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Human T Lymphotropic Virus Type 1 Increases T Lymphocyte Migration by Recruiting the Cytoskeleton Organizer CRMP2 This information is current as of September 28, 2021. Michel Varrin-Doyer, Adeline Nicolle, Romain Marignier, Sylvie Cavagna, Claire Benetollo, Eric Wattel and Pascale Giraudon J Immunol 2012; 188:1222-1233; Prepublished online 6 January 2012; Downloaded from doi: 10.4049/jimmunol.1101562 http://www.jimmunol.org/content/188/3/1222 http://www.jimmunol.org/ Supplementary http://www.jimmunol.org/content/suppl/2012/01/06/jimmunol.110156 Material 2.DC1 References This article cites 83 articles, 31 of which you can access for free at: http://www.jimmunol.org/content/188/3/1222.full#ref-list-1 Why The JI? Submit online. by guest on September 28, 2021 • 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 *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 © 2012 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Human T Lymphotropic Virus Type 1 Increases T Lymphocyte Migration by Recruiting the Cytoskeleton Organizer CRMP2 Michel Varrin-Doyer,*,† Adeline Nicolle,*,† Romain Marignier,*,† Sylvie Cavagna,*,† Claire Benetollo,*,† Eric Wattel,‡ and Pascale Giraudon*,† Recruitment of virus-infected T lymphocytes into the CNS is an essential step in the development of virus-associated neuro- inflammatory diseases, notably myelopathy induced by retrovirus human T leukemia virus-1 (HTLV-1). We have recently shown the key role of collapsin response mediator protein 2 (CRMP2), a phosphoprotein involved in cytoskeleton rearrangement, in the control of human lymphocyte migration and in brain targeting in animal models of virus-induced neuroinflammation. Using lymphocytes cloned from infected patients and chronically infected T cells, we found that HTLV-1 affects CRMP2 activity, Downloaded from resulting in an increased migratory potential. Elevated CRMP2 expression accompanies a higher phosphorylation level of CRMP2 and its more pronounced adhesion to tubulin and actin. CRMP2 forms, a full length and a shorter, cleaved one, are also affected. Tax transfection and extinction strategies show the involvement of this viral protein in enhanced full-length and active CRMP2, resulting in prominent migratory rate. A role for other viral proteins in CRMP2 phosphorylation is suspected. Full-length CRMP2 confers a migratory advantage possibly by preempting the negative effect of short CRMP2 we observe on T lymphocyte migration. In addition, HTLV-1–induced migration seems, in part, supported by the ability of infected cell to increase the http://www.jimmunol.org/ proteosomal degradation of short CRMP2. Finally, gene expression in CD69+ cells selected from patients suggests that HTLV-1 has the capacity to influence the CRMP2/PI3K/Akt axis thus to positively control cytoskeleton organization and lymphocyte migration. Our data provide an additional clue to understanding the infiltration of HTLV-1–infected lymphocytes into various tissues and suggest that the regulation of CRMP2 activity by virus infection is a novel aspect of neuroinflammation. The Journal of Immunology, 2012, 188: 1222–1233. ymphocyte infiltration in the CNS is at the basis of neu- paraparesis (HAM/TSP). This human retrovirus mainly targets roinflammatory diseases, including those associated with CD4 and CD8 T lymphocytes and is the etiological agent of both by guest on September 28, 2021 virus infection. Besides CNS infection, infected immune adult T cell leukemia (ATL) (2) and HAM/TSP (3). The pre- L + + cells can affect neurons and glial cells through the bystander ef- dominant viral reservoir in the peripheral blood is CD4 CD25 fect of secreted inflammatory molecules, leading progressively to T cells (4). How HTLV-1 causes neurologic disorders is not demyelination and axonal loss (1). Dissection of the complex completely understood, but several works have focused on the mechanisms that control infiltration and migration of infected crucial role of infected T cells in pathogenesis. It is thought that, T lymphocytes into the CNS is of importance in understanding in HAM/TSP patients, HTLV-1–infected lymphocytes migrate the pathophysiology of virus-induced neuroinflammatory diseases. from the periphery into the CNS, because lymphocytes of the One prototype of such a disease is human T cell lymphotropic cerebrospinal fluid and PBMC shared the same HTLV-1 integra- virus type-1 (HTLV-1)–associated myelopathy/tropical spastic tion site in cellular DNA (5). Detection of HTLV-1 genome and the viral protein Tax in inflammatory T lymphocytes infiltrated in *INSERM U1028, Centre National de la Recherche Scientifique, Unite´ Mixte de the patients’ CNS has identified T lymphocytes as the main virus Recherche 5292, Centre de Recherche en Neuroscience de Lyon, Equipe Neuroon- reservoir in the brain and the main effectors of pathological cologie-Neuroinflammation, F-69000 Lyon, France; †University of Lyon, F-69000 Lyon, France; and ‡Oncovirologie et Biothe´rapies, Universite´ Claude Bernard Lyon 1, changes (6–10). In fact, Tax has the ability to promote or alter the Centre National de la Recherche Scientifique, Unite´ Mixte de Recherche 5239, expression and activity of several cellular factors involved in Centre Le´on Be´rard, 69008 Lyon, France, France T cell behavior (11–13), as such, may contribute to pathogenic Received for publication May 27, 2011. Accepted for publication November 30, mechanism. In this context, we have previously shown that HTLV- 2011. 1–infected T cells disturb the metabolism of astrocytes through This work was supported by Agence Nationale de la Recherche and Agence de Recherche sur La Scle´rose en Plaques. the activity of TNF-a (14). Secreted by infected T lymphocytes, Address correspondence and reprint requests to Dr. Pascale Giraudon, INSERM TNF-a reduced the glutamate transporter EAAT2/GLT1 at as- U1028, Centre National de la Recherche Scientifique, Unite´ Mixte de Recherche trocyte membrane and, consequently, increased the extracellular 5292, Lyon Neuroscience Research Center, Faculty of Medicine Laennec, Rue Guil- level of glutamate, an excitotoxic amino acid deleterious for laume Paradin, 69372 Lyon, France. E-mail address: [email protected] neurons and oligodendrocytes at a high level. Other virus proteins The online version of this article contains supplemental material. are also involved in pathogenesis, notably the p8 protein, that Abbreviations used in this article: ATL, adult T cell leukemia; BMP, bone morpho- genetic protein; CRMP2, collapsin response mediator protein 2; HAM/TSP, human promote T cell contact and enhance virus transmission (15). Given T leukemia virus 1-associated myelopathy/tropical spastic paraparesis; HD, healthy the crucial role of infected T lymphocytes in pathogenesis, facil- blood donor; HTLV-1, human T leukemia virus 1; pep4, peptide 4; qPCR, quantita- itation of their recruitment into the CNS through changes induced tive PCR; shRNA, short hairpin RNA; siRNA, small interfering RNA. by HTLV-1 in the cell machinery of locomotion could be an im- Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00 portant phase of the disease. www.jimmunol.org/cgi/doi/10.4049/jimmunol.1101562 The Journal of Immunology 1223 During neuroinflammation, leukocytes emigrate from the blood- microbeads. Cells were washed, suspended in 500 ml medium, and put 4 stream to the CNS across the blood–brain barriers through ei- through magnetic columns. Selected cells (1–5 3 10 cells) were used ther endothelial cells of inflamed meningeal veins or epithelial either for RNA amplification and microarray analysis, for quantitative PCR (qPCR) analysis on unamplified RNA, or for transmigration assays. cells of the choroids plexuses (16, 17). The continuous T lym- phocyte extravasation, migration into, and crawling within neural Antibodies tissue are orchestrated by the interplay of multiple adhesion re- CRMP2-A/B, generally termed CRMP2, was detected using purified ceptors whose expression and avidity are modulated by cytokines polyclonal rabbit Abs directed against peptide 4 (pep4) and C-ter epitopes, and chemokines (18–20). Activation of G protein-coupled receptor as previously described (29) and monoclonal C4G Ab from IBL-Japan. by chemokines induces a motile phenotype characterized by the CRMP2-C-ter and C4G Abs recognized only the full-length CRMP2. CRMP2–pep-4 Ab recognized both the full-length (62 kDa) and cleaved formation of lamellipodia at the front of migration and uropod at (58 kDa) CRMP2. Polyclonal anti–CRMP2-A Ab was described previ- the rear of T lymphocyte (21, 22). In parallel, a drastic reconfigu- ously (29). Ab specific of phosphorylated CRMP2 forms CRMP2-pSer522 ration of the microtubule and intermediate filament cytoskeletons and CRMP2-pThr509/514 was from Kinasource (Dundee, U.K.); anti– allows lymphocyte locomotion. However, the mechanism associ-
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  • Discovery of Potent and Selective MRCK Inhibitors with Therapeutic

    Discovery of Potent and Selective MRCK Inhibitors with Therapeutic

    Published OnlineFirst January 30, 2018; DOI: 10.1158/0008-5472.CAN-17-2870 Cancer Translational Science Research Discovery of Potent and Selective MRCK Inhibitors with Therapeutic Effect on Skin Cancer Mathieu Unbekandt1, Simone Belshaw2, Justin Bower2, Maeve Clarke2, Jacqueline Cordes2, Diane Crighton2, Daniel R. Croft2, Martin J. Drysdale2, Mathew J. Garnett3, Kathryn Gill2, Christopher Gray2, David A. Greenhalgh4, James A.M. Hall3, Jennifer Konczal2, Sergio Lilla5, Duncan McArthur2, Patricia McConnell2, Laura McDonald2, Lynn McGarry6, Heather McKinnon2, Carol McMenemy4, Mokdad Mezna2, Nicolas A. Morrice5, June Munro1, Gregory Naylor1, Nicola Rath1, Alexander W. Schuttelkopf€ 2, Mairi Sime2, and Michael F. Olson1,7 Abstract The myotonic dystrophy–related Cdc42-binding kinases an autophosphorylation site, enabling development of a phos- MRCKa and MRCKb contribute to the regulation of actin–myosin phorylation-sensitive antibody tool to report on MRCKa status in cytoskeleton organization and dynamics, acting in concert with tumor specimens. In a two-stage chemical carcinogenesis model the Rho-associated coiled-coil kinases ROCK1 and ROCK2. The of murine squamous cell carcinoma, topical treatments reduced absence of highly potent and selective MRCK inhibitors has MRCKa S1003 autophosphorylation and skin papilloma out- resulted in relatively little knowledge of the potential roles of growth. In parallel work, we validated a phospho-selective anti- these kinases in cancer. Here, we report the discovery of the body with the capability to monitor drug pharmacodynamics. azaindole compounds BDP8900 and BDP9066 as potent and Taken together, our findings establish an important oncogenic selective MRCK inhibitors that reduce substrate phosphorylation, role for MRCK in cancer, and they offer an initial preclinical proof leading to morphologic changes in cancer cells along with inhi- of concept for MRCK inhibition as a valid therapeutic strategy.