Identification of TMEM131L as a Novel Regulator of Thymocyte Proliferation in Humans This information is current as Nesrine Maharzi, Véronique Parietti, Elisabeth Nelson, of September 25, 2021. Simona Denti, Macarena Robledo-Sarmiento, Niclas Setterblad, Aude Parcelier, Marika Pla, François Sigaux, Jean Claude Gluckman and Bruno Canque J Immunol 2013; 190:6187-6197; Prepublished online 20 May 2013; Downloaded from doi: 10.4049/jimmunol.1300400 http://www.jimmunol.org/content/190/12/6187 Supplementary http://www.jimmunol.org/content/suppl/2013/05/21/jimmunol.130040 http://www.jimmunol.org/ Material 0.DC1 References This article cites 44 articles, 20 of which you can access for free at: http://www.jimmunol.org/content/190/12/6187.full#ref-list-1 Why The JI? Submit online. by guest on September 25, 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 © 2013 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Identification of TMEM131L as a Novel Regulator of Thymocyte Proliferation in Humans Nesrine Maharzi,*,†,‡ Ve´ronique Parietti,*,†,‡ Elisabeth Nelson,*,†,‡ Simona Denti,*,†,‡ Macarena Robledo-Sarmiento,*,†,‡ Niclas Setterblad,x Aude Parcelier,*,†,‡ Marika Pla,{ Franc¸ois Sigaux,*,†,‡ Jean Claude Gluckman,*,†,‡ and Bruno Canque*,†,‡ In this study, we identify transmembrane protein 131–like (TMEM131L) as a novel regulator of thymocyte proliferation and demonstrate that it corresponds to a not as yet reported inhibitor of Wnt signaling. Short hairpin RNA–mediated silencing of TMEM131L in human CD34+ hematopoietic progenitors, which were then grafted in NOD-SCID/IL-2rgnull mice, resulted in both thymocyte hyperproliferation and multiple pre– and post–b-selection intrathymic developmental defects. Consistent with deregu- lated Wnt signaling, TMEM131L-deficient thymocytes expressed Wnt target genes at abnormally high levels, and they displayed both constitutive phosphorylation of Wnt coreceptor LRP6 and b-catenin intranuclear accumulation. Using T cell factor reporter Downloaded from assays, we found that membrane-associated TMEM131L inhibited canonical Wnt/b-catenin signaling at the LRP6 coreceptor level. Whereas membrane-associated TMEM131L did not affect LRP6 expression under basal conditions, it triggered lysosome- dependent degradation of its active phosphorylated form following Wnt activation. Genetic mapping showed that phosphorylated LRP6 degradation did not depend on TMEM131L cytoplasmic part but rather on a conserved extracellular domain proximal to the membrane. Collectively, these data indicate that, during thymopoiesis, stage-specific surface translocation of TMEM131L may regulate immature single-positive thymocyte proliferation arrest by acting through mixed Wnt-dependent and -independent http://www.jimmunol.org/ mechanisms. The Journal of Immunology, 2013, 190: 6187–6197. ollowing extravasation from the blood, early thymus mature pro–double-negative (DN)1 human thymocytes (CD34hi 2 immigrants establish lymphostromal synapses with cortical CD7++CD1a ) correlates with both the onset of cell proliferation thymic epithelial cells, which triggers proliferation and and upregulation of T lineage–affiliated genes (5). Downstream F int + + 2 drives their specification along the T cell lineage through the si- transition from the DN1 (CD34 CD7 CD5 CD1a ) to DN2 multaneous activation of the IL-7R, c-Kit, sonic hedgehog, Wnt/ (CD34loCD7+CD5+CD1a+) stage coincides with definitive T lin- LEF/T cell factor (TCF), and Notch signaling pathways (1–3). eage commitment and initiation of TCRb D-J rearrangements (6). by guest on September 25, 2021 2 2 Most current evidence suggests that activation of the Frizzled/ Subsequently, thymocytes reach the DN3a (CD34 CD4intCD8a ) LRP6 receptor complex by its cognate ligands (Wnt1–4) reg- stage, when they stop proliferating to complete V-DJb rearrange- ulates survival and proliferation of early thymus immigrants, ment, and pass through the b-selection checkpoint to become 2 whereas Notch1 ligation by Delta-like 4 also drives commitment DN3b thymocytes (CD34 CD4intCD8a+) (3, 7). Later on, post–b- toward the T lineage (4). Acquisition of CD5 by the most im- selection DN3b thymocytes further upregulate CD4, acquire ex- pression of CD8b-chain, and reach the double-positive (DP) stage where rearrangement of the TCRa locus takes place. Although most *Laboratoire De´veloppement du Syste`me Immunitaire de l’Ecole Pratique des evidence indicates that simultaneous termination of Wnt and Notch Hautes Etudes, Institut Universitaire d’He´matologie, Hoˆpital Saint-Louis, 75571 signaling plays an important role in DN3a proliferation arrest, the Paris, France; †INSERM Unite´ 944, Institut Universitaire d’He´matologie, Hoˆpital Saint-Louis, 75571 Paris, France; ‡Universite´ de Paris 7/Centre National de la underlying mechanisms remain poorly defined. There is evidence Recherche Scientifique Unite´ Mixte de Recherche 7212, Institut Universitaire that E proteins (E2A, HEB) play an important role in DN3a pro- d’He´matologie, Hoˆpital Saint-Louis, 75571 Paris, France; xPlateforme Technologi- que, Institut Universitaire d’He´matologie, Hoˆpital Saint-Louis, 75571 Paris, France; liferation arrest, but whether they interfere with Notch or Wnt and {Unite´ Mixte de Recherche en Sante´ 940, Institut Universitaire d’He´matologie, signaling has not yet been investigated (8–11). Ikaros/IKZF1 also Hoˆpital Saint-Louis, 75571 Paris, France participate in the process through both induction of CDKN1B/ Received for publication February 11, 2013. Accepted for publication April 4, 2013. p27KIP1 (12) and interruption of Notch signaling following binding This work was supported by INSERM, the Association pour la Recherche contre le to proximal RBPJ-responsive elements in the HES1 promoter (13– Cancer, the Comite´ de Paris de la Ligue Nationale contre le Cancer, and by the Ecole 16). The mechanisms of Wnt signaling downmodulation in DN3a Pratique des Hautes Etudes. thymocytes are less well characterized. It has been shown that Address correspondence and reprint requests to Prof. Bruno Canque, Institut Uni- versitaire d’He´matologie, Centre Hayem, 1 Avenue Claude Vellefaux, 75475 Paris a p53-dependent ubiquitin E3 ligase complex involving Siah1, SIP Cedex 10, France. E-mail address: [email protected] (CacyBP), Skp1, and Ebi promotes b-catenin degradation in pre–b- The online version of this article contains supplemental material. selection thymocytes (17). However, that SIP-deficient pre–b- Abbreviations used in this article: BM, bone marrow; CDS, coding sequence; CHD, selection thymocytes display increased susceptibility to apo- conserved homology domain; DN, double-negative; DP, double-positive; ECD, ex- ptosis does not support the view that this pathway could play an tracellular domain; ER, endoplasmic reticulum; HPC, hematopoietic progenitor cell; kd, knocked down; L, long; MA-ICD, membrane-anchored intracellular domain; important role in thymocyte proliferation. Consistent with this NSG, NOD-scid/gc2/2; RT-qPCR, quantitative RT-PCR; S, short; shRNA, short hairpin view, conditional ablation of b-catenin does not affect thymocyte RNA; SP, single-positive; TCF, T cell factor; TMEM131L, transmembrane protein proliferation or differentiation (18, 19). 131–like. In this study, we identify transmembrane protein 131–like Copyright Ó 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00 (TMEM131L) as a negative regulator of thymocyte proliferation www.jimmunol.org/cgi/doi/10.4049/jimmunol.1300400 6188 TMEM131L REGULATES HUMAN INTRATHYMIC PROLIFERATION and provide evidence that it corresponds to a proximal inhibitor of (all from BD Biosciences) or CD8-PE-Cy7 (BioLegend) mAbs. The in- the canonical Wnt pathway with which it interferes through in- tracellular TCRb-chain was detected with the TCRbF1-allophycocyanin duction of lysosome-dependent degradation of the active phos- (Cytognos) mAb and the Cytofix/Cytoperm kit (BD Biosciences). For cell proliferation analysis, mice were injected i.p. with 1 mg BrdU 16 and 10 h phorylated form of the LRP6 coreceptor. before analysis. Cell surface proteins were labeled as above; cells that had incorporated BrdU were detected using a BrdU flow kit (BD Biosciences). Materials and Methods Data were analyzed using FlowJo software. HL60 cells transduced with a lentiviral CMV-Luc reporter (Addgene) Plasmid constructs and cell protein analysis were injected s.c. in nonirradiated NSG mice. Longitudinal follow-up by Full-length TMEM131L coding sequence (CDS) was obtained from the in vivo optical imaging for assessing luciferase activity using the IVIS Deutsches Ressourcenzentrum fu¨r Genomforschung; long (L) and short (S) Spectrum bioluminescence-fluorescence optical imaging system (Caliper isoform CDS were subcloned in frame with the Flag epitope into a