Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Transcription profiling of livers from INFgamma-/- and conditional SOCS3-/- mice treated with different cytokines to study the role of SOCS3 in IL-6 signaling pathway.

ABSTRACT: Changes in mouse liver mRNA profiles following intraperitoneal cytokine injection. Either interferon-gamma-/-, albumin-cre(-) Socs3(w/fl) mice, or albumin-cre(+) Socs3(-/fl) mice were injected with either phosphate-buffered saline, interferon-gamma, or interleukin-6, and livers taken after 4h.

ORGANISM(S): Mus musculus

SUBMITTER: Sam Wormald

PROVIDER: E-GEOD-369 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

SOCS3 negatively regulates IL-6 signaling in vivo.

Croker Ben A BA Krebs Danielle L DL Zhang Jian-Guo JG Wormald Sam S Willson Tracy A TA Stanley Edouard G EG Robb Lorraine L Greenhalgh Christopher J CJ Förster Irmgard I Clausen Björn E BE Nicola Nicos A NA Metcalf Donald D Hilton Douglas J DJ Roberts Andrew W AW Alexander Warren S WS

Nature immunology 20030518 6

Members of the suppressor of cytokine signaling (SOCS) family are potentially key physiological negative regulators of interleukin-6 (IL-6) signaling. To examine whether SOCS3 is involved in regulating this signaling, we have used conditional gene targeting to generate mice lacking Socs3 in the liver or in macrophages. We show that Socs3 deficiency results in prolonged activation of signal transducer and activator of transcription 1 (STAT1) and STAT3 after IL-6 stimulation but normal activation ...[more]

PMID: 12754505

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Project description:Suppressor of cytokine signaling 3 (SOCS3) down-regulates several signaling pathways in multiple cell types, and previous data suggest that SOCS3 may shut off cytokine activation at the early stages of liver regeneration. We developed hepatocyte-specific Socs3 knockout (Socs3 h-KO) mice to directly study the role of SOCS3 during liver regeneration after 2/3 partial hepatectomy (PH). Socs3 h-KO mice demonstrate marked enhancement of DNA replication and liver weight restoration after 2/3 PH in comparison with littermate controls. Without SOCS3, signal transducer and activator of transcription 3 (STAT3) phosphorylation is prolonged, and activation of the mitogenic kinases extracellular signal-regulated kinase 1/2 (ERK1/2) is enhanced after PH. In vitro, we show that SOCS3 deficiency enhances hepatocyte proliferation in association with enhanced STAT3 and ERK activation after epidermal growth factor (EGF) or interleukin 6 (IL-6) stimulation. Microarray analyses show that SOCS3 modulates a distinct set of genes after PH, which fall into diverse physiologic categories. Using a model of chemical-induced carcinogenesis, we found that Socs3 h-KO mice develop hepatocellular carcinoma (HCC) at an accelerated rate. By acting on cytokines and multiple proliferative pathways, SOCS3 modulates both physiologic and neoplastic proliferative processes in the liver, and may act as a tumor suppressor. Experiment Overall Design: Hepatocyte-specific excision of the Socs3 gene was achieved by breeding Socs3 fl/fl mice with mice expressing the Cre recombinase transgene under control of the albumin promoter (Alb-Cre+), yielding Socs3 h-KO mice. Socs3 fl/fl, Alb-Cre- littermates were used as controls for all experiments, and are henceforth referred to as littermates. All mice (C57BL/6) were free of Helicobacter species, housed in a specific pathogen free facility with 12-h light/dark cycles with free access to standard food and water. 2/3 PH and sham operations were performed as previously described (15, 50) (n=3-6 mice per genotype per time point). Liver remnants were weighed after removal of necrotic stumps and sutures, and compared to post-operative body weight. For HCC experiments, a single i.p. injection of DEN (5mg/kg, Sigma) was performed 12-14 d after birth. For short time points, a single injection of DEN (100mg/kg) (31) was given to 4 wk old mice. At indicated time points, mice were sacrificed by CO2 inhalation. All animal studies were carried out under approved IACUC protocols at the University of Washington.

Project description:Interferon-alpha (pegylated interferon and ribavirin) is used as standard-of-care therapeutic for chronic hepatitis C virus infection. Besides good cure in some patients other patients do not benefit from the treatment dependent on the virus type and host factors. One class of putative effector proteins is the family of Suppressors of cytokine signalling (SOCS). They act in a classical negative feedback-loop against the action of interferons and many other cytokines. It has been proven that some of them, in particular SOCS1 and SOCS3, inhibit the expression of interferon induced antiviral proteins. Their mode of action depends on the signal they are interfering with. In relation to the interferon-gamma pathway, they are thought to act on the interferon-alpha receptors by masking its recognition site for the Janus kinases (JAK), by blocking the kinase activity of the JAKs and coincidentally hindering STAT molecules from binding to the kinases. They are also thought to ubiquitinate the JAKs resulting in their proteosomal degradation. The function of SOCS proteins in suppressing the interferon-alpha pathway has not yet been characterized exhaustively. This study should unveil links to understand the resistance in interferon-alpha therapy. As results we got almost complete silencing of JAK-STAT signaling in SOCS1 over-expressing cells and tissue-dependent partially suppressed gene induction in SOCS3 over-expressing cell lines. Two human cancer cell lines (ME-15, HuH-7) were stably transfected with pcDNA3.1-SOCS plasmids in presence of geneticin and daughter cell lines were generated after singularization of cells. Next, original cell lines as well as SOCS1 and SOCS3 over-expressing cell lines were treated with 1000 U/ml interferon-alpha for 4 or 24 hours or in normal culture medium. Cells lines obtained from SOCS4 plasmid transfections were screened as additional control. Gene expression levels of cell cultured in control (0 for 4 hours, 2 for 24 hours) or interferon-alpha supplemented medium for 4 hours (1) or 24 hours (4) were analyzed. mRNA abundance was measured in triplicates using 12x8-sample commercial Illumina microarrays (HumanRef 8, version 3) and scanner system (iScan) as well as reagents recommend by Illumina (IlluminaM-BM-. TotalPrep Kit).

Project description:Oncostatin M (OSM) is a member of the interleukin-6 (IL-6) family of cytokines. OSM has been found to have distinct anti-inflammatory and pro-inflammatory properties in various cellular and disease contexts. OSM signals through a receptor complex that includes OSMRbeta and activates downstream signaling through the JAK/STAT, MAPK/ERK and PI3K/AKT pathways. To investigate the anti- and/or pro-inflammatory roles of OSM signaling in the context of adult hematopoiesis, we utilized the readily available Cre-driven conditional knockout Osmrfl/fl mouse model B6;129-Osmrtm1.1Nat/J. This model contains loxP sites flanking exon 2, which is the first coding exon of the Osmr gene. We crossed Osmrfl/fl mice to an interferon-inducible Mx1-Cre allele that is classically utilized to study adult hematopoiesis given its high efficiency of expression and recombination in adult hematopoietic cells. We developed a PCR assay to evaluate recombination of the Osmrfl allele. In Osmrfl/fl Mx1-Cre mice, we observed complete recombination of the Osmrfl allele and loss of exon 2 in hematopoietic (bone marrow) as well as non-hematopoietic (liver, lung, kidney) tissues. To evaluate Osmr mRNA expression downstream of exon 2, real-time PCR was utilized with TaqMan probes that anneal to an amplicon spanning Osmr exons 17 and 18. Using this assay, reduced Osmr transcript was observed in kidney samples from Osmrfl/fl Mx1-Cre mice compared to Mx1-Cre controls. However, Osmr transcript levels were similar in bone marrow, lung, and liver samples from Osmrfl/fl Mx1-Cre compared to Mx1-Cre control mice, suggesting that transcript is being produced despite loss of exon 2. Western blots revealed that liver cells from Osmrfl/fl Mx1-Cre mice had complete loss of OSMR protein. In contrast, bone marrow, kidney, and lung cells from Osmrfl/fl Mx1-Cre mice had reduced levels of OSMR protein. Together, our data suggest that the B6;129-Osmrtm1.1Nat/J model should be utilized with caution as loss of exon 2 of Osmr variably impacts mRNA and protein expression in a tissue-dependent context.

Dataset Information

Transcription profiling of livers from INFgamma-/- and conditional SOCS3-/- mice treated with different cytokines to study the role of SOCS3 in IL-6 signaling pathway.

Publications

SOCS3 negatively regulates IL-6 signaling in vivo.

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