Histone deacetylase 3 suppresses the expression of SHP-1 via deacetylation of DNMT1 to promote heart failure.

AIMS: Heart failure (HF) is a progressive disease with recurrent hospitalizations and high mortality. However, the mechanisms underlying HF remain unclear. The present study aimed to explore the regulatory mechanism of histone deacetylase 3 (HDAC3) and DNA methyltransferase 1 (DNMT1)/Src homology domain 2-containing tyrosine phosphatase-1 (SHP-1) axis in HF. METHODS: The HF rat models and hypertrophy cell models were established. The characteristic parameters of the heart were detected by echocardiography. A multichannel physiological signal acquisition system was used to detect the hemodynamic parameters. Real-time quantitative polymerase chain reaction (RT-qPCR) was used to detect the expression of HDAC3, DNMT1, and SHP-1 mRNAs, while Western blot was applied to analyze the expression of proteins. Masson staining was used to analyze the degree of collagen fiber infiltration. TdT-mediated DUTP nick end labeling (TUNEL) staining was performed to analyze the apoptosis of myocardial tissue cells. Co-immunoprecipitation (co-IP) was conducted to study the interaction between HDAC3 and DNMT1. Flow cytometry was used to analyze the apoptosis. KEY FINDINGS: HDAC3 and DNMT1 were highly expressed in HF rat and hypertrophy cell models. HDAC3 modified DNMT1 through deacetylation to inhibit ubiquitination-mediated degradation, which promoted the expression of DNMT1. DNMT1 inhibited SHP-1 expression via methylation in the promoter region. In summary, HDAC3 modified DNMT1 by deacetylation to suppress SHP-1 expression, which in turn led to the development of cardiomyocyte hypertrophy-induced HF. SIGNIFICANCE: This study provided potential therapeutic targets for HF treatment.

SHP-1 suppresses the antiviral innate immune response by targeting TRAF3.

Type I interferons play a pivotal role in innate immune response to virus infection. The protein tyrosine phosphatase SHP-1 was reported to function as a negative regulator of inflammatory cytokine production by inhibiting activation of NF-κB and MAPKs during bacterial infection, however, the role of SHP-1 in regulating type I interferons remains unknown. Here, we demonstrated that knockout or knockdown of SHP-1 in macrophages promoted both HSV-1- and VSV-induced antiviral immune response. Conversely, overexpression of SHP-1 in L929 cells suppressed the HSV-1- and VSV-induced immune response; suppression was directly dependent on phosphatase activity. We identified a direct interaction between SHP-1 and TRAF3; the association between these two proteins resulted in diminished recruitment of CK1ε to TRAF3 and inhibited its K63-linked ubiquitination; SHP-1 inhibited K63-linked ubiquitination of TRAF3 by promoting dephosphorylation at Tyr116 and Tyr446. Taken together, our results identify SHP-1 as a negative regulator of antiviral immunity and suggest that SHP-1 may be a target for intervention in acute virus infection.

Regulation of Microtubule Nucleation in Mouse Bone Marrow-Derived Mast Cells by Protein Tyrosine Phosphatase SHP-1.

The antigen-mediated activation of mast cells initiates signaling events leading to their degranulation, to the release of inflammatory mediators, and to the synthesis of cytokines and chemokines. Although rapid and transient microtubule reorganization during activation has been described, the molecular mechanisms that control their rearrangement are largely unknown. Microtubule nucleation is mediated by γ-tubulin complexes. In this study, we report on the regulation of microtubule nucleation in bone marrow-derived mast cells (BMMCs) by Src homology 2 (SH2) domain-containing protein tyrosine phosphatase 1 (SHP-1; Ptpn6). Reciprocal immunoprecipitation experiments and pull-down assays revealed that SHP-1 is present in complexes containing γ-tubulin complex proteins and protein tyrosine kinase Syk. Microtubule regrowth experiments in cells with deleted SHP-1 showed a stimulation of microtubule nucleation, and phenotypic rescue experiments confirmed that SHP-1 represents a negative regulator of microtubule nucleation in BMMCs. Moreover, the inhibition of the SHP-1 activity by inhibitors TPI-1 and NSC87877 also augmented microtubule nucleation. The regulation was due to changes in γ-tubulin accumulation. Further experiments with antigen-activated cells showed that the deletion of SHP-1 stimulated the generation of microtubule protrusions, the activity of Syk kinase, and degranulation. Our data suggest a novel mechanism for the suppression of microtubule formation in the later stages of mast cell activation.

The control of oligodendrocyte bioenergetics by interferon-gamma (IFN-γ) and Src homology region 2 domain-containing phosphatase-1 (SHP-1).

Glycolysis and mitochondrial respiration are essential for oligodendrocyte metabolism in both the developing and adult CNS. Based on recent reports on the effects of the proinflammatory cytokine IFN-γ on metabolism and on oligodendrocytes, we addressed whether IFN-γ may affect oligodendrocyte bioenergetics in ways relevant to CNS disease. Oligodendrocytes of mice treated with IFN-γ showed significant reductions in aerobic glycolysis and mitochondrial respiration. As expected, IFN-γ treatment led to the induction of STAT1 in oligodendrocytes indicating active signaling into these cells. To determine the direct effects of IFN-γ on oligodendrocyte metabolism, cultured oligodendrocytes were treated with IFN-γ in vitro, which resulted in suppression of glycolysis similar to oligodendrocytes of animals treated with IFN-γ in vivo. Mice lacking SHP-1, a key regulator of IFN-γ and STAT1 signaling in CNS glia, had high constitutive levels of STAT1 and decreased aerobic glycolysis and mitochondrial respiration rates relative to wild type mouse oligodendrocytes. Together, these data show that IFN-γ and SHP-1 control oligodendrocyte bioenergetics in ways that may relate to the role of this cytokine in CNS disease.

Dovitinib Acts As a Novel Radiosensitizer in Hepatocellular Carcinoma by Targeting SHP-1/STAT3 Signaling.

PURPOSE: Hepatocellular carcinoma (HCC) is among the most lethal human malignancies, and curative therapy is not an option for most patients. There is growing interest in the potential benefit of combining targeted therapies with radiation therapy (RT). This study aimed to characterize the efficacy and mechanism of an investigational drug, dovitinib, used in combination with RT. METHODS AND MATERIALS: HCC cell lines (PLC5, Hep3B, SK-Hep1, HA59T, and Huh-7) were treated with dovitinib, RT, or both, and apoptosis and signal transduction were analyzed. RESULTS: Dovitinib treatment resulted in Src homology region 2 (SH2) domain-containing phosphatase 1 (SHP-1)-mediated downregulation of p-STAT3 and promoted potent apoptosis of HCC cells. Ectopic expression of STAT3, or inhibition of SHP-1, diminished the effects of dovitinib on HCC cells. By ectopic expression and purified recombinant proteins of various mutant forms of SHP-1, the N-SH2 domain of SHP-1 was found to be required for dovitinib treatment. Overexpression of STAT3 or catalytic-dead mutant SHP-1 restored RT-induced reduction of HCC cell survival. Conversely, ectopic expression of SHP-1 or activation of SHP-1 by dovitinib enhanced the effects of RT against HCC in vitro and in vivo. CONCLUSIONS: SHP-1/STAT3 signaling is critically associated with the radiosensitivity of HCC cells. Combination therapy with RT and the SHP-1 agonist, such as dovitinib, resulted in enhanced in vitro and in vivo anti-HCC effects.

Protein Tyrosine Phosphatase SHP-1 Modulates T Cell Responses by Controlling Cbl-b Degradation.

Previously, we demonstrated that CD28 and CTLA-4 signaling control Casitas-B-lineage lymphoma (Cbl)-b protein expression, which is critical for T cell activation and tolerance induction. However, the molecular mechanism(s) of this regulation remains to be elucidated. In this study, we found that Cbl-b fails to undergo tyrosine phosphorylation upon CD3 stimulation because SHP-1 is recruited to and dephosphorylates Cbl-b, whereas CD28 costimulation abrogates this interaction. In support of this finding, T cells lacking SHP-1 display heightened tyrosine phosphorylation and ubiquitination of Cbl-b upon TCR stimulation, which correlates with decreased levels of Cbl-b protein. The aberrant Th2 phenotype observed in T cell-specific Shp1(-/-) mice is reminiscent of heightened Th2 response in Cblb(-/-) mice. Indeed, overexpressing Cbl-b in T cell-specific Shp1(-/-) T cells not only inhibits heightened Th2 differentiation in vitro, but also Th2 responses and allergic airway inflammation in vivo. Therefore, SHP-1 regulates Cbl-b-mediated T cell responses by controlling its tyrosine phosphorylation and ubiquitination.

Modulating platelet reactivity through control of RGS18 availability.

Most platelet agonists activate platelets by binding to G-protein-coupled receptors. We have shown previously that a critical node in the G-protein signaling network in platelets is formed by a scaffold protein, spinophilin (SPL), the tyrosine phosphatase, Src homology region 2 domain-containing phosphatase-1 (SHP-1), and the regulator of G-protein signaling family member, RGS18. Here, we asked whether SPL and other RGS18 binding proteins such as 14-3-3γ regulate platelet reactivity by sequestering RGS18 and, if so, how this is accomplished. The results show that, in resting platelets, free RGS18 levels are relatively low, increasing when platelets are activated by thrombin. Free RGS18 levels also rise when platelets are rendered resistant to activation by exposure to prostaglandin I2 (PGI2) or forskolin, both of which increase platelet cyclic adenosine monophosphate (cAMP) levels. However, the mechanism for raising free RGS18 is different in these 2 settings. Whereas thrombin activates SHP-1 and causes dephosphorylation of SPL tyrosine residues, PGI2 and forskolin cause phosphorylation of SPL Ser94 without reducing tyrosine phosphorylation. Substituting alanine for Ser94 blocks cAMP-induced dissociation of the SPL/RGS/SHP-1 complex. Replacing Ser94 with aspartate prevents formation of the complex and produces a loss-of-function phenotype when expressed in mouse platelets. Together with the defect in platelet function we previously observed in SPL(-/-) mice, these data show that (1) regulated sequestration and release of RGS18 by intracellular binding proteins provides a mechanism for coordinating activating and inhibitory signaling networks in platelets, and (2) differential phosphorylation of SPL tyrosine and serine residues provides a key to understanding both.

Reversal of Arthritis by Human Monomeric IgA Through the Receptor-Mediated SH2 Domain-Containing Phosphatase 1 Inhibitory Pathway.

OBJECTIVE: Rheumatoid arthritis (RA), one of the most frequent chronic inflammatory rheumatic disorders, is characterized by the presence of autoantibodies and joint infiltration by activated immune cells, leading to cartilage and bone destruction. IgA occurs predominantly as monomers (mIgA) in plasma and regulates many cell responses through interaction with the Fcα receptor type I (FcαRI). FcαRI targeting by anti-FcαRI Fab inhibits activating receptors by inducing an inhibitory immunoreceptor tyrosine-based activation motif (ITAMi) configuration through SH2 domain-containing phosphatase 1 (SHP-1) recruitment. The aim of this study was to investigate the potential utility of mIgA for the treatment of arthritis by acting as an inducer of ITAMi signaling. METHODS: The effect of plasma-derived human mIgA on inhibition of multiple heterologous receptors was evaluated on FcαRI+ cell transfectants, blood phagocytes from healthy individuals, and synovial cells from RA patients. FcαRI-transgenic mice and wild-type mice treated with mIgA were studied in models of collagen antibody-induced arthritis (CAIA) and collagen-induced arthritis (CIA). The mice were assessed for development of arthritis using an arthritis score, and joint tissue samples were evaluated for the extent of leukocyte infiltration and expression of phosphatase. RESULTS: Treatment with mIgA impaired cell activation in an FcαRI-FcRγ-dependent manner, involving ITAMi signaling. Human mIgA or anti-FcαRI Fab were strongly effective in either preventing or attenuating CAIA or CIA in FcαRI-transgenic mice. Administration of mIgA markedly inhibited the recruitment of leukocytes to the inflamed joints of mice, which was associated with induction of SHP-1 phosphorylation in joint tissue cells. Moreover, mIgA reversed the state of inflammation in the synovial fluid of RA patients by inducing an ITAMi configuration. CONCLUSION: These results demonstrate a therapeutic potential of human mIgA in experimental arthritis. The findings support future clinical exploration of mIgA for the treatment of RA.

SHP-1 is a negative regulator of epithelial-mesenchymal transition in hepatocellular carcinoma.

Epithelial-to-mesenchymal transition (EMT) is well known to involve in tumor invasion and metastasis. Src homology region 2 domain-containing phosphatase 1 (SHP-1) functions as a potent tumor suppressor and also acts as a negative regulator of p-STAT3(Tyr705) oncogenic signaling. However, little is known about the molecular mechanism(s) through which SHP-1 regulates EMT during hepatocellular carcinoma (HCC) progression. Here we first reported that endogenous SHP-1 protein levels were significantly downregulated in cells with mesenchymal characteristics and negatively correlated with p-STAT3(Tyr705) and vimentin but positively correlated with E-cadherin. SHP-1 overexpression abolished transforming growth factor-ß1 (TGF-ß1)-induced p-STAT3(Tyr705) and EMT, as well inhibited migration and invasion but further rescued by signal transducer and activator of transcription factor 3 (STAT3) overexpression. Depletion of SHP-1 could induce a more increase in TGF-ß1-induced p-STAT3(Tyr-705) and EMT characteristics, further supporting the mechanism that suppression of TGF-ß1-induced EMT is dependent on SHP-1-mediated STAT3 inactivation. Constitutively overexpressed SHP-1 tyrosine phosphatase activity by D61A-mutated SHP-1 markedly reduced TGF-ß1-induced p-STAT3(Tyr705) and EMT features but was not altered by C453S catalytic-dead mutant SHP-1. Consequently, SHP-1 acted as a powerful suppressor in preventing EMT by exerting its tyrosine phosphatase activity that directly downregulated p-STAT3(Tyr705). Most notably, we discovered a novel SHP-1 agonist SC-43 better than sorafenib to exert more potent anti-EMT effects in vitro as well as anti-metastatic growth in vivo. In conclusion, SHP-1 is a potent suppressor of HCC EMT and metastasis, thus highlighting that SC-43-SHP-1 axis may serve as a potential therapeutic target that antagonized p-STAT3(Tyr705) and thereby prevented HCC EMT and metastasis.

Shifting FcγRIIA-ITAM from activation to inhibitory configuration ameliorates arthritis.

Rheumatoid arthritis-associated (RA-associated) inflammation is mediated through the interaction between RA IgG immune complexes and IgG Fc receptors on immune cells. Polymorphisms within the gene encoding the human IgG Fc receptor IIA (hFcγRIIA) are associated with an increased risk of developing RA. Within the hFcγRIIA intracytoplasmic domain, there are 2 conserved tyrosine residues arranged in a noncanonical immunoreceptor tyrosine-based activation motif (ITAM). Here, we reveal that inhibitory engagement of the hFcγRIIA ITAM either with anti-hFcγRII F(ab')2 fragments or intravenous hIgG (IVIg) ameliorates RA-associated inflammation, and this effect was characteristic of previously described inhibitory ITAM (ITAMi) signaling for hFcαRI and hFcγRIIIA, but only involves a single tyrosine. In hFcγRIIA-expressing mice, arthritis induction was inhibited following hFcγRIIA engagement. Moreover, hFcγRIIA ITAMi-signaling reduced ROS and inflammatory cytokine production through inhibition of guanine nucleotide exchange factor VAV-1 and IL-1 receptor-associated kinase 1 (IRAK-1), respectively. ITAMi signaling was mediated by tyrosine 304 (Y304) within the hFcγRIIA ITAM, which was required for recruitment of tyrosine kinase SYK and tyrosine phosphatase SHP-1. Anti-hFcγRII F(ab')2 treatment of inflammatory synovial cells from RA patients inhibited ROS production through induction of ITAMi signaling. These data suggest that shifting constitutive hFcγRIIA-mediated activation to ITAMi signaling could ameliorate RA-associated inflammation.

Hyperinsulinemia induces insulin resistance and immune suppression via Ptpn6/Shp1 in zebrafish.

Type 2 diabetes, obesity, and metabolic syndrome are pathologies where insulin resistance plays a central role, and that affect a large population worldwide. These pathologies are usually associated with a dysregulation of insulin secretion leading to a chronic exposure of the tissues to high insulin levels (i.e. hyperinsulinemia), which diminishes the concentration of key downstream elements, causing insulin resistance. The complexity of the study of insulin resistance arises from the heterogeneity of the metabolic states where it is observed. To contribute to the understanding of the mechanisms triggering insulin resistance, we have developed a zebrafish model to study insulin metabolism and its associated disorders. Zebrafish larvae appeared to be sensitive to human recombinant insulin, becoming insulin-resistant when exposed to a high dose of the hormone. Moreover RNA-seq-based transcriptomic profiling of these larvae revealed a strong downregulation of a number of immune-relevant genes as a consequence of the exposure to hyperinsulinemia. Interestingly, as an exception, the negative immune modulator protein tyrosine phosphatase nonreceptor type 6 (ptpn6) appeared to be upregulated in insulin-resistant larvae. Knockdown of ptpn6 was found to counteract the observed downregulation of the immune system and insulin signaling pathway caused by hyperinsulinemia. These results indicate that ptpn6 is a mediator of the metabolic switch between insulin-sensitive and insulin-resistant states. Our zebrafish model for hyperinsulinemia has therefore demonstrated its suitability for discovery of novel regulators of insulin resistance. In addition, our data will be very useful in further studies of the function of immunological determinants in a non-obese model system.

The SHP-1 expression is associated with cytokines and psychopathological status in unmedicated first episode schizophrenia patients.

BACKGROUND: Recent lines of research have boosted awareness of the immunological facets of schizophrenia. However, associations with protein tyrosine phosphatase regulators have never been reported. The aim of our study was to investigate the expression and promoter status methylation of phosphatase SHP-1, a key negative regulator of the inflammatory process, in Peripheral blood mononuclear cells (PBMCs) of Schizophrenic patients. METHODS: We enrolled fifty-four (28 men and 26 women) unmedicated first episode subjects (SC) who met DSM-IV and thirty-eight (22 men and 16 women) healthy controls (HC). The SC psychopathological status was assessed using the Positive and Negative Syndrome Scale. We evaluated SHP-1 expression by Quantitative Real-time PCR (qPCR) and Western blotting (WB) methods and promoter status methylation through PCR bisulfate. IKK/NFkB signaling was detected by WB, and medium and plasma levels of pro-inflammatory cytokines (IL-1ß, IL-2, and TNF-α) by the ELISA method. SHP-1 was silenced by treating cells with specific siRNA. RESULTS: We found a significantly lower level of SHP-1 gene expression in PBMCs from SC vs. HC, consistently with which the promoter region analyzed presented significant hypermethylation. Silencing of SHP-1 expression induced higher activation of IKK/NF-kB signaling and pro-inflammatory cytokine production in ex vivo PBMCs from both SC and HC. Linear regression among patients generated a model in which SHP-1 expression explained 30% of the clinical negative symptom variance (adjusted R(2)=0.30, ANOVA p<0.001). CONCLUSIONS: Our findings are the first to suggest that impairment of SHP-1 expression is involved in the physiopathology of schizophrenia, opening fruitful new avenues for ameliorating treatment at least of negative symptoms.

PTPN6 expression is epigenetically regulated and influences survival and response to chemotherapy in high-grade gliomas.

The prognosis of high-grade glioma patients is poor, and the tumors are characterized by resistance to therapy. The aims of this study were to analyze the prognostic value of the expression of the protein tyrosine phosphatase non-receptor type 6 (PTPN6, also referred to as SHP1) in high-grade glioma patients, the epigenetic regulation of the expression of PTPN6, and the role of its expression in chemotherapy resistance in glioma-derived cells. PTPN6 expression was analyzed with immunohistochemistry in 89 high-grade glioma patients. Correlation between PTPN6 expression and overall survival was analyzed with Kaplan-Meier univariate analysis and Cox regression multivariate analysis. Differences in drug sensitivity to a panel of 16 chemotherapeutic drugs between PTPN6-overexpressing clones and control clones were analyzed in vitro with the fluorometric microculture cytotoxicity assay. Cell cycle analysis was done with Krishan staining and flow cytometry. Apoptosis was analyzed with a cell death detection ELISA kit as well as cleaved caspase-3 and caspase-9 Western blotting. Autophagy was analyzed with LC3B Western blotting. Methylation of the PTPN6 promoter was analyzed with bisulfite pyrosequencing, and demethylation of PTPN6 was done with decitabine treatment. The PTPN6 expression correlated in univariate analysis to poor survival for anaplastic glioma patients (p = 0.026). In glioma-derived cell lines, overexpression of PTPN6 caused increase resistance (p < 0.05) to the chemotherapeutic drugs bortezomib, cisplatin, and melphalan. PTPN6 expression did not affect bortezomib-induced cell cycle arrest, apoptosis, or autophagy. Low PTPN6 promoter methylation correlated to protein expression, and the protein expression was increased upon demethylation in glioma-derived cells. PTPN6 expression may be a factor contributing to poor survival for anaplastic glioma patients, and in glioma-derived cells, its expression is epigenetically regulated and influences the response to chemotherapy.

Hepatocyte-specific Ptpn6 deletion promotes hepatic lipid accretion, but reduces NAFLD in diet-induced obesity: potential role of PPARγ.

UNLABELLED: Hepatocyte-specific Shp1 knockout mice (Ptpn6(H-KO)) are protected from hepatic insulin resistance evoked by high-fat diet (HFD) feeding for 8 weeks. Unexpectedly, we report herein that Ptpn6(H-KO) mice fed an HFD for up to 16 weeks are still protected from insulin resistance, but are more prone to hepatic steatosis, as compared with their HFD-fed Ptpn6(f/f) counterparts. The livers from HFD-fed Ptpn6(H-KO) mice displayed 1) augmented lipogenesis, marked by increased expression of several hepatic genes involved in fatty acid biosynthesis, 2) elevated postprandial fatty acid uptake, and 3) significantly reduced lipid export with enhanced degradation of apolipoprotein B (ApoB). Despite more extensive hepatic steatosis, the inflammatory profile of the HFD-fed Ptpn6(H-KO) liver was similar (8 weeks) or even improved (16 weeks) as compared to their HFD-fed Ptpn6(f/f) littermates, along with reduced hepatocellular damage as revealed by serum levels of hepatic enzymes. Interestingly, comparative microarray analysis revealed a significant up-regulation of peroxisome proliferator-activated receptor gamma (PPARγ) gene expression, confirmed by quantitative polymerase chain reaction. Elevated PPARγ nuclear activity also was observed and found to be directly regulated by Shp1 in a cell-autonomous manner. CONCLUSION: These findings highlight a novel role for hepatocyte Shp1 in the regulation of PPARγ and hepatic lipid metabolism. Shp1 deficiency prevents the development of severe hepatic inflammation and hepatocellular damage in steatotic livers, presenting hepatocyte Shp1 as a potential novel mediator of nonalcoholic fatty liver diseases in obesity.

Megakaryocyte-specific deletion of the protein-tyrosine phosphatases Shp1 and Shp2 causes abnormal megakaryocyte development, platelet production, and function.

The SH2 domain-containing protein-tyrosine phosphatases Shp1 and Shp2 have been implicated in regulating signaling from a variety of platelet and megakaryocyte receptors. In this study, we investigate the functions of Shp1 and Shp2 in megakaryocytes and platelets. Megakaryocyte/platelet (MP)-specific deletion of Shp1 in mice resulted in platelets being less responsive to collagen-related peptide due to reduced GPVI expression and signaling via the Src family kinase (SFK)-Syk-PLCγ2 pathway, and fibrinogen due to reduced SFK activity. By contrast, deletion of Shp2 in the MP lineage resulted in macrothrombocytopenia and platelets being hyper-responsive to anti-CLEC-2 antibody and fibrinogen. Shp1- and Shp2-deficient megakaryocytes had partial blocks at 2N/4N ploidy; however, only the latter exhibited reduced proplatelet formation, thrombopoietin, and integrin signaling. Mice deficient in both Shp1 and Shp2 were severely macrothrombocytopenic and had reduced platelet surface glycoprotein expression, including GPVI, αIIbß3, and GPIbα. Megakaryocytes from these mice were blocked at 2N/4N ploidy and did not survive ex vivo. Deletion of the immunoreceptor tyrosine-based inhibition motif-containing receptor G6b-B in the MP lineage phenocopied multiple features of Shp1/2-deficient mice, suggesting G6b-B is a critical regulator of Shp1 and Shp2. This study establishes Shp1 and Shp2 as major regulators of megakaryocyte development, platelet production, and function.

Alterations of negative regulators of cytokine signalling in immunodeficiency-related non-Hodgkin lymphoma.

We investigated immunodeficiency-related non-Hodgkin lymphoma for the presence of molecular alterations affecting negative regulators of the Janus family protein tyrosine kinase/signal transducer and activator of transcription pathway. Protein tyrosine phosphatase, non-receptor type 6/Src homology 2-containing tyrosine phosphatase-1 epigenetic silencing was recurrent in primary effusion lymphoma (100%), and diffuse large B-cell lymphoma (63%), with a higher prevalence in the non-germinal centre subtype, and was associated with the activation of the Janus family protein tyrosine kinase/signal transducer and activator of transcription 3 pathway. Suppressor of cytokine signalling (SOCS)1 and SOCS3 epigenetic silencing were occasionally detected, whereas SOCS1 was frequently mutated in diffuse large B-cell lymphoma and polymorphic post-transplant lymphoproliferative disorders, possibly as a cause of aberrant somatic hypermutation. However, the mutation profile of the coding region of the gene was different from that expected from the aberrant somatic hypermutation process, suggesting that, at least in some cases, SOCS1 mutations may have been selected for their functional activity.

The protein tyrosine phosphatase SHP-1 regulates phagolysosome biogenesis.

The process of phagocytosis and phagosome maturation involves the recruitment of effector proteins that participate in phagosome formation and in the acidification and/or fusion with various endocytic vesicles. In the current study, we investigated the role of the Src homology region 2 domain-containing phosphatase 1 (SHP-1) in phagolysosome biogenesis. To this end, we used immortalized bone marrow macrophages derived from SHP-1-deficient motheaten mice and their wild-type littermates. We found that SHP-1 is recruited early and remains present on phagosomes for up to 4 h postphagocytosis. Using confocal immunofluorescence microscopy and Western blot analyses on purified phagosome extracts, we observed an impaired recruitment of lysosomal-associated membrane protein 1 in SHP-1-deficient macrophages. Moreover, Western blot analyses revealed that whereas the 51-kDa procathepsin D is recruited to phagosomes, it is not processed into the 46-kDa cathepsin D in the absence of SHP-1, suggesting a defect in acidification. Using the lysosomotropic agent LysoTracker as an indicator of phagosomal pH, we obtained evidence that in the absence of SHP-1, phagosome acidification was impaired. Taken together, these results are consistent with a role for SHP-1 in the regulation of signaling or membrane fusion events involved in phagolysosome biogenesis.

Regulation of inflammation by interleukin-4: a review of "alternatives".

Studies of IL-4 have revealed a wealth of information on the diverse roles of this cytokine in homeostatic regulation and disease pathogenesis. Recent data suggest that instead of simple linear regulatory pathways, IL-4 drives regulation that is full of alternatives. In addition to the well-known dichotomous regulation of Th cell differentiation by IL-4, this cytokine is engaged in several other alternative pathways. Its own production involves alternative mRNA splicing, yielding at least two functional isoforms: full-length IL-4, encoded by the IL-4 gene exons 1-4, and IL-4δ2, encoded by exons 1, 3, and 4. The functional effects of these two isoforms are in some ways similar but in other ways quite distinct. When binding to the surface of target cells, IL-4 may differentially engage two different types of receptors. By acting on macrophages, a cell type critically involved in inflammation, IL-4 induces the so-called alternative macrophage activation. In this review, recent advances in understanding these three IL-4-related branch points--alternative splicing of IL-4, differential receptor engagement by IL-4, and differential regulation of macrophage activation by IL-4--are summarized in light of their contributions to inflammation.

A novel feedback mechanism by Ephrin-B1/B2 in T-cell activation involves a concentration-dependent switch from costimulation to inhibition.

Bidirectional signals via Eph receptors/ephrins have been recognized as major forms of contact-dependent cell communications such as cell attraction and repulsion. T cells express EphBs, and their ligands, the ephrin-Bs, have been known as costimulatory molecules for T-cell proliferation. Recently, another remarkable feature of ephrin-As has emerged in the form of a concentration-dependent transition from promotion to inhibition in axon growth. Here we examined whether this modification plays a role in ephrin-B costimulation in murine primary T cells. Low doses of ephrin-B1 and ephrin-B2 costimulated T-cell proliferation induced by anti-CD3, but high concentrations strongly inhibited it. In contrast, ephrin-B3 showed a steadily increasing stimulatory effect. This modulation was virtually preserved in T cells from mice simultaneously lacking four genes, EphB1, EphB2, EphB3, and EphB6. High concentrations of ephrin-B1/B2, but not ephrin-B3, inhibited the anti-CD3-induced phosphorylation of Lck and its downstream signals such as Erk and Akt. Additionally, high doses of any ephrin-Bs could phosphorylate EphB4. However, only ephrin-B1/B2 but not ephrin-B3 recruited SHP1, a phosphatase to suppress the phosphorylation of Lck. These data suggest that EphB4 signaling could engage in negative feedback to TCR signals. T-cell activation may be finely adjusted by the combination and concentration of ephrin-Bs expressed in the immunological microenvironment.

Synthetic peptides containing ITIM-like domains block expression of inflammatory mediators and migration/invasion of cancer cells through activation of SHP-1 and PI3K.

Increasing evidence supports that inflammation is closely associated with the development of cancer. In an effort to develop synthetic peptides that can suppress the inflammatory activation of cancer cells, decapeptides representing immunoreceptor tyrosine-based inhibitory motif (ITIM)-like sequences of immune receptor expressed on myeloid cells-1 (IREM-1) were tested for their anti-inflammatory effects in cancer cell lines. One (named TAT-YADL) out of the five synthetic peptides tested exhibited inhibitory effects on the expression of inflammatory mediators as well as invasion and migration. The inhibitory activities of the synthetic peptides required activation of SH2-containing protein tyrosine phosphatase-1 (SHP-1) and phosphoinositide 3-kinase (PI3K).