論文

Stress or aging of tissue-specific stem cells is considered central to the decline of tissue homeostasis in the elderly, although little is known of molecular mechanisms underlying hematopoietic stem cell (HSC) aging and stress resistance. Here, we report that mice lacking the transcription factor forkhead box O3a (FoxO3a) develop neutrophilia associated with inhibition of the up-regulation of negative regulator of cell proliferation, Sprouty-related Ena/VASP homology 1 domain-containing proteins 2 (Spred2) and AKT and ERK activation, in HSCs during hema-topoietic recovery following myelosup-pressive stress conditions. Compared with aged wild-type mice, more severe neutrophilia was also observed in aged Foxo3a-deficient mice. AKT and ERK activation and inhibition of Spred2 were detected in HSCs from aged FoxO3a-deficient mice. Spred2-deficient mice also developed neutrophilia during hematopoietic recovery following myelosuppressive stress, indicating that FoxO3a plays a pivotal role in maintenance, integrity, and stress resistance of HSCs through negative feedback pathways for proliferation. This will provide new insight into the hematopoietic homeostasis in conditions of aging and stress. © 2008 by The American Society of Hematology.

Suppressor of cytokine signaling 3 (Socs3) has been identified as a mediator of central leptin resistance, but the identity of specific neurons in which Socs3 acts to suppress leptin signaling remains elusive. The ventromedial hypothalamus (VMH) was recently shown to be an important site for leptin action because deleting leptin receptor within VMH neurons causes obesity. To examine the role of VMH Socs3 in leptin resistance and energy homeostasis, we generated mice lacking Socs3 specifically in neurons positive for steroidogenic factor 1 (SF1), which is expressed abundantly in the VMH. These mice had increased phosphorylation of signal transducer and activator of transcription-3 in VMH neurons, suggesting improved leptin signaling, and consistently, food intake and weight-reducing effects of exogenous leptin were enhanced. Furthermore, on either chow or high-fat diets, these mice had reduced food intake. Unexpectedly, energy expenditure was reduced as well. Mice lacking Socs3 in SF1 neurons, despite no change in body weight, had improved glucose homeostasis and were partially protected from hyperglycemia and hyperinsulinemia induced by high-fat diets. These results suggest that Socs3 in SF1 neurons negatively regulates leptin signaling and plays important roles in mediating leptin sensitivity, glucose homeostasis, and energy expenditure. Copyright © 2008 by The Endocrine Society.

Objective: In the present work, we investigate the role of interleukin (IL)27/IL27 receptor α (Rα) (WSX-1) in the development of autoimmune disorders in the MRL/lpr mouse, which is considered as an experimental model of systemic lupus erythaematosus (SLE) in humans. Methods: We generated two strains of WSX-1 transgenic mice in the MRL/lpr background with different expression levels of WSX-1, and investigated the effect of WSX-1 overexpression on survival, glomerulonephritis and immunological properties. Results: In comparison with wild type (WT) MRL/lpr and transgenic (Tg) low (TgL) mice, Tg high (TgH) mice exhibited a prolonged lifespan and no apparent development of autoimmune nephritis. Production of anti-dsDNA antibody and total IgG and lgG2a were significantly lower in TgH mice than those of TgL and WT mice. The expressed amounts of interferon (IFN)y and IL4 mRNA by CD4+ T cells from Tg mice decreased in a dose-dependent fashion. CD4+ splenic lymphocytes in TgH mice were more subject to the IL27-mediated suppression of cytokine production. In vitro stimulation of CD4+ T cells by IL27 resulted in over phosphorylation of STAT3 in TgH cells than in WT cells. Conclusion: WSX-1 overexpression in the MRL/lpr background rendered the autoimmune prone mice protected from the development of autoimmune diseases. Our results suggest that IL27 signalling may be a therapeutic target against autoimmune diseases, including human SLE.

Signal-transducing adaptor protein-2 (STAP-2) is a recently identified adaptor protein as a c-Fms/M-CSF receptor-interacting protein and constitutively expressed in macrophages. In our previous study, we examined the role of STAP-2 in the c-Fms/M-CSF receptor signaling using a murine macrophage tumor cells line, Raw264.7. Overexpression of STAP-2 in Raw264.7 cells markedly suppressed M-CSF-induced activation of extracellular signal regulated kinase and Akt. In addition, Raw264.7 overexpressing STAP-2 affected cell migration in wound-healing process. These results suggest that STAP-2 deficiency influences endogenous c-Fms/M-CSF receptor signaling. Here we show that loss of STAP-2 expression in knockout mouse macrophages results in marked enhancement of the c-Fms/M-CSF receptor signaling and wound-healing process. We therefore propose that STAP-2 acts as an endogenous regulator in normal macrophages functions. © 2008 Pharmaceutical Society of Japan.

Inflammatory cytokines cause tissue dysfunction. We previously reported that retinal inflammation down-regulates rhodopsin expression and impairs visual function by an unknown mechanism. Here, we demonstrate that rhodopsin levels were preserved by suppressor of cytokine signaling 3 (SOCS3), a negative feedback regulator of STAT3 activation. SOCS3 was expressed mainly in photoreceptor cells in the retina. In the SOCS3-deficient retinas, rhodopsin protein levels dropped sooner, and the reduction was more profound than in the wild type. Visual dysfunction, measured by electroretinogram, was prolonged in retina-specific SOCS3 conditional knockout mice. Visual dysfunction and decreased rhodopsin levels both correlated with increased STAT3 activation enhanced by SOCS3 deficiency. Interleukin 6, one of the inflammatory cytokines found during retinal inflammation, activated STAT3 and decreased rhodopsin protein in adult retinal explants. This was enhanced by inhibiting SOCS3 function in vitro, indicating that rhodopsin reduction was not a secondary effect in the mutant mice. Interestingly, in the inflamed SOCS3-deficient adult retina, rhodopsin decreased post-transcriptionally at least partly through ubiquitin-proteasome-dependent degradation accelerated by STAT3 activation and not transcriptionally as in the developing retina, on which we reported previously. A STAT3-dependent E3 ubiquitin ligase, Ubr1, was responsible for rhodopsin degradation and was up-regulated in the inflamed SOCS3-deficient retinas. These results indicate that in wild-type animals, a decrease in rhodopsin during inflammation is minimized by endogenous SOCS3. However, when STAT3 activation exceeds some threshold beyond the compensatory activity of endogenous SOCS3, rhodopsin levels decrease. These findings suggest SOCS3 as a potential therapeutic target molecule for protecting photoreceptor cell function during inflammation. © 2008 by The American Society for Biochemistry and Molecular Biology, Inc.

The signal-transducing adaptor protein 2 (STAP-2) is a recently identified adaptor protein that contains a pleckstrin homology (PH) and Src homology 2 (SH2)-like domains, as well as a proline-rich domain in its C-terminal region. In previous studies, we demonstrated that STAP-2 binds to MyD88 and IKK-α or IKK-β and modulates NF-κB signaling in macrophages. In the present study, we found that ectopic expression of STAP-2 inhibited Epstein-Barr virus (EBV) LMP1-mediated NF-κB signaling and interleukin-6 expression. Indeed, STAP-2 associated with LMP1 through its PH and SH2-like domains, and these proteins interacted with each other in EBV-positive human B cells. We found, furthermore, that STAP-2 regulated LMP1-mediated NF-κB signaling through direct or indirect interactions with the tumor necrosis factor receptor (TNFR)-associated factor 3 (TRAF3) and TNFR-associated death domain (TRADD) proteins. STAP-2 mRNA was induced by the expression of LMP1 in human B cells. Furthermore, transient expression of STAP-2 in EBV-positive human B cells decreased cell growth. Finally, STAP-2 knockout mouse embryonic fibroblasts showed enhanced LMP1-induced cell growth. These results suggest that STAP-2 acts as an endogenous negative regulator of EBV LMP1-mediated signaling through TRAF3 and TRADD. Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Cytokine and glucocorticoid (GC) hormone signaling act in an integrated fashion to control inflammation and immune response. Here we establish a new mode of interaction of these two pathways and propose Suppressor of Cytokine Signaling (SOCS)-1 as an essential player in mediating cross-talk. We observed that glucocorticoid receptor (GR) and SOCS1 form an intracellular complex through an interaction, which required the SH2 domain of SOCS1 and the ligand binding domain of GR. Furthermore, GC stimulation was found to increase the nuclear level of SOCS1. SOCS1 binding to the GR did not require ligand binding of the receptor; however, it was abolished after long term GC stimulation, suggesting a functional role of the interaction for the early phase of GC action. The interaction between GR and SOCS1 appeared to negatively influence the transcription of the two GR-regulated genes, FKBP5 and MKP1, because the GC-dependent expression of these genes was inhibited by the SOCS1 inducer IFNγ and enhanced in SOCS1-deficient murine embryonic fibroblasts as compared with IFNγ treated wild-type cells. Our results suggest a prominent role of SOCS1 in the early phase of cross-talk between GR and cytokine signaling. © 2008 by The American Society for Biochemistry and Molecular Biology, Inc.

Proliferation and differentiation of keratinocytes during wound healing are regulated by cytokines and chemokines, which are secreted by resident and inflammatory cells and activate the transcription factor signal transducer and activator of transcription (STAT)3. However, it is not clear to what extent STAT3 in keratinocytes is activated by gp130-containing receptors. We addressed this question genetically by deleting the suppressor of cytokine signaling (SOCS)3, a negative regulator of gp130-mediated STAT3 activation. Socs3 alleles flanked by loxP sites were deleted in mice with either an MMTV-Cre or K5-Cre transgene. While both transgenes are active in keratinocytes, the MMTV-Cre deletes floxed genes also in immune cells. Deletion of Socs3 using the MMTV-Cre transgene resulted in aberrant STAT3 activation, impaired wound healing, prolonged secretion of chemokines, a hyperproliferative epidermis, and neutrophil infiltration into wounds. Simultaneous deletion of the Socs3 and gp130 genes restored normal wound healing. Moreover, deletion of Socs3 only in keratinocytes caused impaired wound healing. These results demonstrate that wound healing is controlled in keratinocytes by the gp130-SOCS3-STAT3 pathway and an imbalance of this pathway results in delayed wound healing. © 2008 The Society for Investigative Dermatology.

Inflammatory bowel disease (IBD) is a chronic disorder of the gastrointestinal tract. Although the etiology and pathogenesis of IBD remain unknown, pro-inflammatory cytokines including IFN-γ play an important role in the development of IBD. Suppressor of cytokine signaling-1 (SOCS-1) is a crucial inhibitor of cytokine signaling, particularly of IFN-γ. In this study, we investigated the role of SOCS-1 in the development of murine dextran sulfate sodium (DSS)-induced colitis, a model of colitis resembling human IBD. SOCS-1 heterozygous (SOCS-1+/-) and wild-type (WT) mice were given 3% DSS dissolved in drinking water for 5 days. Activation and expression of signal transducers and activators of transcription (STAT) in colonic tissues were assessed by western blot analysis. The expression of CD4, IFN-γ, IL-4, IL-17 and Forkhead box P3 (Foxp3) in colonic lamina propria lymphocytes was analyzed by flow cytometry and cytokine concentrations in serum were measured. DSS-treated SOCS-1+/- mice developed more severe colitis than DSS-treated WT mice. Enhanced activation of STAT1, a higher ratio of CD4+ IFN-γ+T cells and a lower frequency of Foxp3+ regulatory T (Treg) cells, were observed in the colon of DSS-treated SOCS-1+/- mice compared with DSS-treated WT mice. DSS-treated SOCS-1+/- mice showed higher levels of IFN-γ in sera than did DSS-treated WT mice. Furthermore, T cell-specific SOCS-1-conditional knockout mice developed more severe colitis than control mice after DSS administration. Our findings suggest that SOCS-1, particularly in T cells, prevents the development of DSS-induced colitis in mice by inhibiting IFN-γ/STAT1 signaling and by subsequently regulating Treg cell development. © The Japanese Society for Immunology. 2008. All rights reserved.

The cytokine, transforming growth factor-β1 (TGF-β1), converts naive T cells into regulatory T cells that prevent autoimmunity. However, in the presence of interleukin (IL)-6, TGF-β1 has also been found to promote differentiation into IL-17-producing helper T (Th17) cells that are deeply involved in autoimmunity and inflammation. However, it has not been clarified how TGF-β1 and IL-6 determine such a distinct fate. Here we found that a master regulator for Th17, retinoic acid-related orphan receptor γt (RORγt), was rapidly induced by TGF-β1 regardless of the presence of IL-6. IL-6 reduced Foxp3 expression, and overexpression of Foxp3 in a T cell line resulted in a strong reduction of IL-17A expression. We have characterized the IL-17A promoter and found that RORγt binding is sufficient for activation of the minimum promoter in the HEK 293T cells. RORγt-mediated IL-17A promoter activation was suppressed by forced expression of Foxp3. Foxp3 directly interacted with RORγt through exon 2 region of Foxp3. The exon 2 region and forkhead (FKH) domain of Foxp3 were necessary for the suppression of RORγt-mediated IL-17A promoter activation. We propose that induction of Foxp3 is the mechanism for the suppression of Th17 and polarization into inducible Treg. © 2008 by The American Society for Biochemistry and Molecular Biology, Inc.

Acute liver failure is associated with significant mortality. However, the underlying pathophysiological mechanism is not yet fully understood. Suppressor of cytokine signaling-1 (SOCS1), which is a negative-feedback molecule for cytokine signaling, has been shown to be rapidly induced during liver injury. Here, using liver-specific SOCS1-conditional-knockout mice, we demonstrated that SOCS1 deletion in hepatocytes enhanced concanavalin A (ConA)-induced hepatitis, which has been shown to be dependent on activated T and natural killer T (NKT) cells. Although serum cytokine level and NKT cell activation were similar in wild-type (WT) and SOCS1-deficient mice after ConA treatment, proapoptotic signals, including signal transducers and activators of transcription 1 (STAT1) and Jun-terminal kinase (JNK) activation, were enhanced in SOCS1-deficient fivers compared with those in WT livers. SOCS1-deficient hepatocytes had higher expression of Fas antigen and were more sensitive to anti-Fas antibody-induced apoptosis than were WT hepatocytes. Furthermore, SOCS1-deficient hepatocytes were more sensitive to tumor necrosis factor (TNF)-α-induced JNK activation and apoptosis. These data indicate that SOCS1 is important to the prevention of hepatocyte apoptosis induced by Fas and TNF-α. In contrast, SOCS1 overexpression in the liver by adenoviral gene transfer prevented ConA-induced fiver injury. Conclusion: These findings indicate that SOCS1 plays important negative roles in fulminant hepatitis and that forced expression of SOCS1 is therapeutic in preventing hepatitis. Copyright © 2008 by the American Association for the Study of Liver Diseases.

It has been shown that transforming growth factor β1 (TGF-β1) is critical in the generation of CD4+CD25+Foxp3 +-inducible regulatory T cells (iTregs) from naïve CD4 +T cells. However, in contrast to natural Tregs, TGF-β1-induced iTregs rapidly lose both Foxp3 expression and suppression activity. We found that TGF-β1-induced Foxp3 levels were maintained by the addition of the anti-interleukin 4 (IL-4) antibody or by STAT6 gene deletion. Thus, IL-4 is an important suppressor of Foxp3 induction, and T helper 2 development is a major cause for the disappearance of iTreg during long culture. Using promoter analysis in EL4 cells and primary T cells, we identified a silencer region containing a STAT6 binding site. STAT6 binding to this site reduced TGF-β1-mediated Foxp3 promoter activation and chromatin modification. Retinoic acid has also been shown to suppress loss of Foxp3 induced by TGF-β1. Retinoic acid in the presence of TGF-β1 reduced STAT6 binding to the Foxp3 promoter and enhanced histone acetylation, thereby reverting the effect of IL-4. We propose that antagonistic agents for neutralizing IL-4 could be a novel strategy to facilitate inducible Treg cell generation and the promotion of tolerance in Th2-dominated diseases such as allergy. © 2008 by The American Society for Biochemistry and Molecular Biology, Inc.

Suppressor of cytokine signaling 1 (SOCS1) is an important negative regulator for cytokines; however, the role of SOCS1 in Th17 differentiation has not been clarified. We generated T cell-specific SOCS1-deficient mice and found that these mice were extremely resistant to a Th17-dependent autoimmune disease model, experimental autoimmune encephalomyelitis. SOCS1-deficient naive CD4 + T cells were predominantly differentiated into Th1 and poorly into Th17 in vitro. These phenotypes were canceled in IFN-γ-/- background, suggesting that a large amount of IFN-γ in SOCS1-deficient T cells suppressed Th17 differentiation. IL-6 plus TGF-β enhanced retinoic acid receptor-related orphan receptor (ROR)-γt expression and suppressed IFN-γ production in wild-type T cells, whereas these effects were severely impaired in SOCS1-deficient T cells. These phenotypes can be partly explained by STAT3 suppression by enhanced SOCS3 induction through hyper-STAT1 activation in SOCS1-deficient T cells. In addition, SOCS1-deficient T cells were much less sensitive to TGF-β. Suppression of Th1 differentiation by TGF-β was impaired in SOCS1-deficient T cells. TGF-β-mediated Smad transcriptional activity was severely inhibited in SOCS1-deficient cells in the presence of IFN-γ. Such impairment of TGF-γ functions were not observed in SOCS3-overexpressed cells, indicating that suppression of Smads was independent of SOCS3. Therefore, SOCS1 is necessary for Th17 differentiation by suppressing antagonistic effect of IFN-γ on both STAT3 and Smads. Induction of SOCS3 can partly explain IFN-γ-mediated STAT3 suppression, while other mechanism(s) will be involved in IFN-β-mediated Smad suppression. SOCS1-deficient T cells will be very useful to investigate the molecular mechanism for the STAT1-mediated suppression of Th17 development. Copyright © 2008 by The American Association of Immunologists, Inc.

IL-17-producing CD4+ T cells, so called Th17 cells, constitute a newly identified inflammatogenic cell population, which is critically involved in some inflammatory diseases. To explore the role of Th17 cells in murine experimental autoimmune uveoretinitis (EAU), a model of human autoimmune uveitis where Th1 responses predominantly participate in the pathogenesis, IL-17- mice were immunized with interphotoreceptor retinoid-binding protein peptide 1-20 for disease induction. Funduscopic examination revealed that EAU was induced in IL-17- mice just like in wild-type (WT) mice at early phases of the disease. However, at later/maintenance phases, the severity was significantly reduced in IL-17- mice. Expression of IFN-γ and MCP-1 was comparable between WT and IL-17- mice during the time course. In vivo blockade of IFN-γ and IL-4 resulted in exacerbation of EAU at later phases with augmented IL-17 production. Taken together, our data demonstrated that IL-17/Th17 participates in the late phases of EAU and also that Th1 and Th17 responses are differentially required for EAU. © The Japanese Society for Immunology. 2007. All rights reserved.

In macrophages and monocytes, microbial components trigger the production of pro-inflammatory cytokine through Toll-like receptors (TLRs). Although major TLR signaling pathways are mediated by serine/ threonine kinases, including TAK1, IKK and MAP kinases, tyrosine phosphorylation of intracellular proteins by TLR ligands has been suggested in a number of reports. Here, we demonstrated that peptidoglycan (PGN) of a Gram-positive bacterial cell wall component, a TLR2 ligand and lipopoysaccharide (LPS) of a Gram-positive bacterial component, a TLR4 ligand induced tyrosine phosphorylation of phospholipase Cγ-2 (PLCγ2), leading to intracellular free Ca2+ mobilization in bone marrow-derived macrophages (BMMφ) and bone marrow-derived dendritic cells (BMDC). PGN- and LPS-induced Ca2+ mobilization was not observed in BMDC from PLCγ2 knockout mice. Thus, PLCγ2 is essential for TLR2 and TLR4-mediated Ca2+ flux. In PLCγ2-knockdown cells, PGN-induced IκB-α phosphorylation and p38 activation were reduced. Moreover, PLCγ2 was necessary for the full production of TNF-α and IL-6. These data indicate that the PLCγ2 pathway plays an important role in bacterial ligands-induced activation of macrophages and dendritic cells. Journal compilation © 2008 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.

Human immunodeficiency virus type 1 (HIV-1) utilizes the macromolecular machinery of the infected host cell to produce progeny virus. The discovery of cellular factors that participate in HIV-1 replication pathways has provided further insight into the molecular basis of virus-host cell interactions. Here, we report that the suppressor of cytokine signaling 1 (SOCS1) is an inducible host factor during HIV-1 infection and regulates the late stages of the HIV-1 replication pathway. SOCS1 can directly bind to the matrix and nucleocapsid regions of the HIV-1 p55 Gag polyprotein and enhance its stability and trafficking, resulting in the efficient production of HIV-1 particles via an IFN signaling-independent mechanism. The depletion of SOCS1 by siRNA reduces both the targeted trafficking and assembly of HIV-1 Gag, resulting in its accumulation as perinuclear solid aggregates that are eventually subjected to lysosomal degradation. These results together indicate that SOCS1 is a crucial host factor that regulates the intracellular dynamism of HIV-1 Gag and could therefore be a potential new therapeutic target for AIDS and its related disorders. © 2008 by The National Academy of Sciences of the USA.

Human T cell leukemia virus type 1 (HTLV-1) Tax is an oncoprotein that plays a crucial role in the proliferation and transformation of HTLV-1-infected T lymphocytes. It has recently been reported that Tax activates a MAPKKK family, TAK1. However, the molecular mechanism of Tax-mediated TAK1 activation is not well understood. In this report, we investigated the role of TAK1-binding protein 2 (TAB2) in Tax-mediated TAK1 activation. We found that TAB2 physically interacts with Tax and augments Tax-induced NF-κB activity. Tax and TAB2 cooperatively activate TAK1 when they are coexpressed. Furthermore, TAK1 activation by Tax requires TAB2 binding as well as ubiquitination of Tax. We also found that the overexpression of TRAF2, 5, or 6 strongly induces Tax ubiquitination. These results suggest that TAB2 may be critically involved in Tax-mediated activation of TAK1 and that NF-κB-activating TRAF family proteins are potential cellular E3 ubiquitin ligases toward Tax. © 2007 Elsevier Inc. All rights reserved.

Cleft palate is one of the most common craniofacial deformities. The fibroblast growth factor (FGF) plays a central role in reciprocal interactions between adjacent tissues during palatal development, and the FGF signaling pathway has been shown to be inhibited by members of the Sprouty protein family. In this study, we report the incidence of cleft palate, possibly caused by failure of palatal shelf elevation, in Sprouty2-deficient (KO) mice. Sprouty2-deficient palates fused completely in palatal organ culture. However, palate mesenchymal cell proliferation estimated by Ki-67 staining was increased in Sprouty2 KO mice compared with WT mice. Sprouty2-null palates expressed higher levels of FGF target genes, such as Msx1, Etv5, and Ptx1 than WT controls. Furthermore, proliferation and the extracellular signal-regulated kinase (Erk) activation in response to FGF was enhanced in palate mesenchymal cells transfected with Sprouty2 small interfering RNA. These results suggest that Sprouty2 regulates palate mesenchymal cell proliferation via FGF signaling and is involved in palatal shelf elevation. © 2010 Elsevier Inc.

Cleft palate is one of the most common craniofacial deformities. The fibroblast growth factor (FGF) plays a central role in reciprocal interactions between adjacent tissues during palatal development, and the FGF signaling pathway has been shown to be inhibited by members of the Sprouty protein family. In this study, we report the incidence of cleft palate, possibly caused by failure of palatal shelf elevation, in Sprouty2-deficient (KO) mice. Sprouty2-deficient palates fused completely in palatal organ culture. However, palate mesenchymal cell proliferation estimated by Ki-67 staining was increased in Sprouty2 KO mice compared with WT mice. Sprouty2-null palates expressed higher levels of FGF target genes, such as Msx1, Etv5, and Ptx1 than WT controls. Furthermore, proliferation and the extracellular signal-regulated kinase (Erk) activation in response to FGF was enhanced in palate mesenchymal cells transfected with Sprouty2 small interfering RNA. These results suggest that Sprouty2 regulates palate mesenchymal cell proliferation via FGF signaling and is involved in palatal shelf elevation. © 2010 Elsevier Inc.

The glial cell line-derived neurotrophic factor (GDNF)/RET tyrosine kinase signaling pathway plays crucial roles in the development of the enteric nervous system (ENS) and the kidney. Tyrosine 1062 (Y1062) in RET is an autophosphorylation residue that is responsible for the activation of the PI3K/AKT and RAS/MAPK signaling pathways. Mice lacking signaling via Ret Y1062 show renal hypoplasia and hypoganglionosis of the ENS although the phenotype is milder than the Gdnf- or Ret-deficient mice. Sprouty2 (Spry2) was found to be an antagonist for fibroblast growth factor receptor (FGFR) and acts as an inhibitory regulator of ERK activation. Spry2-deficient mice exhibit hearing loss and enteric nerve hyperplasia. In the present study, we generated Spry2-deficient and Ret Y1062F knock-in (tyrosine 1062 is replaced with phenylalanine) double mutant mice to see if abnormalities of the ENS and kidney, caused by loss of signaling via Ret Y1062, are rescued by a deficiency of Spry2. Double mutant mice showed significant recovery of ureteric bud branching and ENS development in the stomach. These results indicate that Spry2 regulates downstream signaling mediated by GDNF/RET signaling complex in vivo. © 2010 Elsevier Inc.