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Cells RI-Dependent Signal Transduction in Mast Ε Rhoh Plays RhoH Plays Critical Roles in Fcε RI-Dependent Signal Transduction in Mast Cells This information is current as Hiroyo Oda, Manabu Fujimoto, Michael S. Patrick, Dai of October 2, 2021. Chida, Yoshinori Sato, Yoshinao Azuma, Hiroki Aoki, Takaya Abe, Harumi Suzuki and Mutsunori Shirai J Immunol 2009; 182:957-962; ; doi: 10.4049/jimmunol.182.2.957 http://www.jimmunol.org/content/182/2/957 Downloaded from References This article cites 45 articles, 13 of which you can access for free at: http://www.jimmunol.org/content/182/2/957.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on October 2, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2009 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology RhoH Plays Critical Roles in Fc␧RI-Dependent Signal Transduction in Mast Cells1 Hiroyo Oda,* Manabu Fujimoto,† Michael S. Patrick,*‡ Dai Chida,* Yoshinori Sato,* Yoshinao Azuma,‡ Hiroki Aoki,§ Takaya Abe,¶ Harumi Suzuki,2* and Mutsunori Shirai‡ RhoH is an atypical small G protein with defective GTPase activity that is specifically expressed in hematopoietic lineage cells. RhoH has been implicated in regulation of several physiological processes including hematopoiesis, integrin activation, and T cell differentiation and activation. In the present study, we investigated the role of RhoH in mast cells by generating RhoH knockout mice. Despite observing normal development of mast cells in vivo, passive systemic anaphylaxis and histamine release were impaired in these mice. We also observed defective degranulation and cytokine production upon Fc␧RI ligation in RhoH-deficient bone marrow-derived mast cells. Furthermore, Fc␧RI-dependent activation of Syk and phosphorylation of its downstream targets, including LAT, SLP76, PLC␥1, and PLC␥2 were impaired, however phosphorylation of the ␥-subunit of Fc␧RI remained intact. Downloaded from We also found RhoH-Syk association that was greatly enhanced by active Fyn. Our results indicate that RhoH regulates Fc␧RI signaling in mast cells by facilitating Syk activation, possibly as an adaptor molecule for Syk. The Journal of Immunology, 2009, 182: 957–962. hoH is a newly identified hematopoietic small G protein, tion of RhoH in other hematopoietic lineage cells is largely originally cloned as one of the genes frequently disrupted unknown. R in diffuse large B cell lymphoma (1, 2). Because RhoH is Mast cells are widely distributed in the body and function as the http://www.jimmunol.org/ defective in GTPase activity and thus constitutively active, the primary effectors in immediate-type hypersensitivity reactions (9, function of this protein was thought to be regulated by its expres- 10). Mast cells recognize Ags via IgE and specific, high-affinity Fc sion level. Overexpression of RhoH inhibited RhoA/Rac/cdc42- receptors, termed Fc␧RI (11–13). Fc␧RI cross-linking triggers ac- ␬ dependent NF- B activation in HEK293 cells (3), and it also in- tivation of Src family kinases Lyn and Fyn, and phosphorylation of hibited SCF-mediated Rac1 activation in bone marrow progenitor ITAM motifs on the ␥ subunit of Fc␧RI complexes (14, 15). Sub- cells (4). Knockdown of RhoH increased spontaneous LFA-1-me- sequently, ZAP-70-related Syk kinase binds to phosphorylated diated adhesion in Jurkat cells (5), and in vitro colony formation in ITAM motifs of the ␥ subunit and is thus activated by Src kinases bone marrow progenitor cells (4). Recent studies, however, have by guest on October 2, 2021 (16–18). Activated Syk, in turn, phosphorylates LAT, LAT-2, and demonstrated that RhoH plays critical roles in T cell development SLP-76 to form the signalosome, which transduces signals down- (6, 7) by functioning as an adaptor for ZAP-70 in TCR signaling stream, initiating Ca2ϩ mobilization, degranulation, and the ex- (7) via its tyrosine-phosphorylated ITAM-like motif (8). In the ␧ absence of RhoH, development of T cells in the thymus is impaired pression of specific genes (19–21). The Fc RI-initiated signal cas- in both ␤-selection and positive selection, resulting in a severe cade in mast cells is analogous to the TCR-initiated signal cascade reduction of mature peripheral T cells (6, 7). RhoH-deficient T in T cells, sharing many common molecules and features (14, 15, ␧ cells showed defective phosphorylation of LAT and ERK upon 22). This prompted us to investigate the function of RhoH in Fc RI TCR stimulation, indicating that RhoH is critical in TCR-depen- signaling in mast cells. dent proximal signal transduction events. The precise function of In this study, we report the critical role of RhoH in mast cell RhoH in TCR signaling, however, remains controversial because activation. We established RhoH-deficient mice to unveil the phys- there is a discrepancy in the phosphorylation status of ZAP-70 iological roles of RhoH in mast cells. RhoH-deficient mice showed between two reports (6, 7). Furthermore, the physiological func- impaired passive systemic anaphylaxis (PSA)3 and histamine re- lease upon challenge with the specific Ag. Our in vitro data show- ing impaired Syk activation with defective degranulation and cy- *Department of Pathology, Research Institute, International Medical Center of Japan, tokine production in RhoHϪ/Ϫ mast cells supports the observed in Tokyo, Japan; †Department of Dermatology, Kanazawa University Graduate School of Medical Science, Kanazawa, Japan; ‡Department of Microbiology, Yamaguchi vivo phenotypes. Furthermore, we demonstrated that RhoH asso- University School of Medicine, Ube, Japan; §Cardiovascular Research Institute Ku- ciates with Syk, and this interaction was greatly enhanced in the rume University School of Medicine, Kurume, Japan; and ¶Laboratory for Animal Resources and Genetic Engineering, Center for Developmental Biology, RIKEN presence of constitutively active Fyn. These results suggest that Kobe, Japan RhoH acts as a positive regulator for Fc␧RI-mediated signal trans- Received for publication January 16, 2008. Accepted for publication November duction by facilitating Syk activation. 6, 2008. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology. 3 Abbreviations used in this paper: PSA, passive systemic anaphylaxis; BMMC, bone 2 Address correspondence and reprint requests to Dr. Harumi Suzuki, Department of marrow-derived mast cell; HSA, human serum albumin; HA, hemagglutinin. Pathology, Research Institute, International Medical Center of Japan, 1-21-1 Toyama, Shinjuku, Tokyo, Japan. E-mail address: [email protected] Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00 www.jimmunol.org 958 RhoH MEDIATES Fc␧RI-DEPENDENT SIGNAL TRANSDUCTION IN MAST CELLS Materials and Methods ctatggtactccagaa3Ј), TNF-␣ (5Јtcttctcattcctgcttgtgg3Ј/5Јggtctgggccatagaactga Ј ␤ Ј Ј Ј Ј Mice 3 ), and -actin (5 aaggccaaccgtgaaaagat3 /5 gtggtacgaccagaggcatac3 ). RhoH knockout mice (Acc.No.CDB0483K) were generated using the pre- Immunoblotting and immunoprecipitation viously described methods (23, 24). In brief, the entire ORF of the RhoH For immunoblotting, BMMCs were sensitized with 1 ␮g/ml anti-DNP IgE gene located in the third exon was replaced with a cassette consisting of a 6 lacZ and neomycin resistance gene (LacZ/neo) by homologous recombi- at 10 cells/ml for 4 h without IL-3, washed three times, and then stimu- nation. The lengths of the homologous regions in the targeting vector were lated with DNP-HSA for the indicated periods. Stimulated BMMCs were 8.2 kb and 4.8 kb at the 5Ј and 3Ј sides of the DT-A/LacZ/neo cassette lysed in lysis/wash buffer (1% Nonidet P-40, 150 mM NaCl, 50 mM Tris-Cl (pH 8.0), 1 mM sodium orthovanadate, 1 mM DTT, proteinase (http://www.cdb.riken.jp/arg/cassette.html), respectively. Two mutant 5 mouse lines were established from two independent homologous recom- inhibitors), subjected to 7.5–12.5% SDS-PAGE and Western blotting (10 ␥ 6 binant ES cell lines; no difference was found in their phenotype. All mice cells per lane). For FcR and Lyn, 10 BMMCs were lysed with lysis/wash ␮ were maintained after five generations of backcrossing to C57BL/6J (B6) buffer and immunoprecipitated with 1 g of the indicated Abs, then sub- and housed under specific pathogen-free conditions in accordance with jected to 12.5% SDS-PAGE in tricine-SDS running buffer (0.1 M Tris, 0.1 institutional guidelines. M Tricine, 0.1% SDS for the upper chamber, 0.2 M Tris-Cl (pH 8.9) for the lower chamber), and Western blotting. For RhoH and Syk, 4.5 ϫ 105 293T Mast cell culture cells were transiently transfected with HA-tagged RhoH, myc-tagged Syk with or without constitutively active Fyn using FuGENE 6 (Roche). After Bone marrow-derived mast cells (BMMCs) were prepared as described 24 h, transfected cells were incubated with or without 30 ␮M PP2 (src ϩ/ϩ (25). In brief, femurs were isolated from 8- to 20-wk-old RhoH and kinase inhibitor) for 2 h before lysis. After 2 h, cells were lysed with RhoHϪ/Ϫ mice, and BM cells were cultured in 2% conditioned RPMI 1640 magnesium containing lysis/wash buffer (1% NP40, 10 mM MgCl2,25 medium from X63-IL-3 cells (gift from Dr.
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