Regulation of Insulin and Leptin Signaling by Muscle Suppressor of Cytokine Signaling 3 (SOCS3)

Skeletal muscle resistance to the key metabolic hormones, leptin and insulin, is an early defect in obesity. Suppressor of cytokine signaling 3 (SOCS3) is a major negative regulator of both leptin and insulin signaling, thereby implicating SOCS3 in the pathogenesis of obesity and associated metabolic abnormalities. Here, we demonstrate that SOCS3 mRNA expression is increased in murine skeletal muscle in the setting of diet-induced and genetic obesity, inflammation, and hyperlipidemia. To further evaluate the contribution of muscle SOCS3 to leptin and insulin resistance in obesity, we generated transgenic mice with muscle-specific overexpression of SOCS3 (MCK/SOCS3 mice). Despite similar body weight, MCK/SOCS3 mice develop impaired systemic and muscle-specific glucose homeostasis and insulin action based on glucose and insulin tolerance tests, hyperinsulinemic-euglycemic clamps, and insulin signaling studies. With regards to leptin action, MCK/SOCS3 mice exhibit suppressed basal and leptin-stimulated activity and phosphorylation of alpha2 AMP-activated protein kinase (α2AMPK) and its downstream target, acetyl-CoA carboxylase (ACC). Muscle SOCS3 overexpression also suppresses leptin-regulated genes involved in fatty acid oxidation and mitochondrial function. These studies demonstrate that SOC3 within skeletal muscle is a critical regulator of leptin and insulin action and that increased SOCS may mediate insulin and leptin resistance in obesity.     

Prerequisites for Functional Interleukin 31 Signaling and Its Feedback Regulation by Suppressor of Cytokine Signaling 3 (SOCS3)

Interleukin-31 (IL-31) is a T helper type 2 cell-derived cytokine tightly associated with inflammatory skin disorders. IL-31-induced signaling is mediated by a receptor complex composed of oncostatin M receptor β and the cytokine-specific receptor subunit IL-31Rα, of which there are several isoforms. The latter can be classified as long or short isoforms with respect to their intracellular domain. At present, the signaling capabilities of the different isoforms remain inchoately understood, and potential mechanisms involved in negative regulation of IL-31Rα signaling have so far not been studied in detail. Here, we show that both the long and short isoforms of IL-31Rα are capable of inducing STAT signaling. However, the presence of a functional JAK-binding box within IL-31Rα is an essential prerequisite for functional IL-31-mediated STAT3 signaling. Moreover, both the long and short isoforms require oncostatin M receptor β for their activity. We also show that IL-31 induces expression of four suppressor of cytokine signaling family members and provide evidence that SOCS3 acts as a potent feedback inhibitor of IL-31-induced signaling. Taken together, this study identifies crucial requirements for IL-31 signaling and shows its counter-regulation by SOCS3.     

Suppressor of Cytokine Signaling 4 (SOCS4) Protects against Severe Cytokine Storm and Enhances Viral Clearance during Influenza Infection

Suppressor of cytokine signaling (SOCS) proteins are key regulators of innate and adaptive immunity. There is no described biological role for SOCS4, despite broad expression in the hematopoietic system. We demonstrate that mice lacking functional SOCS4 protein rapidly succumb to infection with a pathogenic H1N1 influenza virus (PR8) and are hypersusceptible to infection with the less virulent H3N2 (X31) strain. In SOCS4-deficient animals, this led to substantially greater weight loss, dysregulated pro-inflammatory cytokine and chemokine production in the lungs and delayed viral clearance. This was associated with impaired trafficking of influenza-specific CD8 T cells to the site of infection and linked to defects in T cell receptor activation. These results demonstrate that SOCS4 is a critical regulator of anti-viral immunity.     

Expression of Suppressor of Cytokine Signaling 1 (SOCS1) Impairs Viral Clearance and Exacerbates Lung Injury during Influenza Infection

Suppressor of cytokine signaling (SOCS) proteins are inducible feedback inhibitors of cytokine signaling. SOCS1^−/− mice die within three weeks postnatally due to IFN-γ-induced hyperinflammation. Since it is well established that IFN-γ is dispensable for protection against influenza infection, we generated SOCS1^−/−IFN-γ^−/− mice to determine whether SOCS1 regulates antiviral immunity in vivo. Here we show that SOCS1^−/−IFN-γ^−/− mice exhibited significantly enhanced resistance to influenza infection, as evidenced by improved viral clearance, attenuated acute lung damage, and consequently increased survival rates compared to either IFN-γ^−/− or WT animals. Enhanced viral clearance in SOCS1^−/−IFN-γ^−/− mice coincided with a rapid onset of adaptive immune responses during acute infection, while their reduced lung injury was associated with decreased inflammatory cell infiltration at the resolution phase of infection. We further determined the contribution of SOCS1-deficient T cells to antiviral immunity. Anti-CD4 antibody treatment of SOCS1^−/−IFN-γ^−/− mice had no significant effect on their enhanced resistance to influenza infection, while CD8⁺ splenocytes from SOCS1^−/−IFN-γ^−/− mice were sufficient to rescue RAG1^−/− animals from an otherwise lethal infection. Surprisingly, despite their markedly reduced viral burdens, RAG1^−/− mice reconstituted with SOCS1^−/−IFN-γ^−/− adaptive immune cells failed to ameliorate influenza-induced lung injury. In conclusion, in the absence of IFN-γ, the cytoplasmic protein SOCS1 not only inhibits adaptive antiviral immune responses but also exacerbates inflammatory lung damage. Importantly, these detrimental effects of SOCS1 are conveyed through discrete cell populations. Specifically, while SOCS1 expression in adaptive immune cells is sufficient to inhibit antiviral immunity, SOCS1 in innate/stromal cells is responsible for aggravated lung injury.     

Suppressor of Cytokine Signaling (SOCS) 5 Utilises Distinct Domains for Regulation of JAK1 and Interaction with the Adaptor Protein Shc-1

Suppressor of Cytokine Signaling (SOCS)5 is thought to act as a tumour suppressor through negative regulation of JAK/STAT and epidermal growth factor (EGF) signaling. However, the mechanism/s by which SOCS5 acts on these two distinct pathways is unclear. We show for the first time that SOCS5 can interact directly with JAK via a unique, conserved region in its N-terminus, which we have termed the JAK interaction region (JIR). Co-expression of SOCS5 was able to specifically reduce JAK1 and JAK2 (but not JAK3 or TYK2) autophosphorylation and this function required both the conserved JIR and additional sequences within the long SOCS5 N-terminal region. We further demonstrate that SOCS5 can directly inhibit JAK1 kinase activity, although its mechanism of action appears distinct from that of SOCS1 and SOCS3. In addition, we identify phosphoTyr317 in Shc-1 as a high-affinity substrate for the SOCS5-SH2 domain and suggest that SOCS5 may negatively regulate EGF and growth factor-driven Shc-1 signaling by binding to this site. These findings suggest that different domains in SOCS5 contribute to two distinct mechanisms for regulation of cytokine and growth factor signaling.     

Suppressor of Cytokine Signaling-2 (SOCS2) Regulates the Microglial Response and Improves Functional Outcome after Traumatic Brain Injury in Mice

Traumatic brain injury (TBI) is frequently characterized by neuronal, axonal and myelin loss, reactive gliosis and neuroinflammation, often associated with functional deficits. Endogenous repair mechanisms include production of new neurons from precursor cells, but usually the new neurons fail to integrate and survive more than a few weeks. This is in part mediated by the toxic and inflammatory environment present in the injured brain which activates precursor cells to proliferate and differentiate but limits survival of the newborn progeny. Therefore, an understanding of mechanisms that regulate production and survival of newborn neurons and the neuroinflammatory response after brain injury may lead to therapeutic options to improve outcomes. Suppressor of Cytokine Signaling 2 (SOCS2) promotes hippocampal neurogenesis and survival of newborn neurons in the adult brain and regulates anti-inflammatory responses in the periphery, suggesting it may be a useful candidate to improve outcomes of TBI. In this study the functional and cellular responses of SOCS2 over-expressing transgenic (SOCS2Tg) mice were compared to wildtype littermates following mild or moderately severe TBI. Unlike wildtype controls, SOCS2Tg mice showed functional improvement on a ladder test, with a smaller lesion volume at 7d post injury and increased numbers of proliferative CD11b⁺ microglia/macrophages at 35d post-injury in the mild injury paradigm. At 7d post-moderately severe injury there was an increase in the area covered by cells expressing an anti-inflammatory M2 phenotype marker (CD206⁺) but no difference in cells with a pro-inflammatory M1 phenotype marker (CD16/32⁺). No effect of SOCS2 overexpression was observed in production or survival of newborn neurons, even in the presence of the neuroprotective agent erythropoietin (EPO). Therefore, SOCS2 may improve outcome of TBI in mice by regulating aspects of the neuroinflammatory response, promoting a more anti-inflammatory environment, although this was not sufficient to enhance survival of newborn cortical neurons.     

Suppressor of Cytokine Signaling 6 (SOCS6) Negatively Regulates Flt3 Signal Transduction through Direct Binding to Phosphorylated Tyrosines 591 and 919 of Flt3

The receptor tyrosine kinase Flt3 is an important growth factor receptor in hematopoiesis, and gain-of-function mutations of the receptor contribute to the transformation of acute myeloid leukemia. SOCS6 (suppressor of cytokine signaling 6) is a member of the SOCS family of E3 ubiquitin ligases that can regulate receptor tyrosine kinase signal transduction. In this study, we analyzed the role of SOCS6 in Flt3 signal transduction. The results show that ligand stimulation of Flt3 can induce association of SOCS6 and Flt3 and tyrosine phosphorylation of SOCS6. Phosphopeptide fishing indicated that SOCS6 binds directly to phosphotyrosines 591 and 919 of Flt3. By using stably transfected Ba/F3 cells with Flt3 and/or SOCS6, we show that the presence of SOCS6 can enhance ubiquitination of Flt3, as well as internalization and degradation of the receptor. The presence of SOCS6 also induces weaker activation of Erk1/2, but not Akt, in transfected Ba/F3 and UT-7 cells and in OCI-AML-5 cells. The absence of SOCS6 promotes Ba/F3 and UT-7 cell proliferation induced by oncogenic internal tandem duplications of Flt3. Taken together, these results suggest that SOCS6 negatively regulates Flt3 activation, the downstream Erk signaling pathway, and cell proliferation.     

Sites of Acetylation on Newly Synthesized Histone H4 Are Required for Chromatin Assembly and DNA Damage Response Signaling

The best-characterized acetylation of newly synthesized histone H4 is the diacetylation of the NH₂-terminal tail on lysines 5 and 12. Despite its evolutionary conservation, this pattern of modification has not been shown to be essential for either viability or chromatin assembly in any model organism. We demonstrate that mutations in histone H4 lysines 5 and 12 in yeast confer hypersensitivity to replication stress and DNA-damaging agents when combined with mutations in histone H4 lysine 91, which has also been found to be a site of acetylation on soluble histone H4. In addition, these mutations confer a dramatic decrease in cell viability when combined with mutations in histone H3 lysine 56. We also show that mutation of the sites of acetylation on newly synthesized histone H4 results in defects in the reassembly of chromatin structure that accompanies the repair of HO-mediated double-strand breaks. This defect is not due to a decrease in the level of histone H3 lysine 56 acetylation. Intriguingly, mutations that alter the sites of newly synthesized histone H4 acetylation display a marked decrease in levels of phosphorylated H2A (γ-H2AX) in chromatin surrounding the double-strand break. These results indicate that the sites of acetylation on newly synthesized histones H3 and H4 can function in nonoverlapping ways that are required for chromatin assembly, viability, and DNA damage response signaling.     

Protein Interaction Screening for the Ankyrin Repeats and Suppressor of Cytokine Signaling (SOCS) Box (ASB) Family Identify Asb11 as a Novel Endoplasmic Reticulum Resident Ubiquitin Ligase

The ankyrin and SOCS (suppressor of cytokine signaling) box (ASB) family of proteins function as the substrate recognition subunit in a subset of Elongin-Cullin-SOCS (ECS) E3 ubiquitin ligases. Despite counting 18 members in humans, the identity of the physiological targets of the Asb proteins remains largely unexplored. To increase our understanding of the function of ASB proteins, we conducted a family-wide SILAC (stable isotope labeling by amino acids in cell culture)-based protein/protein interaction analysis. This investigation led to the identification of novel as well as known ASB-associated proteins like Cullin 5 and Elongins B/C. We observed that several proteins can be bound by more than one Asb protein. The additional exploration of this phenomenon demonstrated that ASB-Cullin 5 complexes can oligomerize and provides evidence that Cullin 5 forms heterodimeric complexes with the Cullin 4a-DDB1 complex. We also demonstrated that ASB11 is a novel endoplasmic reticulum-associated ubiquitin ligase with the ability to interact and promote the ubiquitination of Ribophorin 1, an integral protein of the oligosaccharyltransferase (OST) glycosylation complex. Moreover, expression of ASB11 can increase Ribophorin 1 protein turnover in vivo. In summary, we provide a comprehensive protein/protein interaction data resource that can aid the biological and functional characterization of ASB ubiquitin ligases.     

Suppressor of Cytokine Signalling 2 (SOCS2) Regulates Numbers of Mature Newborn Adult Hippocampal Neurons and Their Dendritic Spine Maturation

Overexpression of suppressor of cytokine signalling 2 (SOCS2) has been shown to promote hippocampal neurogenesis in vivo and promote neurite outgrowth of neurons in vitro. In the adult mouse brain, SOCS2 is most highly expressed in the hippocampal CA3 region and at lower levels in the dentate gyrus, an expression pattern that suggests a role in adult neurogenesis. Herein we examine generation of neuroblasts and their maturation into more mature neurons in SOCS2 null (SOCS2KO) mice. EdU was administered for 7 days to label proliferative neural precursor cells. The number of EdU-labelled doublecortin⁺ neuroblasts and NeuN⁺ mature neurons they generated was examined at day 8 and day 35, respectively. While no effect of SOCS2 deletion was observed in neuroblast generation, it reduced the numbers of EdU-labelled mature newborn neurons at 35 days. As SOCS2 regulates neurite outgrowth and dentate granule neurons project to the CA3 region, alterations in dendritic arborisation or spine formation may have correlated with the decreased numbers of EdU-labelled newborn neurons. SOCS2KO mice were crossed with Nes-CreER^T2/mTmG mice, in which membrane eGFP is inducibly expressed in neural precursor cells and their progeny, and the dendrite and dendritic spine morphology of newborn neurons were examined at 35 days. SOCS2 deletion had no effect on total dendrite length, number of dendritic segments, number of branch points or total dendritic spine density but increased the number of mature “mushroom” spines. Our results suggest that endogenous SOCS2 regulates numbers of EdU-labelled mature newborn adult hippocampal neurons, possibly by mediating their survival and that this may be via a mechanism regulating dendritic spine maturation.     

X射线辐照后肝癌细胞HepG2 人宫颈癌基因及其相关基因关系的研究

目的:利用不同剂量X射线对体外培养肝癌细胞HepG2进行照射,研究人宫颈癌基因(HCCR)及其相关基因对于细胞增殖的影响及其可能的分子机制。方法:采用0、1.0、2.0、4.0 Gy的吸收剂量X射线照射细胞,用克隆形成法观察细胞的存活情况;光镜下观察细胞形态变化;同时在辐照24 h后用RT-PCR法检测细胞中HCCR、p53、Bax mRNA的表达情况。结果:1.0、2.0、4.0 Gy吸收剂量时克隆形成率分别为(76.13±3.52)%、(62.22±2.17)%、(25.35±3.10)%,随着辐射剂量的增加细胞存活率显著下降(P0.05)。HCCR mRNA相对表达量分别为0.95±0.04、0.8±0.07、0.45±0.03;p53 mRNA相对表达量分别为1.07±0.22、2.02±0.34、2.90±0.52;Bax mRNA相对表达量分别为1.12±0.11、1.63±0.36、2.48±0.27。较空白对照组,2Gy、4Gy剂量照射组HCCR mRNA相对表达量显著降低(P0.05),而p53、Bax mRNA相对表达量显著增加(P0.05)。结论:X射线照射可下调肝癌细胞HepG2的HCCR-1表达,抑制细胞增殖,作用机制可能与其相关基因p53、Bax表达升高有关。     

High Mutability of the Tumor Suppressor Genes RASSF1 and RBSP3 (CTDSPL) in Cancer

Background

Many different genetic alterations are observed in cancer cells. Individual cancer genes display point mutations such as base changes, insertions and deletions that initiate and promote cancer growth and spread. Somatic hypermutation is a powerful mechanism for generation of different mutations. It was shown previously that somatic hypermutability of proto-oncogenes can induce development of lymphomas.

Methodology/Principal Findings

We found an exceptionally high incidence of single-base mutations in the tumor suppressor genes RASSF1 and RBSP3 (CTDSPL) both located in 3p21.3 regions, LUCA and AP20 respectively. These regions contain clusters of tumor suppressor genes involved in multiple cancer types such as lung, kidney, breast, cervical, head and neck, nasopharyngeal, prostate and other carcinomas. Altogether in 144 sequenced RASSF1A clones (exons 1–2), 129 mutations were detected (mutation frequency, MF = 0.23 per 100 bp) and in 98 clones of exons 3–5 we found 146 mutations (MF = 0.29). In 85 sequenced RBSP3 clones, 89 mutations were found (MF = 0.10). The mutations were not cytidine-specific, as would be expected from alterations generated by AID/APOBEC family enzymes, and appeared de novo during cell proliferation. They diminished the ability of corresponding transgenes to suppress cell and tumor growth implying a loss of function. These high levels of somatic mutations were found both in cancer biopsies and cancer cell lines.

Conclusions/Significance

This is the first report of high frequencies of somatic mutations in RASSF1 and RBSP3 in different cancers suggesting it may underlay the mutator phenotype of cancer. Somatic hypermutations in tumor suppressor genes involved in major human malignancies offer a novel insight in cancer development, progression and spread.