In order to construct recombinant adenovirus vector expressing Suppressor of cytokine signaling 3 (SOCS3) and obtain infectious adenoviral particles, SOCS3 gene was amplified from plasmid pcDNA3-SOCS3 and subcloned into the adenovirus shuttle plasmid pAdTrack-CMV. After sequence confirmation, the recombinant shuttle plasmid pAdTrack-CMV-SOCS3 was linearized by Pme I, and then transformed into BJ5183 competent cell, the recombinant plasmid pAd-SOCS3 was obtained by homologous recombination between pAdTrack-CMV-SOCS3 and the adenoviral backbone plasmid pAdEasy-1 in BJ5183. The pAd-SOCS3 was linearized by Pac I and transfected into HEK293 cells via liposome. The recombinant adenovirus was packaged and amplified in HEK293 cells. After purifying, virus titer was determined by tissue culture infectious dose 50 (TCID50). Using the recombinant adenoviruses to infect porcine primary adipocytes, the expression of green fluorescent protein (GFP) was observed by fluorescent microscopy, and SOCS3 gene was identified by RT-PCR and Western blotting. Restriction enzyme and PCR analysis demonstrated that the recombinant adenovirus vector was constructed correctly, and the virus titer reached 1.2x10(9) PFU/mL. The result of RT-PCR and Western blotting showed that SOCS3 mRNA and protein expression was remarkably increased in porcine primary adipocytes infected with recombinant adenovirus. In conclusion, this study successfully constructed the recombinant adenovirus containing SOCS3 gene, and can be helpful for further research on the function of SOCS3.
Adenoviridae ; genetics ; metabolism ; Adipocytes ; metabolism ; Animals ; Cloning, Molecular ; Green Fluorescent Proteins ; biosynthesis ; genetics ; HEK293 Cells ; Humans ; RNA, Messenger ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Suppressor of Cytokine Signaling 3 Protein ; Suppressor of Cytokine Signaling Proteins ; biosynthesis ; genetics ; Swine ; Transfection

In order to investigate the suppressor of cytokine signaling-1 (SOCS-1) expression in peripheral blood mononuclear cells (PBMNC) of patients with acute and chronic myeloid leukemia and analyze its clinical significance, RT-PCR method was used for detecting SOCS-1 mRNA expression in PBMNC of 50 newly diagnosed patients. The result showed that positive expressions of SOCS-1 were observed in 4 of 25 patients with AML (16.00%), in 11 of 25 patients with CML (44.00%) and none in 10 normal controls. The differences between patients with AML and normal controls, and between patients with CML and normal controls were statistically significant. In CML group, 2 out of 12 cases with non-progression (chronic phase), 9 of 13 cases with progression showed the positive expression, the difference between two subgroups was statistically significant. Those CML patients with SOCS-1 mRNA expression had poor response to IFN-alpha. When they transformed into accelerated phase, SOCS-1 mRNA expression was more persistently and frequently observed, and no response to IFN-alpha was observed. Most of them had very poor prognosis. It is concluded that the SOCS-1 mRNA can be detected in the PBMNC of the patients with acute and chronic myeloid leukemia. The SOCS-1 mRNA expression in the patients with CML is higher than that in patients with AML, and it is higher in accelerated phase and blast crisis significantly. This phenomenon is highly related to the reaction of IFN-alpha and prognosis.
Humans ; Interferon-alpha ; metabolism ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; genetics ; metabolism ; Leukemia, Myeloid, Acute ; genetics ; metabolism ; RNA, Messenger ; biosynthesis ; Reverse Transcriptase Polymerase Chain Reaction ; Suppressor of Cytokine Signaling 1 Protein ; Suppressor of Cytokine Signaling Proteins ; biosynthesis ; genetics

BACKGROUNDIdiopathic pulmonary fibrosis (IPF) is a progressive diffuse parenchymal disease with a poor prognosis. A variety of cytokines and chemokines are involved in its pathophysiology. The aim of this study was to evaluate the clinical features in IPF patients with the expression of suppressor of cytokine signaling 1 (SOCS-1), which acts as a negative regulator of cytokine signaling.

METHODSIPF patients (n = 20) and healthy controls (n = 16) were included in this study. The expression of SOCS-1 was analyzed in peripheral blood mononuclear cells (PBMC) of subjects using RT-PCR. Interleukin 4 (IL-4), transforming growth factor β1 (TGF-β1) and type I collagen expression were also analyzed in each individual using enzyme-linked immunosorbent assay (ELISA). The clinical characteristics of IPF patients were delineated. These results were analyzed by SPSS13.0 statistics software.

RESULTSSOCS-1 mRNA expression was significantly decreased in the PBMC of IPF patients compared with healthy controls; serum levels of IL-4 and TGF-β1 were higher in IPF patients. The patients with lower expression of SOCS-1 developed lower percentage of forced vital capacity (FVC%) and DLCO/VA. A patients' SOCS-1 mRNA level was negatively correlated with serum levels of IL-4, and negatively correlated with their high-resolution computed tomography (HRCT) scores.

CONCLUSIONSSOCS-1 mRNA can be detected in PBMC, and it is down-regulated in IPF patients. The expression of SOCS-1 is associated with the severity of IPF patients' symptoms, so it might be the predictor of disease severity. SOCS-1 might play an important role in IPF by reducing the expression of the T helper type 2 (Th2) cell-related cytokine IL-4.

Adult ; Aged ; Aged, 80 and over ; Enzyme-Linked Immunosorbent Assay ; Female ; Humans ; Idiopathic Pulmonary Fibrosis ; metabolism ; Interleukin-4 ; metabolism ; Leukocytes, Mononuclear ; metabolism ; Male ; Middle Aged ; Suppressor of Cytokine Signaling 1 Protein ; Suppressor of Cytokine Signaling Proteins ; genetics ; metabolism ; Transforming Growth Factor beta1 ; metabolism

Swine is an ideal model for diabetes studies. Insulin and insulin resistance are closely related with diabetes. To investigate the effect of SOCS-3 in insulin resistance, porcine primary adipocyte was treated with insulin (100 nmol/L) and dexamethasone (300 nmol/L) to induce insulin resistance. The simi-quantitative PCR results suggested that insulin increased GLUT4, PPARgamma and SOCS-3 gene expression in primary culture porcine adipocytes and no change of OB gene expression. Under insulin resistance conditions, SOCS-3 and OB gene expression were up-regulated, whereas GLUT4 and PPARgamma gene expression were down-regulated in primary porcine adipocytes. The overexpression of PPARgamma gene resulted in the increase of GLUT4 expression by insulin. Different expression levels of SOCS-3 determined the inhibitory effects of insulin signaling. Induction of insulin resistance by dexamethasone was not only due to inhibition of glucose transportation, but also repression of insulin signaling. SOCS-3 might be a potential gene to block the insulin resistance.
Adipocytes ; cytology ; metabolism ; Animals ; Cells, Cultured ; Dexamethasone ; pharmacology ; Glucose Transporter Type 4 ; biosynthesis ; genetics ; Insulin ; pharmacology ; Insulin Resistance ; Leptin ; biosynthesis ; genetics ; PPAR gamma ; biosynthesis ; genetics ; Suppressor of Cytokine Signaling 3 Protein ; Suppressor of Cytokine Signaling Proteins ; biosynthesis ; genetics ; Swine

OBJECTIVEResistin was thought to link the obesity to type 2 diabetes. This study aimed to investigate the effect of resistin on insulinoma cell proliferation.

METHODSpcDNA3.1-resistin was transfected into rat insulinoma cells RINm5F. Cell proliferation was assessed by the MTT assay. The resistin and SOCS3 mRNA levels were assessed by RT-PCR. The total Akt level and the phosphorylation status were assessed by Western blot.

RESULTSThe over-expressed resistin inhibited the RINm5F cell proliferation (p<0.05). SOCS-3 expression was up-regulated by resistin over-expression (3.2 folds over the control; p<0.05). Akt phosphorylation was down-regulated by resistin over-expression (0.6 fold over the control; p<0.05).

CONCLUSIONSResistin impairs the rat insulinoma cell RINm5F proliferation. This might be attributed to a down-regulation of Akt level caused by increased SOCS-3 expression.

Animals ; Cell Line, Tumor ; Cell Proliferation ; Insulinoma ; pathology ; Pancreatic Neoplasms ; pathology ; Phosphorylation ; Proto-Oncogene Proteins c-akt ; metabolism ; Rats ; Resistin ; genetics ; physiology ; Suppressor of Cytokine Signaling 3 Protein ; Suppressor of Cytokine Signaling Proteins ; genetics ; Transfection

OBJECTIVETo 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.

METHODSThe 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.

RESULTSSOCS-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.

CONCLUSIONHBx-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.

Cell Line ; CpG Islands ; DNA Methylation ; Humans ; Plasmids ; Promoter Regions, Genetic ; RNA, Messenger ; genetics ; Suppressor of Cytokine Signaling 1 Protein ; Suppressor of Cytokine Signaling Proteins ; metabolism ; Trans-Activators ; genetics ; metabolism ; Transfection

Cells, Cultured ; Enzyme-Linked Immunosorbent Assay ; Female ; Fetal Blood ; Humans ; Infant, Newborn ; Lipopolysaccharides ; Lymphocytes ; metabolism ; Male ; RNA, Messenger ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Suppressor of Cytokine Signaling 1 Protein ; Suppressor of Cytokine Signaling 3 Protein ; Suppressor of Cytokine Signaling Proteins ; genetics ; metabolism ; Time Factors ; Toll-Like Receptor 2 ; genetics ; metabolism ; Toll-Like Receptor 4 ; genetics ; metabolism ; Tumor Necrosis Factor-alpha ; metabolism

OBJECTIVETo investigate the expression of SOCS3 and Pyk2 and their correlations in non-small cell lung cancer (NSCLC).

METHODSThe expression of SOCS3 and Pyk2 was detected in 100 cases of NSCLC, human bronchial epithelial cells (HBE) and 6 lung cancer cell lines by immunohistochemistry and immunofluorescence staining. The methylation status of SOCS3 was investigated in A549 cells by methylation-specific PCR. A549 cells were either treated with a demethylation agent 5-aza-2'-deoxycytidine (5-aza) or transfected with three SOCS3 mutants with various functional domains deleted. Besides, the cells were pretreated with a proteasome inhibitor β-lactacystin where indicated. The effects of SOCS3 on Pyk2 expression, Pyk2 Tyr 402 and ERK1/2 phosphorylations were assessed by Western blot. RT-PCR was used to estimate Pyk2 mRNA levels. Transwell experiments were performed to evaluate cell migration.

RESULTSSOCS3 (43.0%, 43/100) and Pyk2 (65.0%, 65/100) were expressed in NSCLC. A significant negative correlation was found between SOCS3 and Pyk2 in both NSCLC tissues and cell lines. SOCS3 was aberrantly methylated and 5-aza restored SOCS3 expression. Transfection studies indicated that exogenous SOCS3 interacted with Pyk2, and both Src homology 2 (SH2) and kinase inhibitory region (KIR) domains contributed to Pyk2 binding. Furthermore, SOCS3 was found to inhibit Pyk2-associated ERK1/2 activity in A549 cells. SOCS3 possibly promoted degradation of Pyk2 in a SOCS-box-dependent manner and interfered with cell migration.

CONCLUSIONSThe data indicates that SOCS3 definitely plays roles in regulating Pyk2 signaling, and cell motility. Decreased SOCS3 induced by methylation may confer a migration advantage to A549 cells.

Adenocarcinoma ; genetics ; metabolism ; pathology ; Carcinoma, Non-Small-Cell Lung ; genetics ; metabolism ; pathology ; Carcinoma, Squamous Cell ; genetics ; metabolism ; pathology ; Cell Line, Tumor ; Cell Movement ; DNA Methylation ; Focal Adhesion Kinase 2 ; metabolism ; Humans ; Lung Neoplasms ; genetics ; metabolism ; pathology ; Lymphatic Metastasis ; Mutation ; Phosphorylation ; Signal Transduction ; Suppressor of Cytokine Signaling 3 Protein ; Suppressor of Cytokine Signaling Proteins ; genetics ; metabolism

OBJECTIVE: To screen the inflammatory mediators genes regulated by HSF1, and explore the mechanism of downstream genes regulated by HSF1. METHODS: HSF-/- and HSF1+/+ mice were injected with 15 mg/kg LPS intraperitoneally (ip), respectively, and were treated as previous after HSR. The total RNA of lung tissues were extracted and filtrated by SuperArray gene Microarry. The promoter of candidate genes were analyzed by transcription element search software to search for heat shock element (HSE). Select the suppressor of cytokine signaling 3 (SOCS3) with HSE. Macrophage cells were stimulated with 400 ng/mL LPS, and were treated as previous after HSR, then the total RNA was extracted respectively. RT-PCR and northern blot assay were performed to detect the expression levels of SOCS3 mRNA. RESULTS: Fifteen genes were repressed by HSF1, including 9 genes with complete HSE. Eleven genes were accelerated by HSF1 possibly, including 8 genes with complete HSE. The promoter of SOCS3 gene contained one complete HSE. LPS stimulation obviously increased the levels of SOCS3 mRNA in macrophages of RAW264.7 mice, which was inhibited by HSR and over-expression of HSF1. CONCLUSION HSR or HSF1 inhibits LPS induced expression of SOCS3 mRNA; HSF1 might inhibit LPS-induced expression of SOCS3 mRNA by binding to HSE in the promoter of SOCS3 gene.
Animals ; DNA-Binding Proteins ; genetics ; pharmacology ; Endotoxemia ; chemically induced ; genetics ; Heat Shock Transcription Factors ; Heat-Shock Proteins ; genetics ; Inflammation ; genetics ; Lipopolysaccharides ; Macrophages ; metabolism ; Mice ; Mice, Inbred BALB C ; Mice, Knockout ; Mutation ; RNA, Messenger ; biosynthesis ; genetics ; Signal Transduction ; Suppressor of Cytokine Signaling 3 Protein ; Suppressor of Cytokine Signaling Proteins ; biosynthesis ; genetics ; Transcription Factors ; genetics ; pharmacology

To investigate the relationship between socs3 mRNA expression and jak2v617f point mutation in bcr-abl negative patients with myelo-proliferative disease (MPD), 62 bcr-abl negative MPD patients (26 cases of PV, 26 cases of ET, 9 cases of IMF, and one case of CNL) were arranged as experiment group, and the others (20 cases of CML, 10 cases of AL and 15 healthy volunteers) were arranged as control group. All the diagnosis had been made according to the 2001 WHO criteria. jak2v617f point mutation was detected by AS-PCR and confirmed by direct sequencing. The expression level of socs3 mRNA was measured by RT-PCR. The association jak2v617f point mutation with socs3 mRNA expression in bcr-abl negative MPD patients was observed and analyzed. The results showed that among 62 bcr-abl negative MPD patients, the somatic jak2v617f point mutation was positive in 44 patients (PV 23, ET 15, IMF 5, CNL 1) while negative in the other 18 patients and control group. The expression of socs3 mRNA could be detected in 39 patients from 44 jak2v617f mutation positive patients and in 10 patients from 18 jak2v617f mutation-negative patients. There was a statistically significant difference in socs3 mRNA expression rates between jak2v617f mutation positive and negative group (chi(2) = 8.44, p < 0.005). There was a statistically significant difference in socs3 mRNA expression levels between jak2v617f mutation positive and negative groups (p = 0.035). It is concluded that there is a statistically significant difference in the expression level of socs3 mRNA in the patients between jak2v617f mutation positive group and negative group.
Adult ; Aged ; Aged, 80 and over ; Base Sequence ; Female ; Fusion Proteins, bcr-abl ; metabolism ; Humans ; Janus Kinase 2 ; genetics ; Male ; Middle Aged ; Molecular Sequence Data ; Myeloproliferative Disorders ; genetics ; metabolism ; Point Mutation ; RNA, Messenger ; genetics ; metabolism ; Suppressor of Cytokine Signaling 3 Protein ; Suppressor of Cytokine Signaling Proteins ; genetics ; metabolism