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Calreticulin Controlling the Membrane Translocation of Immunogenicity Of ERP57 Membrane Translocation Dictates the Immunogenicity of Tumor Cell Death by Controlling the Membrane Translocation of Calreticulin This information is current as of September 25, 2021. Michel Obeid J Immunol 2008; 181:2533-2543; ; doi: 10.4049/jimmunol.181.4.2533 http://www.jimmunol.org/content/181/4/2533 Downloaded from References This article cites 26 articles, 11 of which you can access for free at: http://www.jimmunol.org/content/181/4/2533.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 September 25, 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 © 2008 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology ERP57 Membrane Translocation Dictates the Immunogenicity of Tumor Cell Death by Controlling the Membrane Translocation of Calreticulin1 Michel Obeid2 Several pieces of experimental evidence indicate the following: 1) the most efficient antitumor treatments (this principle applies on both chemotherapy and radiotherapy) are those that induce immunogenic cell death and are able to trigger a specific antitumor immune response; and 2) the immunogenicity of cell death depends very closely on the plasma membrane quantity of calreticulin (CRT), an endoplasmic reticulum (ER) stress protein exposed to the cell membrane after immunogenic treatment. Nevertheless, the mechanisms implicated in CRT translocation are unknown. CRT is known to interact in the ER with ERP57, another ER stress protein. I sought to determine whether ERP57 would have any role in tumor immunogenicity. In this article I report that CRT Downloaded from exposure is controlled by ERP57 exposure. CRT and ERP57 are translocated together in the same molecular complex. ERP57 knockdown suppressed CRT exposure as well as phagocytosis by dendritic cells and abolished the immunogenicity in vivo. Knockdown or the absence of CRT abolishes ERP57 exposure. Administration of recombinant ERP57, unlike the admin- istration of recombinant CRT, did not restore the immunogenicity of CRT or ERP57 small interfering RNA-transfected tumor cells. Together, these studies identify ERP57 as a key protein that controls immunogenicity by controlling CRT exposure and illustrate the ability of ERP57 to serve as a new molecular marker of immunogenicity. The Journal of http://www.jimmunol.org/ Immunology, 2008, 181: 2533–2543. e have recently shown that the exposure of calreticu- in particular with the family of protein disulfide isomerases and lin (CRT)3 dictates the immunogenicity of cancer cell especially with the ERP57 protein (9). W death induced by chemotherapy compounds (1, 2), CRT has been reported to be secreted from cells (10, 11) and is gamma irradiation (3), or UVC (3). The cell surface (ecto)CRT present at low levels in human plasma (12). will act as an “eat me” signal and mediate the phagocytosis of Nevertheless, CRT has been found to be localized in various tumor cell death by dendritic cells (DCs). Thus, the principal con- subcellular compartments, including the cytosol, the nucleus, and sequences of this uptake will be the following: 1) an increase in Ag the cell surface membrane. The role of CRT at the cell surface is by guest on September 25, 2021 cross-presentation; 2) an increase in the number of specific CTLs; more and more clear. It has been reported that ectoCRT orches- 3) an increase in the production of IFN-␥ by T cells; and 4) the trates a number of cellular events including the modulation of cell eradication of implanted tumors (1, 2). adhesion and migration through interaction with integrins (13, 14) CRT is a 60-kDa chaperone Ca2ϩ-binding protein and is mainly and with the extracellular matrix proteins fibrinogen (15) and lami- ubiquitous in the endoplasmic reticulum (ER). CRT has also been nin (16). We have shown lately that ectoCRT on dying or dead recognized as a multifunctional protein involved in a wide variety tumor cells represent an “eat me signal” for DCs as well as a major of cellular processes including modulation of cell adhesion (4), the molecular determinant that makes the difference between immu- folding of newly synthesized glycoproteins (5–7), modulation of nogenic cell death and nonimmunogenic cell death (1, 3, 17). gene expression (5), the regulation of Ca2ϩ homeostasis and Ca2ϩ- However, the mechanisms by which CRT is translocated to the dependent pathways (8), and lectin-like chaperone activity (7). In- surface is still unknown. Because endoCRT can interact in the ER tracellular (endo)CRT can interact with many ER resident proteins, lumen with ERP57 and because ERP57 membrane translocation was induced by anthracyclines, I examined whether CRT and ERP57 are translocated together to the cell surface in the same GenCal, Paris, France molecular complex and whether ERP57 has any role in the induc- Received for publication April 15, 2008. Accepted for publication June 11, 2008. ing of immunogenic cell death. At this point, I identified another The costs of publication of this article were defrayed in part by the payment of page particular biochemical alteration in the plasma membrane of im- charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. munogenic dying cells, the surface exposure of ERP57, as a con- 1 This work was supported in part by a grant from GenCal and by a fellowship to comitant and determining event to CRT exposure that only occurs M.O. from Association pour la Recherche sur le Cancer (ARC). in immunogenic tumor cell death. Accordingly, I show that the M.O. performed the in vivo and in vitro experiments, conducted the data analysis, ERP57 protein does not itself possess any immunogenic propri- conceived and designed the study and wrote the manuscript. eties but could dictate indirectly the immunogenicity of tumor cell 2 Address correspondence and reprint requests to Dr. Michel Obeid, GenCal, 43 Rue death by controlling the translocation of CRT. Violet, F-75015 Paris, France. E-mail address: [email protected] 3 Abbreviations used in this paper: CRT, calreticulin; BM, bone marrow; DC, den- dritic cell; ecto, cell surface; endo, intracellular; ER, endoplasmic reticulum; Materials and Methods GADD34, growth arrest and DNA damage inducible protein 34; PP1, protein phos- Cell lines and cell death inducers phatase 1; siRNA, small interfering RNA; z-VAD-fmk, N-benzyloxycarbonyl-Val- Ala-Asp-fluoromethyl ketone. The concentrations and times used in vitro for the treatment of CT26 cells with the different substances were established in preliminary dose-response ϳ Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00 experiments designed to determine the LC75, the dose that kills 75% of www.jimmunol.org 2534 ERP57 IN IMMUNOGENIC CANCER CELL DEATH the cells. Therefore, CT26 cells were cultured at 37°C under 5% CO2 in densitometry. Anti-actin or anti-GAPDH Ab (Chemicon) was used to con- RPMI 1640 medium supplemented with 10% FCS, penicillin, streptomy- trol equal loading. cin, 1 mM pyruvate, and 10 mM HEPES in the presence of various sub- stances to establish a dose-response analysis and a time kinetic. After that, Immunoprecipitation I determined the LC . The substances, concentrations, and culture periods 75 CT26 cells were lysed in lysis buffer (20 mM Tris ⅐ HCl (pH 7.5), 150 mM used are as follows: doxorubicin at 25 ␮M for 24 h (Sigma-Aldrich); mi- NaCl, 1 mM Na EDTA, 1 mM EGTA, 2.5 mM sodium pyrophosphate, 1 toxantrone at 1 ␮M for 24 h (Sigma-Aldrich); idarubicin at 1 ␮M for 24 h 2 mM ␤-glycerophosphate, and 1 mM Na VO with 1% Triton X-100) con- (Aventis); mitomycin C at 30 ␮M for 48 h (Sanofi-Synthelabo) and/or 3 4 taining a protease inhibitor mixture (Roche). Whole cell extracts were cen- N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (z-VAD-fmk) at trifuged at 14,000 rpm for 20 min to remove the debris. Immunoprecipi- 50 ␮M for 24 h (Bachem); tunicamycin at 65 ␮M for 24 h; thapsigargin at tations were performed by incubating whole cell extracts with the indicated 30 ␮M for 24 h; brefeldin A at 50 ␮M for 24 h; etoposide at 25 ␮M for Ab preincubated with recombinant protein G agarose (Invitrogen) while 48 h; MG132 at 10 ␮M for 48 h; N-acetyl-leucinyl-leucinyl-norleucinal rocking at 4°C overnight. Immunoprecipitates were washed three times (ALLN) at 45 ␮M for 48 h); betulinic acid at 10 ␮M for 24 h; Hoechst with lysis buffer, resuspended in 50 ␮lof1ϫ Laemmli sample buffer, and 33342 at 0.2 ␮M for 24 h; camptothecin for 15 ␮M for 24 h; lactacystin at then resolved by electrophoresis with 4–15% polyacrylamide Tris ⅐ HCl 60 ␮M for 48 h; BAY 11-8072 at 30 ␮M for 24 h; staurosporine at 1.5 ␮M gel. In some experiments, I performed an immunoprecipitation on a plasma for 24 h; bafilomycin A1 at 300 ␮M for 48 h; arsenic trioxide at 30 ␮M for membrane protein isolated by biotinylation on CT26 cell surface mem- 24 h; C2-ceramide at 60 ␮M for 24 h; calyculin A at 30 nM for 48 h or brane proteins.
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