Suppressor of cytokine signaling 2 regulates neuronal differentiation by inhibiting growth hormone signaling

The intracellular mechanisms that determine the response of neural progenitor cells to growth factors and regulate their differentiation into either neurons or astrocytes remain unclear. We found that expression of SOCS2, an intracellular regulator of cytokine signaling, was restricted to mouse progenitor cells and neurons in response to leukemia inhibitory factor (LIF)-like cytokines. Progenitors lacking SOCS2 produced fewer neurons and more astrocytes in vitro, and Socs2(-/-) mice had fewer neurons and neurogenin-1 (Ngn1)-expressing cells in the developing cortex, whereas overexpression of SOCS2 increased neuronal differentiation. We also report that growth hormone inhibited Ngn1 expression and neuronal production, and this action was blocked by SOCS2 overexpression. These findings indicate that SOCS2 promotes neuronal differentiation by blocking growth hormone-mediated downregulation of Ngn1.

Differential regulation of suppressor of cytokine signaling-3 in the liver and adipose tissue of the sheep fetus in late gestation

Differential regulation of suppressor of cytokine signaling-3 in the liver and adipose tissue of the sheep fetus in late gestation. Am J Physiol Regul Integr Comp Physiol 290: R1044 - R1051, 2006. First published November 10, 2005; doi: 10.1152/ajpregu. 00573.2005. - It is unknown whether the JAK/STAT/suppressor of cytokine signaling-3 (SOCS-3) intracellular signaling pathway plays a role in tissue growth and metabolism during fetal life. We investigated whether there is a differential profile of SOCS-3 expression in the liver and perirenal adipose tissue during the period of increased fetal growth in late gestation and the impact of fetal growth restriction on SOCS-3 expression in the fetal liver. We also determined whether basal SOCS-3 expression in the fetal liver and perirenal adipose tissue is regulated by endogenous fetal prolactin (PRL). SOCS-3 mRNA abundance was higher in the liver than in the pancreas, spleen, and kidney of the sheep fetus during late gestation. In the liver, SOCS-3 mRNA expression was increased (P < 0.05) between 125 (n < 4) and 145 days (n < 7) gestation and lower (P < 0.05) in growth-restricted compared with normally grown fetal sheep in late gestation. The relative expression of SOCS-3 mRNA in the fetal liver was directly related to the mean plasma PRL concentrations during a 48-h infusion of either a dopaminergic agonist, bromocriptine (n < 7), or saline (n < 5), such that SOCS-3 mRNA expression was lower when plasma PRL concentrations decreased below similar to 20 ng/ml [y = 0.99 - (2.47/x) + (4.96/x(2)); r(2) = 0.91, P < 0.0001, n < 12]. No relationship was shown between the abundance of phospho-STAT5 in the fetal liver and circulating PRL. SOCS-3 expression in perirenal adipose tissue decreased (P < 0001) between 90 - 91 (n < 6) and 140 - 145 days (n < 9) gestation and was not related to endogenous PRL concentrations. Thus SOCS-3 is differentially expressed and regulated in key fetal tissues and may play an important and tissue-specific role in the regulation of cellular proliferation and differentiation before birth.

Prolactin and the expression of suppressor of cytokine signaling-3 in the sheep adrenal gland before birth

Prolactin and the expression of suppressor of cytokine signaling-3 in the sheep adrenal gland before birth. Am J Physiol Regul Integr Comp Physiol 291: R1399-R1405, 2006. First published June 29, 2006; doi: 10.1152/ajpregu.00252.2006.-The fetal pituitary-adrenal axis plays a key role in the fetal response to intrauterine stress and in the timing of parturition. The fetal sheep adrenal gland is relatively refractory to stimulation in midgestation (90-120 days) before the prepartum activation, which occurs around 135 days gestation (term = 147 +/- 3 days). The mechanisms underlying the switch from adrenal quiescence to activation are unclear. Therefore, we have investigated the expression of suppressor of cytokine signaling-3 (SOCS-3), a putative inhibitor of tissue growth in the fetal sheep adrenal between 50 and 145 days gestation and in the adrenal of the growth-restricted fetal sheep in late gestation. SOCS-3 is activated by a range of cytokines, including prolactin (PRL), and we have, therefore, determined whether PRL administered in vivo or in vitro stimulates SOCS-3 mRNA expression in the fetal adrenal in late gestation. There was a decrease (P < 0.005) in SOCS-3 expression in the fetal adrenal between 54 and 133 days and between 141 and 144 days gestation. Infusion of the dopaminergic agonist, bromocriptine, which suppressed fetal PRL concentrations but did not decrease adrenal SOCS-3 mRNA expression. PRL administration, however, significantly increased adrenal SOCS-3 mRNA expression (P < 0.05). Similarly, there was an increase (P < 0.05) in SOCS-3 mRNA expression in adrenocortical cells in vitro after exposure to PRL (50 ng/ml). Placental and fetal growth restriction had no effect on SOCS-3 expression in the adrenal during late gestation. In summary, the decrease in the expression of the inhibitor SOCS-3 after 133 days gestation may be permissive for a subsequent increase in fetal adrenal growth before birth. We conclude that factors other than PRL act to maintain adrenal SOCS-3 mRNA expression before 133 days gestation but that acute elevations of PRL can act to upregulate adrenal SOCS-3 expression in the sheep fetus during late gestation.

Suppressor of cytokine signaling 3 limits protection of leukemia inhibitory factor receptor signaling against central demyelination

Enhancement of oligodendrocyte survival through activation of leukemia inhibitory factor receptor (LIFR) signaling is a candidate therapeutic strategy for demyelinating disease. However, in other cell types, LIFR signaling is under tight negative regulation by the intracellular protein suppressor of cytokine signaling 3 (SOCS3). We, therefore, postulated that deletion of the SOCS3 gene in oligodendrocytes would promote the beneficial effects of LIFR signaling in limiting demyelination. By studying wild-type and LIF-knockout mice, we established that SOCS3 expression by oligodendrocytes was induced by the demyelinative insult, that this induction depended on LIF, and that enclogenously produced LIF was likely to be a key determinant of the CNS response to oligodendrocyte loss. Compared with wild-type controls, oligo-dendrocyte-specific SOCS3 conditional-knockout mice displayed enhanced c-fos activation and exogenous LIF-induced phosphorylation of signal transducer and activator of transcription 3. Moreover, these SOCS3-deficient mice were protected against cupri-zone-induced oligodendrocyte loss relative to wild-type animals. These results indicate that modulation of SOCS3 expression could facilitate the endogenous response to CNS injury.

Suppressor of cytokine signalling gene expression is elevated in breast carcinoma

Cytokines are important for breast cell function, both as trophic hormones and as mediators of host defense mechanisms against breast cancer. Recently, inducible feedback suppressors of cytokine signalling (SOCS/JAB/SSI) have been identified, which decrease cell sensitivity to cytokines. We examined the expression of SOCS genes in 17 breast carcinomas and 10 breast cancer lines, in comparison with normal tissue and breast lines. We report elevated expression of SOCS-1-3 and CIS immunoreactive proteins within in situ ductal carcinomas and infiltrating ductal carcinomas relative to normal breast tissue. Significantly increased expression of SOCS-1-3 and CIS transcripts was also shown by quantitative in situ hybridisation within both tumour tissue and reactive stroma. CIS transcript expression was elevated in all 10 cancer lines, but not in control lines. However, there was no consistent elevation of other SOCS transcripts. CIS protein was shown by immunoblot to be present in all cancer lines at increased levels, mainly as the 47 kDa ubiquitinylated form. A potential proliferative role for CIS overexpression is supported by reports that CIS activates ERK kinases, and by strong induction in transient reporter assays with an ERK-responsive promoter. The in vivo elevation of SOCS gene expression may be part of the host/tumour response or a response to autocrine/paracrine GH and prolactin. However, increased CIS expression in breast cancer lines appears to be a specific lesion, and could simultaneously shut down STAT 5 signalling by trophic hormones, confer resistance to host cytokines and increase proliferation through ERK kinases.

Non-response to antiviral therapy is associated with obesity and increased hepatic expression of suppressor of cytokine signalling 3 (SOCS-3) in patients with chronic hepatitis C, viral genotype 1

Background: Interferon alpha (IFN-alpha) activated cellular signalling is negatively regulated by inhibitory factors, including the suppressor of cytokine signalling (SOCS) family. The effects of host factors such as obesity on hepatic expression of these inhibitory factors in subjects with chronic hepatitis C virus (HCV) are unknown. Objectives: To assess the independent effects of obesity, insulin resistance, and steatosis on response to IFN-alpha therapy and to determine hepatic expression of factors inhibiting IFN-alpha signalling in obese and nonobese subjects with chronic HCV. Methods: A total of 145 subjects were analysed to determine host factors associated with non-response to antiviral therapy. Treatment comprised IFN-alpha or peginterferon alpha, either alone or in combination with ribavirin. In a separate cohort of 73 patients, real time-polymerase chain reaction was performed to analyse hepatic mRNA expression. Immunohistochemistry for SOCS-3 was performed on liver biopsy samples from 38 patients with viral genotype 1 who had received antiviral treatment. Results: Non-response (NR) to treatment occurred in 55% of patients with HCV genotypes 1 or 4 and 22% with genotypes 2 or 3. Factors independently associated with NR were viral genotype 1/4 (p < 0.001), cirrhosis on pretreatment biopsy (p = 0.025), and body mass index >= 30 kg/m(2) (p = 0.010). Obese subjects with viral genotype 1 had increased hepatic mRNA expression of phosphoenolpyruvate carboxy kinase (p = 0.01) and SOCS-3 (p = 0.047), in comparison with lean subjects. Following multivariate analysis, SOCS-3 mRNA expression remained independently associated with obesity (p = 0.023). SOCS-3 immunoreactivity was significantly increased in obesity (p = 0.013) and in non-responders compared with responders (p = 0.014). Conclusions: In patients with chronic HCV viral genotype 1, increased expression of factors that inhibit interferon signalling may be one mechanism by which obesity reduces the biological response to IFN-alpha.

Cooperation of cytokine signaling with chimeric transcription factors in leukemogenesis: PML-retinoic acid receptor alpha blocks growth factor-mediated differentiation

We utilized a mouse model of acute promyelocytic leukemia (APL) to investigate how aberrant activation of cytokine signaling pathways interacts with chimeric transcription factors to generate acute myeloid leukemia. Expression in mice of the APL-associated fusion, PML-RARA, initially has only modest effects on myelopoiesis. Whereas treatment of control animals with interleukin-3 (IL-3) resulted in expanded myelopoiesis without a block in differentiation, PML-RARA abrogated differentiation that normally characterizes the response to IL-3. Retroviral transduction of bone marrow with an IL-3-expressing retrovirus revealed that IL-3 and promyelocytic leukemia-retinoic acid receptor alpha (PML-RARalpha) combined to generate a lethal leukemia-like syndrome in

Mechanisms underlying the diminished sensitivity to prolactin negative feedback during lactation: Reduced STAT5 signaling and up-regulation of cytokine-inducible SH2 domain-containing protein (CIS) expression in tuberoinfundibular dopaminergic neurons

Hyperprolactinaemia during lactation is a consequence of the sucking stimulus and in part due to reduced prolactin (PRL) negative feedback. To date, the mechanisms involved in this diminished sensitivity to PRL feedback are unknown but may involve changes in PRL signal transduction within tuberoinfundibular dopaminergic (TIDA) neurons. Therefore, we investigated signal transducers and activators of transcription (STAT) 5 signaling in the TIDA neurons of lactating rats. Dual-label confocal immunofluorescence studies were used to determine the intracellular distribution of STAT5 within TIDA neurons in the dorsomedial arcuate nucleus. In lactating rats with pups removed for 16 h, injection of ovine PRL significantly (P < 0.05) increased the STAT5 nuclear/cytoplasmic ratio compared with vehicle-treated mothers. In contrast, ovine PRL injection did not increase the STAT5 nuclear/cytoplasmic ratio in lactating mothers with pups, demonstrating that PRL signal transduction through STAT5 is reduced in TIDA neurons in the presence of pups. To investigate possible mechanisms involved in reduced PRL signaling, we examined the expression of suppressors of cytokine signaling (SOCS) proteins. Northern analysis on whole hypothalamus showed that CIS (cytokine-inducible SH2 domain-containing protein), but not SOCS1 or SOCS3, mRNA expression was significantly (P < 0.01) up-regulated in suckled lactating rats. Semiquantitative RT-PCR on arcuate nucleus micropunches also showed up-regulation of CIS transcripts. Immunofluorescence studies demonstrated that CIS is expressed in all TIDA neurons in the dorsomedial arcuate nucleus, and the intensity of CIS staining in these neurons is significantly (P < 0.05) increased in lactating rats with sucking pups. Together, these results support the hypothesis that loss of sensitivity to PRL-negative feedback during lactation is a result of increased CIS expression in TIDA neurons.

Alterations in ciliary neurotrophic factor signaling in rapsyn deficient mice

Rapsyn is a key molecule involved in the formation of postsynaptic specializations at the neuromuscular junction, in its absence there are both pre- and post-synaptic deficits including failure to cluster acety]choline receptors. Recently we have documented increases in both nerve-muscle branching and numbers of motoneurons, suggesting alterations in skeletal muscle derived trophic support for motoneurons. The aim of the present study was to evaluate the contribution of target derived trophic factors to increases in motoneuron branching and number, in rapsyn deficient mice that had their postsynaptic specializations disrupted, We have used reverse transcription-polymerase chain reaction and Western blot to document the expression of known trophic factors and their receptors in muscle, during the period of synapse formation in rapsyn deficient mouse embryos. We found that the mRNA levels for ciliary neurotrophic factor (CNTF) was decreased in the rapsyn deficient muscles compared with litter mate controls although those for NGF, BDNF, NT-3 and TGF-beta2 did not differ. We found that both the mRNA and the protein expression for suppressor of cytokine signaling 3 (SOCS3) decreased although janus kinase 2 (JAK2) did not change in the rapsyn deficient muscles compared with litter mate controls. These results suggest that failure to form postsynaptic specializations in rapsyn deficient mice has altered the CNTF cytokine signaling pathway within skeletal muscle, the target for motoneurons. This alteration may in part, account for the increased muscle nerve branching and motoneuron survival seen in rapsyn deficient mice. (C) 2001 Wiley-Liss, Inc.

Overlapping motifs in the membrane-proximal region of cytokine receptor accessory and signaling subunits

The membrane-proximal cytoplasmic region of cytokine receptors (CRs) is highly conserved and essential for receptor activation. In particular this region is essential for the activation of members of the Janus family of protein kinases (JAK) which results in initiation of receptor signaling. We have examined the sequence of this region in a number of CR signaling and accessory subunits with a view to better delineating motifs that play an important role in initiating receptor activity. Here, we have delineated two distinct proline-rich motifs in the membrane-proximal domains of cytokine receptors. Their configuration and distribution among CR subunits strongly suggest a model in which the two motifs act in a concerted manner to induce full receptor and JAK activation. (C) 2004 Elsevier Ltd. All rights reserved.

A prostaglandin F2a Analog induces suppressors of the cytokine signalling-3 expression n the corpus luteum of the pregnant rat: A potential new mechanism in luteolysis

PRL and placental lactogen (PL) play key roles in maintaining the rodent corpus luteum through pregnancy. Suppressors of cytokine signaling (SOCS) have been shown to decrease cell sensitivity to cytokines, including PRL, and so here we have addressed the issue of whether luteolysis induced by prostaglandin F-2alpha (PGF(2alpha)) might up-regulate SOCS proteins to inhibit PRL signaling. In d 19 pregnant rats, cloprostenol, a PGF(2alpha) analog, rapidly induced transcripts for SOCS-3 and, to a lesser extent, SOCS-1. We also found increased SOCS-3 protein in the ovary by immunoblot and in the corpus luteum by immunohistochemistry. Increased SOCS-3 expression was preceded by an increase in STAT3 tyrosine phosphorylation 10 min after cloprostenol injection and was maintained for 4 h, as determined by gel shift and immunohistochemistry. Induction of SOCS-3 was accompanied by a sharp decrease in active STAT5, as determined by gel-shift assay and by loss of nuclear localized STAT5. Four hours after cloprostenol administration, the corpus luteum was refractory to stimulation of STAT5 by PRL administration, and this was not due to down-regulation of PRL receptor. Therefore, induction of SOCS-3 by PGF(2alpha) may be an important element in the initiation of luteolysis via rapid suppression of luteotropic support from PL.

Inhibition of interferon signaling by the New York 99 strain and Kunjin subtype of West Nile virus involves blockage of STAT1 and STAT2 activation by nonstructural proteins

The interferon (IFN) response is the first line of defense against viral infections, and the majority of viruses have developed different strategies to counteract IFN responses in order to ensure their survival in an infected host. In this study, the abilities to inhibit IFN signaling of two closely related West Nile viruses, the New York 99 strain (NY99) and Kunjin virus (KUN), strain MRM61C, were analyzed using reporter plasmid assays, as well as immunofluorescence and Western blot analyses. We have demonstrated that infections with both NY99 and KUN, as well as transient or stable transfections with their replicon RNAs, inhibited the signaling of both alpha/beta IFN (IFN-alpha/beta) and gamma IFN (IFN-gamma) by blocking the phosphorylation of STAT1 and its translocation to the nucleus. In addition, the phosphorylation of STAT2 and its translocation to the nucleus were also blocked by KUN, NY99, and their replicons in response to treatment with IFN-alpha. IFN-alpha signaling and STAT2 translocation to the nucleus was inhibited when the KUN nonstructural proteins NS2A, NS2B, NS3, NS4A, and NS4B, but not NS1 and NS5, were expressed individually from the pcDNA3 vector. The results clearly demonstrate that both NY99 and KUN inhibit IFN signaling by preventing STAT1 and STAT2 phosphorylation and identify nonstructural proteins. responsible for this inhibition.

A family of cytokine-inducible inhibitors of signalling

Cytokines are secreted proteins that regulate important cellular responses such as proliferation and differentiation(1). Key events in cytokine signal transduction are well defined: cytokines induce receptor aggregation, leading to activation of members of the JAK family of cytoplasmic tyrosine kinases. In turn, members af the STAT family of transcription factors are phosphorylated, dimerize and increase the transcription of genes with STAT recognition sites in their promoters(1-4). Less is known of how cytokine signal transduction is switched off. We have cloned a complementary DNA encoding a protein SOCS-1, containing an SH2-domain, by its ability to inhibit the macrophage differentiation of M1 cells in response to interleukin-6. Expression of SOCS-1 inhibited both interleukin-6-induced receptor phosphorylation and STAT activation. We have also cloned two-relatives of SOCS-1, named SOCS-2 and SOCS-3, which together with the previously described CIS (ref. 5) form a new family of proteins. Transcription of all four SOCS genes is increased rapidly in response to interleukin-6, in vitro and in vivo, suggesting they may act in a classic negative feedback loop to regulate cytokine signal transduction.

Interrelationship between Epstein-Barr virus infection in gastric carcinomas and the expression of apoptosis-associated proteins

Aims: Epstein-Barr virus (EBV) and its associated proteins may be protective against the occurrence of apoptosis that would normally inhibit cancer development and progression. Alternatively, the viral infection may cause altered or mutated expression of oncogenes or tumour suppressor genes that are necessary for tumour development. an action that may also involve apoptosis, In this study, a relationship was sought between occurrence of EBV infection, expression of apoptosis-associated proteins (tumour suppressor gene p53 and oncogenes c-myc and bcl-2) and levels of cell death (apoptosis or necrosis) in 119 cases of gastric carcinoma. Methods and results: The EBV status of the gastric carcinomas (using the EBV-encoded small RNA I (EBER-1) and in-situ hybridization), stage and grade of tumour and sex of patients were compared for bcl-2, p53 and c-myc expression patterns. EBER-1 was detected in approximately 20% of cases studied. There was no significant correlation between levels of cell death in the tumour tissue and EBV status. In the protein analyses, development and progression of gastric carcinoma, with or without EBV infection. was independent of bcl-2 expression. However, in gastric cancers with EBV infection, p53 overexpression was inhibited and c-myc expression was increased in early stage cancers, in comparison with decreased c-myc expression in late stage cancers. Conclusions: The p53 and c-myc expression patterns indicate that EBV-infected gastric carcinomas are less likely to have a natural regression via apoptosis at an early stage and explain, in part, the resistance to treatment of late stage of gastric cancers.