Published papers

Inflammation promotes regeneration of injured tissues through poorly understood mechanisms, some of which involve interleukin (IL)-6 family members, the expression of which is elevated in many diseases including inflammatory bowel diseases and colorectal cancer. Here we show in mice and human cells that gp130, a co-receptor for IL-6 cytokines, triggers activation of YAP and Notch, transcriptional regulators that control tissue growth and regeneration, independently of the gp130 effector STAT3. Through YAP and Notch, intestinal gp130 signalling stimulates epithelial cell proliferation, causes aberrant differentiation and confers resistance to mucosal erosion. gp130 associates with the related tyrosine kinases Src and Yes, which are activated on receptor engagement to phosphorylate YAP and induce its stabilization and nuclear translocation. This signalling module is strongly activated upon mucosal injury to promote healing and maintain barrier function.

Chronic graft-versus-host disease (GVHD) is a major cause of late death and morbidity after allogeneic hematopoietic cell transplantation. Recently, in addition to Th2 cells, Th1 and Th17 cells have been shown to contribute to chronic GVHD progression. IL-12 induces Th1 cells and IL-23 plays a role in stabilizing and/or amplifying Th17 cells, as well as in inducing IFN-γ/IL-17 double-producing cells. Because mAb targeting the p40 subunit common to both IL-12 and IL-23 can inhibit both IL-12R and IL-23R-mediated signaling, we investigated the effects of anti-p40 mAb on a well-defined chronic GVHD mice model. Treatment of anti-p40 mAb in allogeneic recipients significantly reduced the severity of clinical and pathological chronic GVHD. Intracellular staining revealed that IFN-γ single-positive (IL-17-) and IFN-γ/IL-17 double-positive cells were suppressed in anti-p40 mAb-treated allogeneic recipients compared with control recipients. The cytokine levels of IFN-γ and IL-17 were also decreased in serum from anti-p40 mAb-treated allogeneic recipients. T-bet expression of donor IL-17+ CD4+ T cells was reduced significantly in anti-p40 mAb-treated recipients, and this reduction in T-bet expression was associated with IL-22 production by donor T cells. These results suggested that anti-p40 mAb attenuated chronic GVHD via suppression of IFN-γ/IL-17-producing cells, and that targeting the IL-12/IL-23 pathway may represent a promising therapeutic strategy for preventing and treating chronic GVHD.

Chronic graft-versus-host disease (GVHD) is a major cause of late death and morbidity after allogeneic hematopoietic cell transplantation. Recently, in addition to Th2 cells, Th1 and Th17 cells have been shown to contribute to chronic GVHD progression. IL-12 induces Th1 cells and IL-23 plays a role in stabilizing and/or amplifying Th17 cells, as well as in inducing IFN-γ/IL-17 double-producing cells. Because mAb targeting the p40 subunit common to both IL-12 and IL-23 can inhibit both IL-12R and IL-23R-mediated signaling, we investigated the effects of anti-p40 mAb on a well-defined chronic GVHD mice model. Treatment of anti-p40 mAb in allogeneic recipients significantly reduced the severity of clinical and pathological chronic GVHD. Intracellular staining revealed that IFN-γ single-positive (IL-17-) and IFN-γ/IL-17 double-positive cells were suppressed in anti-p40 mAb-treated allogeneic recipients compared with control recipients. The cytokine levels of IFN-γ and IL-17 were also decreased in serum from anti-p40 mAb-treated allogeneic recipients. T-bet expression of donor IL-17+ CD4+ T cells was reduced significantly in anti-p40 mAb-treated recipients, and this reduction in T-bet expression was associated with IL-22 production by donor T cells. These results suggested that anti-p40 mAb attenuated chronic GVHD via suppression of IFN-γ/IL-17-producing cells, and that targeting the IL-12/IL-23 pathway may represent a promising therapeutic strategy for preventing and treating chronic GVHD.

Transplantation of endothelial cells (ECs) is a promising therapeutic approach for ischemic disorders. In addition, the generation of ECs has become increasingly important for providing vascular plexus to regenerated organs, such as the liver. Although many attempts have been made to generate ECs from pluripotent stem cells and nonvascular cells, the minimum number of transcription factors that specialize in directly inducing vascular ECs remains undefined. Here, by screening 18 transcription factors that are important for both endothelial and hematopoietic development, we demonstrate that ets variant 2 (ETV2) alone directly converts primary human adult skin fibroblasts into functional vascular endothelial cells (ETVECs). In coordination with endogenous FOXC2 in fibroblasts, transduced ETV2 elicits expression of multiple key endothelial development factors, including FLI1, ERG, and TAL1, and induces expression of endothelial functional molecules, including EGFL7 and von Willebrand factor. Consequently, ETVECs exhibits EC characteristics in vitro and forms mature functional vasculature in Matrigel plugs transplanted in NOD SCID mice. Furthermore, ETVECs significantly improve blood flow recovery in a hind limb ischemic model using BALB/c-nu mice. Our study indicates that the creation of ETVECs provides further understanding of human EC development induced by ETV2.

Transplantation of endothelial cells (ECs) is a promising therapeutic approach for ischemic disorders. In addition, the generation of ECs has become increasingly important for providing vascular plexus to regenerated organs, such as the liver. Although many attempts have been made to generate ECs from pluripotent stem cells and nonvascular cells, the minimum number of transcription factors that specialize in directly inducing vascular ECs remains undefined. Here, by screening 18 transcription factors that are important for both endothelial and hematopoietic development, we demonstrate that ets variant 2 (ETV2) alone directly converts primary human adult skin fibroblasts into functional vascular endothelial cells (ETVECs). In coordination with endogenous FOXC2 in fibroblasts, transduced ETV2 elicits expression of multiple key endothelial development factors, including FLI1, ERG, and TAL1, and induces expression of endothelial functional molecules, including EGFL7 and von Willebrand factor. Consequently, ETVECs exhibits EC characteristics in vitro and forms mature functional vasculature in Matrigel plugs transplanted in NOD SCID mice. Furthermore, ETVECs significantly improve blood flow recovery in a hind limb ischemic model using BALB/c-nu mice. Our study indicates that the creation of ETVECs provides further understanding of human EC development induced by ETV2.

Background: The role of interleukin (IL)-23 in asthma pathophysiology is still controversial. We examined its role in allergic airway inflammation in response to two distinct antigens using IL-23-deficient mice. Methods: Allergic airway inflammation was evaluated in wild-type and IL-23p19 -/- mice. Mice were sensitized to ovalbumin (OVA) or house dust mite (HDM) by intraperitoneal injection of antigen and their airways were then exposed to the same antigen. Levels of antigen-specific immunoglobulins in serum as well as cytokines in bronchoalveolar or peritoneal lavage fluid and lung tissue were determined by enzyme-linked immunosorbent assay and/or quantitative polymerase chain reaction. Results: Deficiency of IL-23p19 decreased eosinophils and Th2 cytokines in bronchoalveolar lavage fluid (BALF) of OVA-treated mice, while it increased BALF eosinophils of HDM-treated mice. Peritoneal injection of OVA with alum, but not of HDM, induced local synthesis of IL-6, IL-10, and IL-23. Systemic production of antigen-specific IgG1 was partially dependent on IL-23. In contrast, airway exposure to HDM, but not to OVA, induced IL-23p19 mRNA expression in the lungs. In IL-23p19-deficient mice, HDM-exposed lungs did not exhibit the induction of IL-17A, which negatively regulates eosinophilic inflammation. Conclusions: Different antigens induced IL-23 at different part of the body in our similar asthma models. Endogenous IL-23 production at the site of antigen sensitization facilitates type-2 immune responses, whereas IL-23 production and subsequent IL-17A synthesis in the airways suppresses allergic inflammation.

Background: The role of interleukin (IL)-23 in asthma pathophysiology is still controversial. We examined its role in allergic airway inflammation in response to two distinct antigens using IL-23-deficient mice. Methods: Allergic airway inflammation was evaluated in wild-type and IL-23p19 -/- mice. Mice were sensitized to ovalbumin (OVA) or house dust mite (HDM) by intraperitoneal injection of antigen and their airways were then exposed to the same antigen. Levels of antigen-specific immunoglobulins in serum as well as cytokines in bronchoalveolar or peritoneal lavage fluid and lung tissue were determined by enzyme-linked immunosorbent assay and/or quantitative polymerase chain reaction. Results: Deficiency of IL-23p19 decreased eosinophils and Th2 cytokines in bronchoalveolar lavage fluid (BALF) of OVA-treated mice, while it increased BALF eosinophils of HDM-treated mice. Peritoneal injection of OVA with alum, but not of HDM, induced local synthesis of IL-6, IL-10, and IL-23. Systemic production of antigen-specific IgG1 was partially dependent on IL-23. In contrast, airway exposure to HDM, but not to OVA, induced IL-23p19 mRNA expression in the lungs. In IL-23p19-deficient mice, HDM-exposed lungs did not exhibit the induction of IL-17A, which negatively regulates eosinophilic inflammation. Conclusions: Different antigens induced IL-23 at different part of the body in our similar asthma models. Endogenous IL-23 production at the site of antigen sensitization facilitates type-2 immune responses, whereas IL-23 production and subsequent IL-17A synthesis in the airways suppresses allergic inflammation.

Pigment epithelium-derived factor (PEDF) is the most potent inhibitor of angiogenesis, suggesting that loss of PEDF contributes to proliferative diabetic retinopathy. However, the role of PEDF against retinal vascular hyperpermeability remains to be elucidated. We investigated here whether and how PEDF could inhibit the advanced glycation end product (AGE) signaling to vascular hyperpermeability. Intravenous administration of AGEs to normal rats not only increased retinal vascular permeability by stimulating vascular endothelial growth factor (VEGF) expression but also decreased retinal PEDF levels. Simultaneous treatments with PEDF inhibited the AGE-elicited VEGF-mediated permeability by down-regulating mRNA levels of p22phox and gp91phox, membrane components of NADPH oxidase, and subsequently decreasing retinal levels of an oxidative stress marker, 8- hydroxydeoxyguanosine. PEDF also inhibited the AGE-induced vascular hyperpermeability evaluated by transendothelial electrical resistance by suppressing VEGF expression. Furthermore, PEDF decreased reactive oxygen species (ROS) generation in AGE-exposed endothelial cells by suppressing NADPH oxidase activity via down-regulation of mRNA levels of p22PHOX and gp91 PHOX. This led to blockade of the AGE-elicited Ras activation and NF-κB-dependent VEGF gene induction in endothelial cells. These results indicate that the central mechanism for PEDF inhibition of the AGE signaling to vascular permeability is by suppression of NADPH oxidase-mediated ROS generation and subsequent VEGF expression. Substitution of PEDF may offer a promising strategy for halting the development of diabetic retinopathy. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.

It has been estimated that >20% of all malignancies are initiated or exacerbated by inflammation. Until recently, the molecular basis of this process has not been clarified. However, recent studies have uncovered the molecular mechanism of intracellular signaling pathways of inflammatory cytokines such as tumor necrosis factor (TNF)-α, interferon (IFN)-γ and interleukin (IL)-6. Three major transcription factors including NF-κB, STAT1 and STAT3 have been shown to play major roles in transmitting inflammatory cytokine signals to the nucleus. One function of NF-κB and STAT3 in tumor cells is the promotion of cell growth and cell survival through the induction of target genes, whose products promote cell division and inhibit apoptosis. In addition, NF-κB and STAT1 are important transcription factors that induce inflammatory mediators from inflammatory cells, especially macrophages, while STAT3 often antagonizes this process. STAT1 is generally believed to be an anti-oncogene because it promotes apoptosis through p53, but it could promote inflammation-mediated tumor development by enhancing tissue injury, remodeling, fibrosis and inflammation. Hence, the inhibition of NF-κB and STATs offers a strategy for treatment of a variety of malignancies and can convert inflammation-induced tumor growth into inflammation-induced tumor regression. © 2006 Japanese Cancer Association.

Approximately 20% of human cancers are estimated to develop from chronic inflammation. Recently, the NF-κB pathway was shown to play an essential role in promoting inflammation-associated cancer, but the role of the JAK/STAT pathway, another important signaling pathway of proinflammatory cytokines, remains to be investigated. Suppressor of cytokine signaling-1 (SOCS1) acts as an important physiological regulator of cytokine responses, and silencing of the SOCS1 gene by DNA methylation has been found in several human cancers. Here, we demonstrated that SOCS1-deficient mice (SOCS1-/-Tg mice), in which SOCS1 expression was restored in T and B cells on a SOCS1-/- background, spontaneously developed colorectal carcinomas carrying nuclear β-catenin accumulation and p53 mutations at 6 months of age. However, interferon (IFN)γ-/-SOCS1-/- mice and SOCS1 -/-Tg mice treated with anti-IFNγ antibody did not develop such tumors. STAT3 and NF-κB activation was evident in SOCS1-/-Tg mice, but these were not sufficient for tumor development because these are also activated in IFNγ-/-SOCS1-/- mice. However, colons of SOCS1-/-Tg mice, but not IFNγ-/-SOCS1 -/- mice, showed hyperactivation of STAT1, which resulted in the induction of carcinogenesis-related enzymes, cyclooxygenase-2 and inducible nitric oxide synthase. These data strongly suggest that SOCS1 is a unique antioncogene which prevents chronic inflammation-mediated carcinogenesis by regulation of the IFNγ/STAT1 pathways. JEM © The Rockefeller University Press.

Toll-like receptors (TLRs) play an important role as a sensor of microbial pathogens in the innate immune response. TLRs transmit signals through the recruitment of adaptor proteins including tumor necrosis factor-associated factor 6 (TRAF6), which mediates the activation of IκB kinase (IKK). TIFA (TRAF-interacting protein with a forkhead-associated (FHA) domain) has been shown to bind to TRAF6 and activate IKK by promoting the oligomerization and ubiquitin-ligase activity of TRAF6. FHA domains preferentially bind to phospho-threonine residues in their targets. Here, we identified a novel zinc finger protein, ZCCHC11, that interacts with TIFA from phosphoproteins of a macrophage cell line, RAW 264.7, by using affinity purification with GST-TIFA and mass spectrometric analysis. By a search of the EST database, we found a 200 kDa full-length form (ZCCHC11L). ZCCHC11L was mostly located to the nucleus, but translocated into the cytoplasm in response to LPS and bound to TIFA. Overexpression and knockdown by siRNA indicated that ZCCHC11 functions as a negative regulator of TLR-mediated NF-κB activation. The N-terminal region (ZCCHC11S) including CCHC-type Zn-finger motif was sufficient for suppression of NF-κB. We propose that ZCCHC11 is a unique TLR signal regulator, which interacts with TIFA after LPS treatment and suppresses the TRAF6-dependent activation of NF-κB. © 2006 Elsevier Inc. All rights reserved.

Neuromedin U (NMU) is a neuropeptide expressed not only in the central nervous system but also in various organs, including the gastrointestinal tract and lungs. NMU interacts with two G protein-coupled receptors, NMU-R1 and NMU-R2. Although NMU-R2 is expressed in a specific region of the brain, NMU-R1 is expressed in various peripheral tissues, including immune and hematopoietic cells. Our recent study demonstrated an important role of NMU in mast cell-mediated inflammation. In this study, we showed that airway eosinophilia was reduced in NMU-deficient mice in an allergen-induced asthma model. There were no differences in the antigen-induced Th2 responses between wild-type and NMU knockout mice. NMU-R1 was highly expressed in the eosinophil cell line, and NMU directly induced Ca2+ mobilization and extracellular/signal- regulated kinase phosphorylation. NMU also induced cell adhesion to components of the extracellular matrix (fibronectin and collagen type I), and chemotaxis in vitro. Furthermore, NMU-R1 was also expressed in human peripheral blood eosinophils, and NMU induced cell adhesion in a dose-dependent manner. These data indicate that NMU promotes eosinophil infiltration into inflammatory sites by directly activating eosinophils. Our study suggests that NMU receptor antagonists could be novel targets for pharmacological inhibition of allergic inflammatory diseases, including asthma. Copyright © 2006 the American Physiological Society.

Suppressor of cytokine signaling (Socs) 3 is a cytokine-inducible inhibitor with critical but selective cell-specific effects. We show that deficiency of Socs3 in T cells had minimal effects on differentiation of T cells to the T helper (Th) 1 or Th2 subsets; accordingly, Socs3 had no effect on IL-12-dependent signal transducer and activator of transcription (Stat) 4 phosphorylation or IL-4-dependent Stat6 phosphorylation. By contrast, Socs3 was found to be a major regulator of IL-23-mediated Stat3 phosphorylation and Th17 generation, and Stat3 directly binds to the IL-17A and IL-17F promoters. We conclude that Socs3 is an essential negative regulator of IL-23 signaling, inhibition of which constrains the generation of Th17 differentiation. © 2006 by The National Academy of Sciences of the USA.

Recently, DNA methylation and reduced expression of the suppressor of the cytokine signaling-3 (SOCS3) gene in human hepatocellular carcinoma (HCC) patients have been reported. However, the roles of SOCS3 in HCC development in vivo have not been clarified. Using RT-PCR analysis and Western blotting, we confirmed that SOCS3 expression was reduced in HCC patients. However, reduced expression of SOCS3 occurred not only in HCC but also in nontumor regions, and this reduction was stronger as the fibrosis grade increased. Furthermore, SOCS3 levels were inversely correlated with signal transducers and activators of transcription-3 (STAT3) activation as well as transforming growth factor (TGF)-β1 levels in the non-HCC region. To define the molecular consequences of SOCS3 silencing/STAT3 hyperactivation and liver fibrosis, we examined liver-specific SOCS3-deficient mice. We demonstrated that SOCS3 deletion in the liver resulted in hyperactivation of STAT3 and promoted ConA- and chemical-induced liver fibrosis. The expression of TGF-β1, a mediator of fibrosis, was enhanced by SOCS3 gene deletion, but suppressed by the overexpression of a dominant-negative STAT3 or SOCS3 both in vivo and in vitro. These data suggest that TGF-β1 is a target gene of STAT3 and could be one of the mechanisms for enhanced fibrosis in SOCS3-deficient mice. Thus, our present study provides a novel role of SOCS3 and STAT3 in HCC development: in addition to the previously characterized oncogenic potentials, STAT3 enhances hepatic fibrosis through the upregulation of TGF-β1 expression, and SOCS3 prevents this process. © 2006 Nature Publishing Group All rights reserved.

Suppressor of cytokine signaling (SOCS)3 is a major negative feedback regulator of signal transducer and activator of transcription (STAT)3-activating cytokines. Transgenic mouse studies indicate that high levels of SOCS3 in T cells result in type 2 T helper cell (Th2) skewing and lead to hypersensitivity to allergic diseases. To define the physiological roles of SOCS3 in T cells, we generated T cell-specific SOCS3 conditional knockout mice. We found that the mice lacking SOCS3 in T cells showed reduced immune responses not only to ovalbumin-induced airway hyperresponsiveness but also to Leishmania major infection. In vitro, SOCS3-deficient CD4+ T cells produced more transforming growth factor (TGF)-β1 and interleukin (IL)-10, but less IL-4 than control T cells, suggesting preferential Th3-like differentiation. We found that STAT3 positively regulates TGF-β1 promoter activity depending on the potential STAT3 binding sites. Furthermore, chromatin immunoprecipitation assay revealed that more STAT3 was recruited to the TGF-β1 promoter in SOCS3-deficient T cells than in control T cells. The activated STAT3 enhanced TGF-β1 and IL-10 expression in T cells, whereas the dominant-negative form of STAT3 suppressed these. From these findings, we propose that SOCS3 regulates the production of the immunoregulatory cytokines TGF-β1 and IL-10 through modulating STAT3 activation. © The Rockefeller University Press.

Neuromedin U (NMU) is a neuropeptide involved in appetite, circadian rhythm, and pronociception. However, the NMU receptor NMU-R1 has been shown to be expressed in immune cells and NMU promotes mast cell-dependent inflammation. In this study, we demonstrated that NMU plays an important role in IL-6 production in macrophages. NMU-deficient mice were resistant against cecal ligation puncture- as well as LPS-induced septic shock. IL-6 but not TNF-α levels were markedly reduced in LPS-treated NMU-deficient mice compared with wild type mice. Both NMU and NMU-R1 were expressed in wild type peritoneal macrophages, and treatment with LPS resulted in up-regulation of NMU but down-regulation of NMU-R1 expression, however, no down-regulation of NMU-R1 was observed in NMU-deficient macrophages where LPS-induced IL-6 production was severely reduced. These data suggest that LPS-induced IL-6 expression is partly dependent on autocrine/paracrine activation of the NMU-NMU-R1 signals in macrophages. © 2006 Elsevier Inc. All rights reserved.

The cytokine interleukin-10 (IL-10) potently inhibits macrophage function through activation of the transcription factor STAT3. The expression of SOCS3 (suppressor of cytokine signaling-3) has been shown to be induced by IL-10 in a STAT3-dependent manner. However, the relevance of SOCS3 expression to the anti-inflammatory effect of IL-10 on macrophages has been controversial. Through kinetic analysis of the requirement for SOCS3 in IL-10 inhibition of lipopolysaccharide (LPS)-stimulated tumor necrosis factor-α (TNFα) transcription and translation, SOCS3 was found to be necessary for TNFα expression during the early phase, but not the late phase of IL-10 action. SOCS3 was essential for IL-10 inhibition of LPS-stimulated production of iNOS (inducible nitricoxide synthase) protein and nitric oxide (NO). To determine the domains of SOCS3 protein important in mediating these effects, SOCS3 -/- macrophages were reconstituted with SOCS3 mutated for the SH2, KIR, SOCS box domains, and tyrosines 204 (Tyr204) and 221 (Tyr 221). The SH2 domain, SOCS box, and both Tyr204 and Tyr221 were required for IL-10 inhibition of TNFα mRNA and protein expression, but interestingly the KIR domain was necessary only for IL-10 inhibition of TNFα protein expression. In contrast, Tyr 204 and Tyr221 were the only structural features of SOCS3 that were necessary in mediating IL-10 inhibition of iNOS protein expression and NO production. These data define SOCS3 as an important mediator of IL-10 inhibition of macrophage activation and that SOCS3 interferes with distinct LPS-stimulated signal transduction events through differing mechanisms. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.