Mannan-Binding Lectin Deficiency Limits Inflammation-induced Myeloid-Derived Suppressor Cells Expansion via Modulating Tumor Necrosis Factor Alpha-triggered Apoptosis

Background: Mannan-binding lectin (MBL), a soluble pattern recognition molecule in the innate immune system, is reported to be associated with the function of immune cells. Myeloid-derived suppressor cells (MDSCs) are mainly characterized by immunosuppressive activities involving several inflammatory diseases such as cancer, infection, and arthritis. Some of the factors inducing their apoptosis are known, however, mechanisms have not been identified. The underlying impact of MBL on the MDSCs especially under inflammatory conditions remains unknown. This study was designed to investigate whether MBL affects MDSCs survival during inflammation conditions.Methods: WT and MBL-deficient (MBL^-/-) mice were induced on day 0 of the experiment by subcutaneous injection of complete Freund''s adjuvant and then injected with incomplete Freund''s adjuvant into the knee joint space under general anesthesia on day 14 to induce inflammatory arthritis. The proportions of MDSCs in the spleen and blood and the serum level of the inflammatory cytokines were measured. In vitro study, MDSCs were isolated from the bone marrow of WT and MBL^-/- mice and cultured in the presence of interleukin-6 (IL-6) and granulocyte-macrophage colony-stimulating factor (GM-CSF) for 5 days with or without tumor necrosis factor-alpha (TNF-α).Results: After adjuvant treatment, MBL^-/- mice had a significantly lower frequency of MDSCs as well as elevated serum inflammatory cytokines levels compared to WT mice. MBL deficiency markedly inhibited the MDSCs frequency from mice bone marrow induced by IL-6 and GM-CSF in the presence of TNF-α in vitro. Mechanistic studies established that MBL inhibited MDSCs apoptosis via down-regulation of TNF-α/tumor necrosis factor-alpha receptor 1 (TNFR1) signaling pathway and subsequent caspase 3-dependent manner.Conclusion: Mannan-binding lectin deficiency inhibits myeloid-derived suppressor cells expansion via modulating TNF-α triggered apoptosis.     

Sequestering HMGB1 via DNA-Conjugated Beads Ameliorates Murine Colitis

Inflammatory bowel disease (IBD) is chronic inflammation of the gastrointestinal tract that affects millions of people worldwide. Although the etiology of IBD is not clear, it is known that products from stressed cells and enteric microbes promote intestinal inflammation. High mobility group box 1 (HMGB1), originally identified as a nuclear DNA binding protein, is a cytokine-like protein mediator implicated in infection, sterile injury, autoimmune disease, and IBD. Elevated levels of HMGB1 have been detected in inflamed human intestinal tissues and in feces of IBD patients and mouse models of colitis. Neutralizing HMGB1 activity by administration of anti-HMGB1 antibodies or HMGB1-specific antagonist improves clinical outcomes in animal models of colitis. Since HMGB1 binds to DNA with high affinity, here we developed a novel strategy to sequester HMGB1 using DNA immobilized on sepharose beads. Screening of DNA-bead constructs revealed that B2 beads, one linear form of DNA conjugated beads, bind HMGB1 with high affinity, capture HMGB1 ex vivo from endotoxin-stimulated RAW 264.7 cell supernatant and from feces of mice with colitis. Oral administration of B2 DNA beads significantly improved body weight, reduced colon injury, and suppressed colonic and circulating cytokine levels in mice with spontaneous colitis (IL-10 knockout) and with dextran sulfate sodium-induced colitis. Thus, DNA beads reduce inflammation by sequestering HMGB1 and may have therapeutic potential for the treatment of IBD.     

Protein Tyrosine Phosphatase 1B in Hematopoiesis

Protein tyrosine phosphatase 1B (PTP-1B) is a ubiquitously expressed cytosolicphosphatase best known for its role in insulin signaling. Despite the fact that it is highlyexpressed in hematopoietic tissues and has been shown to downregulate cytokinereceptor signaling, no physiological role for PTP-1B in immune regulation had beenreported. Our recent results show that the absence of PTP-1B affects murinemyelopoiesis through increased phosphorylation of the CSF-1 receptor tyrosine kinase.Here we further discuss the role of PTP-1B in monocyte/macrophage differentiation aswell as the implications of our findings in the context of PTP-1B inhibitors.     

Mast Cells Mobilize Myeloid-Derived Suppressor Cells and Treg Cells in Tumor Microenvironment via IL-17 Pathway in Murine Hepatocarcinoma Model

Tumor immunosuppression is commonly braided with chronic inflammation during tumor development. However, the relationship between immunosuppression and inflammation in tumor microenvironment is still unclear. We have demonstrated that mast cells are accumulated and exacerbate the inflammation and immunosuppression in tumor microenvironment via SCF/c-kit signaling pathway. Here, we further elucidate the underlying mechanism, which involves both myeloid-derived suppressor cells (MDSCs) and regulatory T (Treg) cells. Our data showed that mast cells mobilized the infiltration of MDSCs to tumor and induced the production of IL-17 by MDSCs; MDSCs-derived IL-17 indirectly attracted Treg cells, enhanced their suppressor function, and induced the IL-9 production by Treg cells; in turn, IL-9 strengthened the survival and protumor effect of mast cells in tumor microenvironment. Our findings disclose a closed loop among mast cells, MDSCs and Treg cells in tumor microenvironment, which provides a new insight into the paralleled developments of inflammation and immunosuppression in tumor microenvironment. Based on these findings, we propose that targeting tumor inflammation might be a potential strategy to reverse the immunosuppression of tumor microenvironment, thus facilitating cancer immunotherapy.     

Expansion of Monocytic Myeloid-Derived Suppressor Cells Dampens T Cell Function in HIV-1-Seropositive Individuals

T lymphocyte dysfunction contributes to human immunodeficiency virus type 1 (HIV-1) disease progression by impairing antivirus cellular immunity. However, the mechanisms of HIV-1 infection-mediated T cell dysfunction are not completely understood. Here, we provide evidence that expansion of monocytic myeloid-derived suppressor cells (M-MDSCs) suppressed T cell function in HIV-1-infected individuals. We observed a dramatic elevation of M-MDSCs (HLA-DR^−/low CD11b⁺ CD33^+/high CD14⁺ CD15⁻ cells) in the peripheral blood of HIV-1-seropositive subjects (n = 61) compared with healthy controls (n = 51), despite efficacious antiretroviral therapy for nearly 2 years. The elevated M-MDSC frequency in HIV-1⁺ subjects correlated with prognostic HIV-1 disease markers, including the HIV-1 load (r = 0.5957; P < 0.0001), CD4⁺ T cell loss (r = −0.5312; P < 0.0001), and activated T cells (r = 0.4421; P = 0.0004). Functional studies showed that M-MDSCs from HIV-1⁺ subjects suppressed T cell responses in both HIV-1-specific and antigen-nonspecific manners; this effect was dependent on the induction of arginase 1 and required direct cell-cell contact. Further investigations revealed that direct HIV-1 infection or culture with HIV-1-derived Tat protein significantly enhanced human MDSC generation in vitro, and MDSCs from healthy donors could be directly infected by HIV-1 to facilitate HIV-1 replication and transmission, indicating that a positive-feedback loop between HIV-1 infection and MDSC expansion existed. In summary, our studies revealed a novel mechanism of T cell dysfunction in HIV-1-infected individuals and suggested that targeting MDSCs may be a promising strategy for HIV-1 immunotherapy.     

Protein Tyrosine Phosphatase 1B Regulates Pyruvate Kinase M2 Tyrosine Phosphorylation

Protein-tyrosine phosphatase 1B (PTP1B) is a physiological regulator of glucose homeostasis and adiposity and is a drug target for the treatment of obesity and diabetes. Here we identify pyruvate kinase M2 (PKM2) as a novel PTP1B substrate in adipocytes. PTP1B deficiency leads to increased PKM2 total tyrosine and Tyr¹⁰⁵ phosphorylation in cultured adipocytes and in vivo. Substrate trapping and mutagenesis studies identify PKM2 Tyr-105 and Tyr-148 as key sites that mediate PTP1B-PKM2 interaction. In addition, in vitro analyses illustrate a direct effect of Tyr-105 phosphorylation on PKM2 activity in adipocytes. Importantly, PTP1B pharmacological inhibition increased PKM2 Tyr-105 phosphorylation and decreased PKM2 activity. Moreover, PKM2 Tyr-105 phosphorylation is regulated nutritionally, decreasing in adipose tissue depots after high-fat feeding. Further, decreased PKM2 Tyr-105 phosphorylation correlates with the development of glucose intolerance and insulin resistance in rodents, non-human primates, and humans. Together, these findings identify PKM2 as a novel substrate of PTP1B and provide new insights into the regulation of adipose PKM2 activity.     

The Mechanism of Allosteric Inhibition of Protein Tyrosine Phosphatase 1B

As the prototypical member of the PTP family, protein tyrosine phosphatase 1B (PTP1B) is an attractive target for therapeutic interventions in type 2 diabetes. The extremely conserved catalytic site of PTP1B renders the design of selective PTP1B inhibitors intractable. Although discovered allosteric inhibitors containing a benzofuran sulfonamide scaffold offer fascinating opportunities to overcome selectivity issues, the allosteric inhibitory mechanism of PTP1B has remained elusive. Here, molecular dynamics (MD) simulations, coupled with a dynamic weighted community analysis, were performed to unveil the potential allosteric signal propagation pathway from the allosteric site to the catalytic site in PTP1B. This result revealed that the allosteric inhibitor compound-3 induces a conformational rearrangement in helix α7, disrupting the triangular interaction among helix α7, helix α3, and loop11. Helix α7 then produces a force, pulling helix α3 outward, and promotes Ser190 to interact with Tyr176. As a result, the deviation of Tyr176 abrogates the hydrophobic interactions with Trp179 and leads to the downward movement of the WPD loop, which forms an H-bond between Asp181 and Glu115. The formation of this H-bond constrains the WPD loop to its open conformation and thus inactivates PTP1B. The discovery of this allosteric mechanism provides an overall view of the regulation of PTP1B, which is an important insight for the design of potent allosteric PTP1B inhibitors.     

Inhibitors of Protein Tyrosine Phosphatase 1B from Marine Natural Products

The ocean is a capacious area with the most abundant biological resources on the earth. The particularity of the marine ecological environment (high pressure, high salt, and hypoxia) makes the marine species survival competition fiercely, forcing many marine organisms in the process of life to produce a great deal of secondary metabolites with special structures and biological activities. In this article, 118 natural products which were isolated from four kinds of marine organisms, sponges, algae, soft corals and fungus, showing PTP1B inhibitory activity were summarized from 2010 to 2016, which may become the leading compounds towards treating Diabetes mellitus (DM). What's more, we briefly summarized the structure–activity relationship of PTP1B inhibitors.     

Suppression of Proteoglycan-Induced Autoimmune Arthritis by Myeloid-Derived Suppressor Cells Generated In Vitro from Murine Bone Marrow

Background

Myeloid-derived suppressor cells (MDSCs) are innate immune cells capable of suppressing T-cell responses. We previously reported the presence of MDSCs with a granulocytic phenotype in the synovial fluid (SF) of mice with proteoglycan (PG)-induced arthritis (PGIA), a T cell-dependent autoimmune model of rheumatoid arthritis (RA). However, the limited amount of SF-MDSCs precluded investigations into their therapeutic potential. The goals of this study were to develop an in vitro method for generating MDSCs similar to those found in SF and to reveal the therapeutic effect of such cells in PGIA.

Methods

Murine bone marrow (BM) cells were cultured for 3 days in the presence of granulocyte macrophage colony-stimulating factor (GM-CSF), interleukin-6 (IL-6), and granulocyte colony-stimulating factor (G-CSF). The phenotype of cultured cells was analyzed using flow cytometry, microscopy, and biochemical methods. The suppressor activity of BM-MDSCs was tested upon co-culture with activated T cells. To investigate the therapeutic potential of BM-MDSCs, the cells were injected into SCID mice at the early stage of adoptively transferred PGIA, and their effects on the clinical course of arthritis and PG-specific immune responses were determined.

Results

BM cells cultured in the presence of GM-CSF, IL-6, and G-CSF became enriched in MDSC-like cells that showed greater phenotypic heterogeneity than MDSCs present in SF. BM-MDSCs profoundly inhibited both antigen-specific and polyclonal T-cell proliferation primarily via production of nitric oxide. Injection of BM-MDSCs into mice with PGIA ameliorated arthritis and reduced PG-specific T-cell responses and serum antibody levels.

Conclusions

Our in vitro enrichment strategy provides a SF-like, but controlled microenvironment for converting BM myeloid precursors into MDSCs that potently suppress both T-cell responses and the progression of arthritis in a mouse model of RA. Our results also suggest that enrichment of BM in MDSCs could improve the therapeutic efficacy of BM transplantation in RA.     

蛋白质酪氨酸磷酸酶1B（PTP1B）与2型糖尿病及肥胖的关系

蛋白质酪氨酸磷酸酶1B（PTP1B）是一种在体内广泛表达的胞内蛋白质酪氨酸磷酸酶,在调节胰岛素敏感性和能量代谢的过程中起着重要作用。通过抑制PTP1B可增加胰岛素和瘦蛋白（leptin）的活性, 为寻找2型糖尿病、肥胖的治疗提供了光明前景。     

基于天然产物的蛋白酪氨酸磷酸酶1B 抑制剂的虚拟筛选

蛋白酪氨酸磷酸酶1B(protein tyrosine phosphatase 1B,PTP1B)是治疗Ⅱ型糖尿病的靶点之一,筛选PTP1B抑制剂具有十分重要的意义.本文采用分子对接虚拟筛选方法,构建共含有42 296个小分子的天然产物库,分别与PTP1B靶点蛋白进行分子对接,以原配体的结合能量为阈值,经过三轮筛选选取打分值高于阈值的小分子进行药代动力学参数和毒性参数预测,最终筛选出3个PTP1B抑制剂,对苯醌类化合物7、异香豆素类衍生物10和Clavepictine类似物11.结合方式研究表明,3个候选抑制剂类药性良好,均具有较好的PTP1B抑制活性,其中化合物10和11的PTP1B抑制活性未见报道.对化合物10进行体外抑制活性检测,其IC50为(74.58±1.23)μmol/L,可作为潜在Ⅱ型糖尿病治疗药物.     

Protein Tyrosine Phosphatase 1B Variant Associated with Fat Distribution and Insulin Metabolism

Protein tyrosine phosphatase 1B (PTPN1) affects the regulation of insulin signaling and energy metabolism. We studied whether polymorphisms in the PTPN1 gene impact body fat distribution in the HERITAGE Family Study cohort in 502 white and 276 black subjects. Insulin sensitivity index, glucose disappearance index, acute insulin response to glucose (AIR_glucose), and the disposition index (DI) were obtained from the frequently sampled intravenous glucose tolerance test. White subjects with the G82G at the PTPN1 IVS6+G82A polymorphism had higher body fat levels (p = 0.031) and sum of eight skinfolds (p = 0.003) and highest subcutaneous fat on the limbs (p = 0.002). G82A subjects had the lowest AIR_glucose (p = 0.005) and disposition index (p = 0.040). Interaction effects between PTPN1 and leptin receptor gene variants influenced insulin sensitivity index and AIR_glucose (p from 0.006 to 0.010). The variant PTPN1 Pro387Leu was associated with lower fasting insulin level (p = 0.035) and glucose disappearance index (p = 0.038). In summary, PTPN1 IVS6+G82G homozygotes showed higher levels of all measures of adiposity. G82 allele heterozygotes are potentially at higher risk for type 2 diabetes. Gene‐gene interactions between the PTPN1 and leptin receptor genes contributed to the phenotypic variability of insulin sensitivity. The PTPN1 Pro387Leu variant was associated with lower glucose tolerance.     

Neuronal Protein Tyrosine Phosphatase 1B Deficiency Results in Inhibition of Hypothalamic AMPK and Isoform-Specific Activation of AMPK in Peripheral Tissues

PTP1B^−/− mice are resistant to diet-induced obesity due to leptin hypersensitivity and consequent increased energy expenditure. We aimed to determine the cellular mechanisms underlying this metabolic state. AMPK is an important mediator of leptin''s metabolic effects. We find that α1 and α2 AMPK activity are elevated and acetyl-coenzyme A carboxylase activity is decreased in the muscle and brown adipose tissue (BAT) of PTP1B^−/− mice. The effects of PTP1B deficiency on α2, but not α1, AMPK activity in BAT and muscle are neuronally mediated, as they are present in neuron- but not muscle-specific PTP1B^−/− mice. In addition, AMPK activity is decreased in the hypothalamic nuclei of neuronal and whole-body PTP1B^−/− mice, accompanied by alterations in neuropeptide expression that are indicative of enhanced leptin sensitivity. Furthermore, AMPK target genes regulating mitochondrial biogenesis, fatty acid oxidation, and energy expenditure are induced with PTP1B inhibition, resulting in increased mitochondrial content in BAT and conversion to a more oxidative muscle fiber type. Thus, neuronal PTP1B inhibition results in decreased hypothalamic AMPK activity, isoform-specific AMPK activation in peripheral tissues, and downstream gene expression changes that promote leanness and increased energy expenditure. Therefore, the mechanism by which PTP1B regulates adiposity and leptin sensitivity likely involves the coordinated regulation of AMPK in hypothalamus and peripheral tissues.Protein tyrosine phosphatase 1B (PTP1B) belongs to a family of tyrosine phosphatases with diverse roles in eukaryotes (2, 4). PTP1B attenuates insulin signaling by dephosphorylating the insulin receptor (19, 22, 61) and possibly IRS-1 (9, 23) and leptin signaling by dephosphorylating JAK2, which phosphorylates the leptin receptor and associated substrates (10, 45, 67). PTP1B-deficient mice are insulin hypersensitive, lean, and resistant to diet-induced obesity (20, 36) due, at least in part, to increased energy expenditure (36). The leanness can be explained by the absence of PTP1B in neurons, because neuron-specific PTP1B^−/− mice also have reduced body weight and adiposity and increased energy expenditure (6). In contrast, muscle- and liver-specific PTP1B-deficient mice have normal body weight with improved insulin sensitivity, whereas adipose-PTP1B-deficient mice have increased body weight (6, 15, 16). These data suggest that PTP1B in peripheral tissues such as muscle and liver is an important mediator of peripheral insulin sensitivity, whereas PTP1B in the nervous system plays a critical role in regulating energy expenditure and adiposity (6).The adipocyte-derived hormone leptin plays an essential role in regulating energy homeostasis by acting on multiple tissues, most importantly the hypothalamus, to regulate food intake and energy expenditure (1). PTP1B^−/− mice have enhanced basal and leptin-stimulated hypothalamic STAT3 phosphorylation and are hypersensitive to leptin''s effect on food intake and body weight (10, 67). The overexpression of PTP1B in heterologous cells dose dependently reduces the leptin-induced phosphorylation of JAK2 and STAT3 and inhibits leptin-stimulated STAT3-dependent reporter gene activation (10, 35, 39, 67). These and other data established that enhanced leptin sensitivity contributes to the leanness in PTP1B^−/− mice. We sought to determine the cellular mechanisms underlying the altered energy homeostasis in the setting of PTP1B deficiency.AMP-activated protein kinase (AMPK) is a major mediator of leptin''s metabolic effects (43, 44). AMPK is a fuel-sensing enzyme complex activated by cellular stresses that increase AMP or deplete ATP, including hypoxia, ischemia, glucose deprivation, uncouplers of oxidative phosphorylation, exercise, and muscle contraction (66). AMPK also is activated by the antidiabetic drugs metformin (68) and the thiazolidinediones (21). Mechanisms involved in AMPK activation include (i) the binding of AMP to an allosteric site on the γ subunit, which renders the holoenzyme resistant to inactivating serine phosphatases and also may have direct allosteric effects on kinase activity (55), and (ii) phosphorylation by upstream AMPK kinases of the α (catalytic) subunits on Thr172, which is essential for kinase activity (29). Once activated, AMPK phosphorylates multiple downstream substrates, leading to the inhibition of ATP-utilizing pathways, such as fatty acid synthesis, and the activation of ATP-generating pathways, including fatty acid oxidation (34).The phosphorylation of acetyl coenzyme A (acetyl-CoA) carboxylase (ACC) by AMPK results in the inhibition of ACC activity, decreased malonyl-CoA content, and a subsequent increase in fatty acid oxidation in skeletal muscle caused by the disinhibition of carnitine palmitoyltransferase 1 (27, 52, 62). The leptin stimulation of muscle fatty acid oxidation is mediated by AMPK (44). AMPK also is an important regulator of muscle mitochondrial biogenesis and function (7, 37, 48, 58, 63). This may, in part, be mediated by peroxisome proliferator-activated receptor γ (PPARγ)-coactivator 1α (PGC-1α), because AMPK induces the expression and phosphorylation of PGC-1α, which regulates mitochondrial biogenesis and muscle fiber type (31).In addition to a role for AMPK in leptin action in peripheral tissues, the inhibition of hypothalamic AMPK activity by leptin plays an important role in mediating leptin''s effect on food intake and energy homeostasis (43). This appears to involve neurons that express neuropeptide Y (NPY) and agouti-related peptide (AgRP), since the expression of constitutively active AMPK in the basomedial hypothalamus augments NPY/AgRP expression (43). Furthermore, the deletion of the AMPK α2 catalytic subunit specifically in these neurons results in leanness, whereas deletion in proopiomelanocortin (POMC)-expressing neurons results in mild obesity (13).To determine whether alterations in AMPK contribute to increased energy expenditure and leanness in PTP1B^−/− mice, we investigated the AMPK pathway in peripheral tissues and hypothalamus. We demonstrate that the global absence of PTP1B alters AMPK and downstream biological processes in multiple tissues, and that neuronal PTP1B regulates AMPK activity in peripheral tissues in an isoform-specific manner. Our data establish a novel link between PTP1B and AMPK, two signaling molecules that are critical in the regulation of energy homeostasis.     

阿勒泰黄芪提取物对蛋白酪氨酸磷酸酯酶1B的抑制作用

本文探讨了阿勒泰黄芪不同提取物对蛋白酪氨酸磷酸酯酶1B(PTP1B)的抑制作用.采用分光光度法测定了提取物中的黄酮和皂苷含量;通过体外酶促动力学方法检测了不同提取物对PTP1B的影响,并确定了抑制类型;并采用氧化酶法检测了阿勒泰黄芪提取物对细胞利用葡萄糖能力的作用.结果表明,阿勒泰黄芪8种提取物(E1 ～8)中黄酮含量分别为5.09、10.46、3.58、3.23、53.91、21.77、5.76和7.49 mg/mL,其中E1、E2、E6、E7、E8皂苷含量分别为16.53、27.45、21.90、10.21和8.96 mg/mL;各提取物对PTP1B活性均表现出抑制作用,其中E1、E2、E7、E8的IC50分别为34.8、4.7、7.35和7.15 μg/mL,E1、E7和E8是竞争性抑制,E2是混合型竞争性抑制.E1、E2、E5、E7和E8较明显的提高了CHO-K1细胞对葡萄糖的利用.提示皂苷可能是阿勒泰黄芪抑制PTP1B活性的主要物质,通过PTP1B途径有效了提高细胞利用葡萄糖的能力.本研究为阿勒泰黄芪开发为防治糖尿病及改善胰岛素抵抗的药物或保健品提供实验依据.     

Protein Tyrosine Phosphatase 1B is Impaired in Skeletal Muscle of Diabetic Psammomys Obesus

Protein tyrosine phosphatases (PTPases) have been suggested to modulate the insulin receptor signal transduction pathways.We studied PTPases in Psammomys obesus, an animal model of nutritionally induced insulin resistance. No changes in the protein expression level of src homology PTPase 2 (SHP-2) (muscle, liver) or leukocyte antigen receptor (LAR) (liver) were detected. In contrast, the expression level of PTPase 1B (PTP 1B) in the skeletal muscle, but not in liver, was increased by 83% in the diabetic animals, compared with a diabetes-resistant line. However, PTP 1B– specific activity (activity/protein) significantly decreased (50% to 56%) in skeletal muscle of diabetic animals, compared with both the diabetes-resistant line and diabetes-prone animals. In addition, PTP 1B activity was inversely correlated to serum glucose level (r = –.434, P < .02). These findings suggest that PTP 1B, though overexpressed, is not involved in the susceptibility to insulin resistance in Psammomys obesus and is secondarily attenuated by hyperglycemia or other factors in the diabetic milieu.     

Transformation Suppression by Protein Tyrosine Phosphatase 1B Requires a Functional SH3 Ligand

We have recently shown that protein tyrosine phosphatase 1B (PTP1B) associates with the docking protein p130^Cas in 3Y1 rat fibroblasts. This interaction is mediated by a proline-rich sequence on PTP1B and the SH3 domain on p130^Cas. Expression of wild-type PTP1B (WT-PTP1B), but not a catalytically competent, proline-to-alanine point mutant that cannot bind p130^Cas (PA-PTP1B), causes substantial tyrosine dephosphorylation of p130^Cas (F. Liu, D. E. Hill, and J. Chernoff, J. Biol. Chem. 271:31290–31295, 1996). Here we demonstrate that WT-, but not PA-PTP1B, inhibits transformation of rat 3Y1 fibroblasts by v-crk, -src, and -ras, but not by v-raf. These effects on transformation correlate with the phosphorylation status of p130^Cas and two proteins that are associated with p130^Cas, Paxillin and Fak. Expression of WT-PTP1B reduces formation of p130^Cas-Crk complexes and inhibits mitogen-activated protein kinase activation by Src and Crk. These data show that transformation suppression by PTP1B requires a functional SH3 ligand and suggest that p130^Cas may represent an important physiological target of PTP1B in cells.     

Protein Tyrosine Phosphatase PTP1B Is Involved in Hippocampal Synapse Formation and Learning

ER-bound PTP1B is expressed in hippocampal neurons, and accumulates among neurite contacts. PTP1B dephosphorylates ß-catenin in N-cadherin complexes ensuring cell-cell adhesion. Here we show that endogenous PTP1B, as well as expressed GFP-PTP1B, are present in dendritic spines of hippocampal neurons in culture. GFP-PTP1B overexpression does not affect filopodial density or length. In contrast, impairment of PTP1B function or genetic PTP1B-deficiency leads to increased filopodia-like dendritic spines and a reduction in mushroom-like spines, while spine density is unaffected. These morphological alterations are accompanied by a disorganization of pre- and post-synapses, as judged by decreased clustering of synapsin-1 and PSD-95, and suggest a dynamic synaptic phenotype. Notably, levels of ß-catenin-Tyr-654 phosphorylation increased ∼5-fold in the hippocampus of adult PTP1B^−/− (KO) mice compared to wild type (WT) mice and this was accompanied by a reduction in the amount of ß-catenin associated with N-cadherin. To determine whether PTP1B-deficiency alters learning and memory, we generated mice lacking PTP1B in the hippocampus and cortex (PTP1B^fl/fl–Emx1-Cre). PTP1B^fl/fl–Emx1-Cre mice displayed improved performance in the Barnes maze (decreased time to find and enter target hole), utilized a more efficient strategy (cued), and had better recall compared to WT controls. Our results implicate PTP1B in structural plasticity within the hippocampus, likely through modulation of N-cadherin function by ensuring dephosphorylation of ß-catenin on Tyr-654. Disruption of hippocampal PTP1B function or expression leads to elongation of dendritic filopodia and improved learning and memory, demonstrating an exciting novel role for this phosphatase.     

Characterization of Liver Monocytic Myeloid-Derived Suppressor Cells and Their Role in a Murine Model of Non-Alcoholic Fatty Liver Disease

Myeloid-derived suppressor cells (MDSCs) are potent suppressors of T cell immunity in tumors and inflammatory diseases. They are identified by surface expression of CD11b⁺Gr1⁺ in mice, and CD11b⁺Gr1⁺ cells accumulate in the livers of obese mice. However, many myeloid cells share these CD11b⁺Gr1⁺ markers. Accordingly, the aim of this study was to identify the authentic phenotype of MDSCs and investigate their functions in non-alcoholic fatty liver disease (NAFLD). C57BL/6J mice were divided into 2 diet groups: a normal control group and high-fat group to induce NAFLD. We demonstrated that monocytic CD11b⁺Gr1^dim cells could be further divided into 2 populations based on side scatter (SSC) during flow cytometry. We found that SSC^lowCD11b⁺Gr1^dim cells accumulated in the livers of NAFLD mice over time, and that these cells were recruited by the chemokine CCL2 and its receptor CCR2 and might expand in the liver via macrophage colony-stimulating factor stimulation. Furthermore, SSC^lowCD11b⁺Gr1^dim cells had a strong suppressive ability on T cells; this effect was not observed for SSC^highCD11b⁺Gr1^dim cells, and was dependent on nitric oxide production by inducible nitric oxide synthase. Our findings demonstrate that SSC^lowCD11b⁺Gr1^dim cells represent authentic MDSCs in NAFLD livers, and might serve an important negative feedback function in liver inflammation.