcription factor controlling inflammation, cell proliferation, survival, and differentiation in normal tissue as well as in tumor growth. One of its most important negative regulators is the suppressor of cytokine signaling 3 (SOCS3). Here, we analyzed SOCS3 and other tumor-associated local immune regulators in human clear cell renal cell carcinoma (ccRCC). Analyses were performed in tumor and adjacent tumor-free healthy renal tissue from 35 patients with ccRCC. For functional analysis, ccRCC Caki-1 cell lines were stimulated with IL-6 and IFNγ in cell culture assays. We observed significantly lower SOCS3 messenger RNA (mRNA) levels in tumor tissue compared to healthy tissue. SOCS3 mRNA strongly correlated within tumor and healthy tissue. Interestingly vice versa, SOCS3 protein levels were significantly higher in tumor tissue than in healthy tissue. IL-22 and IL-22R1 mRNA displayed no differences in tumor and healthy tissue. Stimulation of Caki-1 cells with IFNγ resulted in markedly increased SOCS3 mRNA levels. We conclude that SOCS3 along with STAT3 participates in regulatory mechanisms in ccRCC, which certainly features only one of multiple factors involved but nevertheless merits further attention.

Seasonal fluctuations in the steady-state mRNA levels of suppressor of cytokine signaling-3 (SOCS-3) in the mammary gland of lactating and non-lactating ewes

Prolactin (PRL) is a hormone that plays a crucial role during pregnancy and lactation. The Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway, which is an important pathway in signal transduction from PRL to target cells, can be modulated by various factors, including suppressor of cytokine signaling-3 (SOCS-3). PRL may increase and decrease SOCS-3 expression, and its actions are mostly dependent on additional factors such as the photoperiod. There is also evidence for a relationship between suckling and SOCS-3 expression in the mammary gland. The aim of this study was to examine the influence of seasonality and physiological status on SOCS-3 expression levels in ovine mammary glands. Mammary gland fragments were obtained from 18 Polish Longwool ewes, and the fine-needle aspiration biopsy technique was used on 4 groups of animals under specific physiological and photoperiodic conditions: non-lactating ewes during long days (LD; n=6), ewes on day 30 (n=6) or day 56 of lactation (n=6) and non-lactating ewes during short days (SD; n=6). SOCS-3 mRNA levels were measured by real-time PCR. The results of this study confirmed that SOCS-3 mRNA expression in sheep is dependent on the season and stage of lactation. The highest expression of the examined factors was noted in the mammary glands of non-lactating sheep during SD, and the lowest levels were observed in ewes on day 30 of lactation. The transcript levels observed in the tissues collected from non-lactating sheep during SD and LD were higher (74% and 41%, respectively) compared with the expression observed on day 30 of lactation. On day 56 of lactation, the SOCS-3 mRNA level was similar to that observed in the udders of non-lactating sheep during LD. These results indicate that SOCS-3 could play an integrating role between hormones and other factors that are involved in the physiological functions of the mammary gland.

Serum levels of suppressor of cytokine signaling 3, tumor necrosis factor-alpha and interleukin-10 in preeclampsia mothers and their clinical values

To explore the serum levels of suppressor of cytokine signaling 3 (SOCS3), tumor necrosis factor-alpha (TNF-α) and interleukin-10 (IL-10) in preeclampsia mothers and determine their clinical values. A total of 40 preeclampsia mothers at International Peace Maternity and Child Health Hospital of China Welfare Institute Affiliated to Shanghai Jiaotong University from March 2011 to August 2013 were enrolled into observation group. Another 40 cases of mothers without pregnancy complications were selected as control group. Then the serum contents of SOCS3, TNF-α and IL-10 were detected and their correlations analyzed. (1) The mRNA levels of SOCS3 of observation and control groups were 38 ± 6and 100 ± 16while the mRNA levels of TNF-α 226 ± 40 and 100 ± 19 respectively; (2)the proteins levels of SOCS3 of observation group were lower than those of control group (P < 0.05) while the proteins levels of TNF-α of observation group were higher than those of control group (P < 0.05); (3)the mRNA and protein contents of SOCS3 were negatively correlated with mRNA and protein contents of TNF-α and positively correlated with IL-10. The serum content of SOCS3 decreases significantly in preeclampsia so that there is a down-regulation of Th1 type cytokine TNF-α and an up-regulation of Th2 type cytokine IL-10. And it may play an important role in the occurrence of disease.

Suppressor of cytokine signaling-3 improves proliferation and migration of human trophoblast cells during pre-eclampsia

Objective To investigate the expression of suppressor of cytokine signaling-3 (SOCS-3) gene in placenta,its role in the pathogenesis of pre-eclampsia and its effect on proliferation and migration of HTR-8/SVneo cells.Methods Fifteen women with severe pre-eclampsia hospitalized in the First Affiliated Hospital of Nanjing Medical University from October 2010 to March 2011 and t 5 normal pregnant women during the same time period were investigated.Cultured HTR-8/SVneo cells were transfected with SOCS-3 specific small interfering RNA (siRNA) or negative siRNA as the controls.The expression of SOCS-3 mRNA and protein in placenta and these cells was detected by real-time quantitative reverse transcription-polymerase chain reaction and Western blot.Cell proliferation was detected by methyl thiazolyl tetrazolium,cell cycle by flow cytometry and migration by the Transwell test.Two independent t tests were used for statistical analysis.Results The SOCS-3 mRNA and protein levels in the severe pre-eclampsia group were lower than those in the normal group (0.25±0.03 vs 0.71±0.08 and 0.21±0.05 vs 0.75±0.12,t=15.94 and 14.29,respectively,both P＜0.05).SOCS-3 mRNA and protein levels in the transfection group at 24 hours were lower than those in the negative control group (0.39±0.02 vs 1.00±0.04 and 0.003 7±0.001 4 vs 1.514 9±0.035 7,t=27.58 and 73.35,respectively,both P＜0.05).The integral absorbance values of cell proliferation in the transfection group at 48,72 and 96 hours after transfection were 0.23 ± 0.01,0.32±0.02 and 0.37± 0.02,respectively,which were lower than those in the negative control group (0.39± 0.02,0.55 ± 0.04 and 0.86± 0.04,t=2.60,6.64 and 42.44,respectively,all P＜0.05).The cell clonal formation was lower in the transfection group compared with the negative group (116± 15 vs 312±24,t=9.96,P＜0.05).The ratios of G1/G0 and S phase cells in the transfection group were (55.75±2.21) ％ and (31.59±0.83) ％,respectively,and were significantly different from those in the negative control group [(47.88± 1.87) ％ and (37.38± 1.34) ％,t=45.43 and 20.06,respectively,P＜0.05].After 48 hours,cell migration in the transfection group was lower than that in the negative control group (93 ± 11 vs 167± 17,t=21.36,P＜O.05).Conclusion SOCS-3 expression is probably involved in the pathogenesis of pre-eclampsia by being down-regulated and therefore impeding proliferation and migration of the trophoblast. Key words: Pre-eclampsia; Suppressor of cytokine signaling proteins; Trophoblasts; Cell proliferation; Cell movement

Expression change of SH2B1, SOCS3, PTP1B and NPY in mice hypothalamus and its relation with obesity

To investigate the expression pattern of adapter protein with a Src-homology 2 domain (SH2B1), the suppressor of cytokine signaling-3 (SOCS3), protein-tyrosine phosphatase 1B (PTP1B) and neturopetide Y (NPY) in obese and normal mice hypothalamus and its relation with serum leptin and insulin levels. The obesity animal model was prepared with healthy C57/bl6 mice. Lee's index and Homeostasis model assessment-insulin resistance (HOMA-IR) were calculated. The mRNA levels of SH2B1, SOCS3, PTP1B and NPY were measured by fluorescent quantitation RT-PCR. The SH2B1 and NPY protein expressions were detected by Western blot. Compared with the normal mice of the same age, SH2B1 mRNA expression in the obese mice hypothalamus decreased. SOCS3 and PTP1B mRNA expression increased. Western blot showed that SH2B1 protein expression decreased, while NPY protein expression increased in the obese mice. Linear correlation analysis showed that the serum leptin and fasting insulin levels were negatively correlated with SH2B1mRNA expression and positively correlated with SOCS3 and PTP1B mRNA expression. SH2B1, SOCS3, PTP1B and NPY are key factors for obesity development.

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.

Effects of aging and caloric restriction on brainstem satiety center signals in rats

Age-related increases of body weight and adiposity, indicating dysregulation of food intake/energy expenditure, can be prevented in rodents by long-term 40% caloric restriction. The dorsal vagal complex (DVC), the brainstem center mediating the satiety reflex, has recently emerged as a determinant effector of long-term feeding adaptation. To study the effects of aging and caloric restriction on satiety circuits, leptin and brain-derived neurotrophic factor (BDNF) signaling systems were studied in 2- and 19-month-old ad libitum-fed (AL) and 19-month-old calorie-restricted (CR) rats. Age-induced hyperleptinemia in AL rats was correlated with elevated DVC BDNF immunoreactive concentrations and satiety threshold stability, suggesting functional desensitization of the DVC to these signals. To better understand this phenomenon, mRNA levels of receptor and post-receptor signaling effectors were measured by real-time RT-PCR. Aging selectively increased BDNF receptors and suppressor of cytokine signaling-3 (SOCS-3) mRNA levels. Caloric restriction prevented age-related increases of serum leptin, DVC BDNF and SOCS-3 mRNA levels, but not those of BDNF receptors. In CR rats, prevention of leptin resistance-promoting SOCS-3 induction was also observed at the protein level. This study suggests that leptin post-receptor targets and BDNF signaling play a role in the establishment of age-related DVC dysfunction.

Porphyromonas gingivalis Induction of MicroRNA-203 Expression Controls Suppressor of Cytokine Signaling 3 in Gingival Epithelial Cells

Porphyromonas gingivalis is a pathogen in severe periodontal disease. Able to exploit an intracellular lifestyle within primary gingival epithelial cells (GECs), a reservoir of P. gingivalis can persist within the gingival epithelia. This process is facilitated by manipulation of the host cell signal transduction cascades which can impact cell cycle, cell death, and cytokine responses. Using microarrays, we investigated the ability of P. gingivalis 33277 to regulate microRNA (miRNA) expression in GECs. One of several miRNAs differentially regulated by GECs in the presence of P. gingivalis was miRNA-203 (miR-203), which was upregulated 4-fold compared to uninfected controls. Differential regulation of miR-203 was confirmed by quantitative reverse transcription-PCR (qRT-PCR). Putative targets of miR-203, suppressor of cytokine signaling 3 (SOCS3) and SOCS6, were evaluated by qRT-PCR. SOCS3 and SOCS6 mRNA levels were reduced >5-fold and >2-fold, respectively, in P. gingivalis-infected GECs compared to controls. Silencing of miR-203 using a small interfering RNA construct reversed the inhibition of SOCS3 expression. A dual luciferase assay confirmed binding of miR-203 to the putative target binding site of the SOCS3 3' untranslated region. Western blot analysis demonstrated that activation of signal transducer and activator of transcription 3 (Stat3), a downstream target of SOCS, was diminished following miR-203 silencing. This study shows that induction of miRNAs by P. gingivalis can modulate important host signaling responses.

Nuclear localization of pyruvate dehydrogenase complex-E2 (PDC-E2), a mitochondrial enzyme, and its role in signal transducer and activator of transcription 5 (STAT5)-dependent gene transcription

STAT (signal transducer and activator of transcription) proteins play a critical role in cellular response to a wide variety of cytokines and growth factors by regulating specific nuclear genes. STAT-dependent gene transcription can be finely tuned through the association with co-factors in the nucleus. We showed previously that STAT5 (including 5a and 5b) specifically interacts with a mitochondrial enzyme PDC-E2 (E2 subunit of pyruvate dehydrogenase complex) in both leukemic T cells and cytokine-stimulated cells. However, the functional significance of this novel association remains largely unknown. Here we report that PDC-E2 may function as a co-activator in STAT5-dependent nuclear gene expression. Subcellular fractionation analysis revealed that a substantial amount of PDC-E2 was constitutively present in the nucleus of BaF3, an interleukin-3 (IL-3)-dependent cell line. IL-3-induced tyrosine-phosphorylated STAT5 associated with nuclear PDC-E2 in co-immunoprecipitation analysis. These findings were confirmed by confocal immunofluorescence microscopy showing constant nuclear localization of PDC-E2 and its co-localization with STAT5 after IL-3 stimulation. Similar to mitochondrial PDC-E2, nuclear PDC-E2 was lipoylated and associated with PDC-E1. Overexpression of PDC-E2 in BaF3 cells augmented IL-3-induced STAT5 activity as measured by reporter assay with consensus STAT5-binding sites. Consistent with the reporter data, PDC-E2 overexpression in BaF3 cells led to elevated mRNA levels of endogenous SOCS3 (suppressor of cytokine signaling 3) gene, a known STAT5 target. We further identified two functional STAT5-binding sites in the SOCS3 gene promoter important for its IL-3-inducibility. The observation that both cis-acting elements were essential to detect the stimulatory effect by PDC-E2 strongly supports the role of PDC-E2 in up-regulating the transactivating ability of STAT5. All together, our results reveal a novel function of PDC-E2 in the nucleus. It also raises the possibility of nuclear-mitochondrial crosstalk through the interaction between STAT5 and PDC-E2.

M5.2 Down-regulation of SOCS-3 signaling is associated with increased endothelial inflammatory response in women with preeclampsia

Prolactin (PRL) is a hormone that plays a crucial role during pregnancy and lactation. The Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway, which is an important pathway in signal transduction from PRL to target cells, can be modulated by various factors, including suppressor of cytokine signaling-3 (SOCS-3). PRL may increase and decrease SOCS-3 expression, and its actions are mostly dependent on additional factors such as the photoperiod. There is also evidence for a relationship between suckling and SOCS-3 expression in the mammary gland. The aim of this study was to examine the influence of seasonality and physiological status on SOCS-3 expression levels in ovine mammary glands. Mammary gland fragments were obtained from 18 Polish Longwool ewes, and the fine-needle aspiration biopsy technique was used on 4 groups of animals under specific physiological and photoperiodic conditions: non-lactating ewes during long days (LD; n = 6), ewes on day 30 (n = 6) or day 56 of lactation (n = 6) and non-lactating ewes during short days (SD; n = 6). SOCS-3 mRNA levels were measured by real-time PCR. The results of this study confirmed that SOCS-3 mRNA expression in sheep is dependent on the season and stage of lactation. The highest expression of the examined factors was noted in the mammary glands of non-lactating sheep during SD, and the lowest levels were observed in ewes on day 30 of lactation. The transcript levels observed in the tissues collected from non-lactating sheep during SD and LD were higher (74% and 41%, respectively) compared with the expression observed on day 30 of lactation. On day 56 of lactation, the SOCS-3 mRNA level was similar to that observed in the udders of non-lactating sheep during LD. These results indicate that SOCS-3 could play an integrating role between hormones and other factors that are involved in the physiological functions of the mammary gland.

Suppressor of cytokine signaling 3 (SOCS3) is not an independent biomarker of colorectal adenoma risk

BackgroundInflammation and its associated pathologies are increasingly suggested as risk factors for colorectal cancer (CRC) development. Previous research from our group has shown that increased levels of circulating, pro-inflammatory cytokines IL-6 and TNFα promote colorectal adenoma risk. Emerging data in mice and humans suggest that Suppressor of Cytokine Signaling 3 (SOCS3) may act as a tumor suppressor in the intestine, and decreased SOCS3 expression may promote CRC. As SOCS3 has been shown to inhibit the actions of IL-6 and TNFα in the intestine, we hypothesized that decreased SOCS3 expression in normal mucosa may predispose to adenomas and thus increase risk for CRC.FindingsWe examined SOCS3 mRNA levels in normal mucosa biopsies of 322 screening colonoscopy patients (93 with adenoma and 229 without adenoma) using real-time qRT-PCR. Logistic regression analysis was used to generate odds ratios (OR) and 95% confidence intervals to determine if low SOCS3 expression was associated with adenoma status. Median SOCS3 values did not differ between patients with or without adenoma. Logistic regression analysis showed no association (unadjusted or adjusted for age and sex) between SOCS3 and colorectal adenomas.ConclusionsLow SOCS3 mRNA expression is not an independent biomarker of colorectal adenoma risk in the normal mucosa. SOCS3 silencing likely occurs later in CRC progression.

Abstract LB-24: Nuclear localization of PDC-E2, a mitochondrial enzyme, and its role in STAT5-dependent gene transcription

Abstract STAT (signal transducer and activator of transcription) proteins play a critical role in cellular response to a wide variety of cytokine and growth factors by regulating specific nuclear genes. Many STAT-target genes are involved in cell proliferation, resistance to apoptosis, angiogenesis, and immune evasion. As a result, numerous cancer cells exhibit constitutive activation of STAT proteins. Among seven STAT family members, STAT3, STAT5a and STAT5b are most widely implicated in tumorigenesis. STAT-dependent gene transcription can be finely tuned through the association with co-factors in the nucleus. Therefore, detailed understanding of how different STAT family member acts in concert with distinct co-factor in the nucleus is essential in elucidating the mechanisms underlying STAT-dependent oncogenesis. We showed previously that STAT5 (including 5a and 5b), but not STAT3, specifically interacts with PDC-E2 (E2 subunit of pyruvate dehydrogenase complex), which is a nuclearly encoded mitochondrial enzyme. However, the functional significance of this novel association remains unknown. Here we report that PDC-E2 may function as a co-activator in STAT5-dependent gene transcription. Our earlier studies indicated that interleukin-3 (IL-3) induced mitochondrial translocation of STAT5 in BaF3, an IL-3-dependent mouse pro-B cell line. Further subcellular fractionation analysis revealed that a substantial amount of PDC-E2 could be detected in the nucleus in addition to mitochondrion. In contrast to IL-3-induced STAT5 nuclear translocation, the nuclear presence of PDC-E2 was constitutive and independent of IL-3 stimulation. This finding was confirmed by immunofluoresence analysis showing nuclear localization of PDC-E2 and its co-localization with STAT5 after IL-3 stimulation. Overexpression of PDC-E2 in BaF3 cells augmented IL-3-induced STAT5 activity as measured by reporter assay with consensus STAT5-binding sites. Consistent with the reporter data, PDC-E2 overexpression in BaF3 cells led to elevated mRNA levels of endogenous SOCS3 (suppressor of cytokine signaling 3) gene, a known STAT5 target. We further identified two functional STAT5-binding sites in the SOCS3 gene promoter important for its IL-3-inducibility. The observation that both cis-acting elements were essential for PDC-E2's synergistic effect strongly supports the role of PDC-E2 in up-regulating the transactivating ability of STAT5. All together, our results clearly demonstrate a unique function of PDC-E2 in the nucleus through STAT5. Functional interaction between STAT5 and PDC-E2 in the nucleus, therefore, may represent a novel molecular target in modulating STAT5 activity in tumor cells and shed new light on targeted cancer therapy. Furthermore, these findings will provide important insights into dysregulated nuclear-mitochondrial crosstalk in human cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-24.

Inhibition of SOCS-3 in adipocytes of rats with diet-induced obesity increases leptin-mediated fatty acid oxidation

Rats with diet-induced obesity (DIO) usually experience hyperleptinemia. Thus, leptin produced by adipocytes does not deplete adipocyte fat, which implying a leptin resistance in adipocytes during overnutrition. Here, we induced hyperleptinemia in rats by feeding them a diet consisting of 45% fat. In epididymal adipose tissues, the mRNA and protein levels of a putative leptin resistant factor, suppressor of cytokine signaling 3 (SOCS-3), were increased. The mRNA levels of SOCS-3 in adipocytes differentiated from adipose-derived stromal cells (ADSCs) were higher in DIO rats than in rats on a 10% fat diet. Using SOCS-3 short hairpin RNA lentivirus interference, we found decreased expression of acetyl-CoA carboxylase mRNA (a marker of de novo lipogenesis) and increased expression of acetyl-CoA oxidase mRNA (a marker of fat oxidation) in SOCS-3-knockdown adipocytes after incubation with 50nM leptin for 6h. We conclude that the SOCS-3 knockdown may have increased the leptin-mediated in situ fatty acid oxidation in the DIO adipocytes, and therefore, SOCS-3 might be an excellent target for therapeutic intervention for obesity.

Expression of STAT3 and SOCS3 in Pancreatic Acinar Cells 1,2

The signal transducer and activator of transcription (STAT) and suppressor of cytokine signaling 3 (SOCS3) pathways are involved in organ inflammation. In the pancreas, however, the STAT3 and SOCS3 response to inflammatory stimuli is unknown. Therefore, we hypothesized that mRNA expression for these signaling proteins would be induced in response to pro-inflammatory mediators TNFalpha and LPS. Because activation of STAT3 and SOCS3 is also linked to cytokine stimulation, we tested TNFalpha and LPS, either alone or in combination with IL-1beta or IL-6 in an in vitro model using rat pancreatic acinar cells. Rat pancreatic acinar cells (AR42J) were treated with combinations of LPS (10 microg/ml) or TNFalpha (10, 100, or 200 ng/ml) in the presence or absence of IL-1beta or IL-6 for 15, 30, 45, 60, 180, or 360 min. At each time point, total RNA was purified and analyzed via RT-PCR for STAT3 and SOCS3 mRNA expression. LPS and TNFalpha activated STAT3 and SOCS3 in pancreatic acinar cells. STAT3 mRNA expression was significantly (P < 0.01) increased above controls without further stimulation by IL-6 or IL1-beta. Significant increases in SOCS3 expression were observed with LPS + IL-6 at 30, 45, 60, 180, min (P < 0.05). SOCS3 mRNA expression with LPS + IL-1beta treatment was maximal by 1 h (P < 0.05). Enhanced STAT3 expression was evident by 3 h with TNFalpha alone (P < 0.01). The addition of cytokines kept STAT3 mRNA levels high. At 200 ng/ml TNFalpha, SOCS3 mRNA levels were increased, but significantly reduced in the presence of IL-1beta or IL-6 (P < 0.05). We have shown that LPS and TNFalpha are potent mediators of STAT3 and SOCS3 expression in the pancreas and that the cytokines IL-6 and IL-1beta are indirectly involved in the signaling pathway. Alteration of the STAT pathway should be explored as a therapeutic target for treatment of pancreatic inflammation.

SOCS-3 Induces Myoblast Differentiation

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.

Suppressor of cytokine signaling 3 expression and insulin resistance in skeletal muscle of obese and type 2 diabetic patients.

Interleukin-6 (IL-6) could be a possible mediator of insulin resistance. We investigated whether IL-6 could inhibit insulin signaling in human skeletal myotubes and whether suppressor of cytokine signaling 3 (SOCS-3) could be related to insulin resistance in vivo in humans. IL-6 inhibited insulin signaling and induced SOCS-3 expression in differentiated myotubes. SOCS-3 mRNA levels were significantly increased in the skeletal muscle of type 2 diabetic patients compared with control subjects and correlated with reduced insulin-stimulated glucose uptake. In contrast, SOCS-3 mRNA levels were reduced in muscle of obese nondiabetic subjects compared with type 2 diabetic patients, despite similar circulating concentrations of IL-6. Increased SOCS-3 mRNA levels in diabetes were not attributable to hyperglycemia, as type 1 diabetic patients had normal SOCS-3 mRNA expression in muscle. However, the combination of high glucose and IL-6 levels in type 2 diabetic patients may induce SOCS-3 expression, as has been seen in human muscle cells. In subcutaneous adipose tissue, SOCS-3 mRNA levels were increased in obese individuals and strongly correlated with IL-6 expression, supporting a paracrine effect of IL-6 on SOCS-3 expression in fat. Taken together, our results showed that SOCS-3 expression in human skeletal muscle in vivo is not related to insulin resistance in the presence of elevated IL-6 concentrations and suggest that cytokine action could differ in type 2 diabetic patients and nondiabetic obese subjects.

Correlation of diagnoses by confocal laser-endoscopy with histology: Histological imaging by endoscopy

Prolactin (PRL) is a hormone that plays a crucial role during pregnancy and lactation. The Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway, which is an important pathway in signal transduction from PRL to target cells, can be modulated by various factors, including suppressor of cytokine signaling-3 (SOCS-3). PRL may increase and decrease SOCS-3 expression, and its actions are mostly dependent on additional factors such as the photoperiod. There is also evidence for a relationship between suckling and SOCS-3 expression in the mammary gland. The aim of this study was to examine the influence of seasonality and physiological status on SOCS-3 expression levels in ovine mammary glands. Mammary gland fragments were obtained from 18 Polish Longwool ewes, and the fine-needle aspiration biopsy technique was used on 4 groups of animals under specific physiological and photoperiodic conditions: non-lactating ewes during long days (LD; n = 6), ewes on day 30 (n = 6) or day 56 of lactation (n = 6) and non-lactating ewes during short days (SD; n = 6). SOCS-3 mRNA levels were measured by real-time PCR. The results of this study confirmed that SOCS-3 mRNA expression in sheep is dependent on the season and stage of lactation. The highest expression of the examined factors was noted in the mammary glands of non-lactating sheep during SD, and the lowest levels were observed in ewes on day 30 of lactation. The transcript levels observed in the tissues collected from non-lactating sheep during SD and LD were higher (74% and 41%, respectively) compared with the expression observed on day 30 of lactation. On day 56 of lactation, the SOCS-3 mRNA level was similar to that observed in the udders of non-lactating sheep during LD. These results indicate that SOCS-3 could play an integrating role between hormones and other factors that are involved in the physiological functions of the mammary gland.