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263.Full.Pdf The HTLV-I Tax Protein Transcriptionally Modulates OX40 Antigen Expression Rüdiger Pankow, Horst Dürkop, Ute Latza, Hans Krause, Ulrich Kunzendorf, Thomas Pohl and Silvia Bulfone-Paus This information is current as of October 2, 2021. J Immunol 2000; 165:263-270; ; doi: 10.4049/jimmunol.165.1.263 http://www.jimmunol.org/content/165/1/263 Downloaded from References This article cites 48 articles, 23 of which you can access for free at: http://www.jimmunol.org/content/165/1/263.full#ref-list-1 Why The JI? Submit online. http://www.jimmunol.org/ • 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 by guest on October 2, 2021 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 © 2000 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The HTLV-I Tax Protein Transcriptionally Modulates OX40 Antigen Expression1 Ru¨diger Pankow,* Horst Du¨rkop,† Ute Latza,†‡ Hans Krause,§ Ulrich Kunzendorf,¶ Thomas Pohl,* and Silvia Bulfone-Paus2* OX40 is a member of the TNF receptor family, expressed on activated T cells. It is the only costimulatory T cell molecule known to be specifically up-regulated in human T cell leukemia virus type-I (HTLV-I)-producing cells. In a T cell line, OX40 surface expression was shown to be induced by HTLV-I Tax alone. To understand molecular mechanisms of OX40 gene regulation and modulation by HTLV-I Tax, we have cloned the human OX40 gene and analyzed its 5؅-flanking region. By reporter gene analysis with progressive 5؅ deletions from nucleotides ؊1259 to ؊64, we have defined a 157-bp DNA fragment as a minimal promoter for constitutive expression. In addition, we show that in the OX40؉ cell line, Co, Tax is able to further increase OX40 surface expression. Up-regulation of OX40 promoter activity by Tax requires two upstream NF-␬B sites, which are not active in the Downloaded from constitutive OX40 expression. Their deletion abrogates Tax responsiveness in reporter gene analysis. The site-directed mutagenesis of each NF-␬B site demonstrates that cooperative NF-␬B binding is a prerequisite for Tax-directed activity as neither site alone is sufficient for a full Tax responsiveness of the OX40 promoter. Upon Tax expression, both sites bind p65 and c-Rel. These data provide new insight into the direct regulation of OX40 by Tax and add to our understanding of the possible role of the OX40/OX40 .ligand system in the proliferation of HTLV-I؉ T cells. The Journal of Immunology, 2000, 165: 263–270 he T cell costimulatory receptor OX40 and its ligand genes, most of which are associated with cell growth. They include http://www.jimmunol.org/ (OX40L)3 are both members of the superfamilies of TNF growth factors, like GM-CSF (16) or cytokines and their receptors T receptors and ligands, respectively (1–4). Interactions of such as IL-2 (17), IL-15 (18), or IL-2R␣ (CD25) (19–21). This ac- these members with their ligands have been shown to be involved tivation occurs via the interaction of Tax with cellular transcription in cell proliferation, activation, as well as induction of apoptosis. factors, which include the serum response factor (22, 23), members of However, unlike the other known TNF receptor family members, the activating transcription factor/cAMP response element binding OX40 has a restricted cellular expression only on activated lym- protein (24, 25), and NF-␬B (26). phocytes, predominantly CD4ϩ T cells (3, 5, 6). Furthermore, it NF-␬B defines a family of transcription factors that result from has thus far not been associated with a cell death-inducing func- the combination of its protein members, such as p50, p65 (RelA), by guest on October 2, 2021 tion. Rather, signaling through OX40 generates strong costimula- c-Rel, and RelB (27). Inactive NF-␬B is retained in the cytoplasm, tory effects, which induce T cell proliferation (7, 8), modulate either by complexing to the I␬B inhibitor or as a precursor mol- cytokine production (9), and influence T cell migration into tissues ecule of the active protein (27). NF-␬B activation and subsequent (10, 11). Both OX40 and OX40L are constitutively expressed on translocation to the nucleus requires phosphorylation and degra- human T cell leukemia virus type I (HTLV-I)-producing T cell dation of I␬B or proteolytic cleavage of the precursor molecule. lines (3, 10–12). Tax is one of the signals known to cause liberation of NF-␬B from HTLV-I infection is associated with the aggressive and lethal adult I␬B (26, 28). It can also bind NF-␬B precursors as well as active T cell leukemia (ATL), as well as chronic inflammatory disorders, nuclear NF-␬B proteins (23, 29–31). such as the tropical spastic paraparesis/HTLV-I-associated myelopa- The OX40L promoter was shown to be activated by the Tax thy and others (13–15). T cell transformation requires the HTLV-I oncoprotein (32) and expression of Tax in the OX40-negative T Tax oncoprotein, which is the activator of viral gene expression. Tax cell line, Jurkat, results in OX40 surface expression (33). These also acts as a transactivator of an increasing number of host cellular findings suggest that auto or paracrine OX40/OX40L interactions on HTLV-I-producing cells may provide necessary costimulatory signals for transformation or survival and proliferation of these *Institute of Immunology, Departments of †Pathology and §Urology, University Hos- pital Benjamin Franklin, Free University Berlin, Berlin, Germany; ‡Berufsgenossen- cells. Thus far, however, little is known about the transcriptional schaftliches Forschungsinstitut fu¨r Arbeitsmedizin, Bochum, Germany; and ¶Depart- regulation of OX40 gene expression and its modulation by the Tax ment of Internal Medicine IV, Friedrich-Alexander-University, Erlangen, Germany protein. Therefore, we have cloned the human OX40 gene, ana- Received for publication December 22, 1999. Accepted for publication April lyzed its promoter region, and defined basal promoter activity. 11, 2000. Moreover, we describe here that Tax further up-regulates OX40 The costs of publication of this article were defrayed in part by the payment of page ϩ ␬ charges. This article must therefore be hereby marked advertisement in accordance gene expression in an OX40 cell line via two NF- B elements, with 18 U.S.C. Section 1734 solely to indicate this fact. which recruit at least three members of the NF-␬B family: c-Rel, 1 This study was supported in part by grants from the Deutsche Forschungsgemein- p65, and p50 in a Tax-dependent manner. schaft (SFB 506 to S.B.P., T.P., and H.D.; SFB 366 to H.D.) and from the Deutsche Krebshilfe (W76-93-Du¨1 to H.D.). Materials and Methods 2 Address correspondence and reprint requests to Dr. Silvia Bulfone-Paus, Institute of Isolation and characterization of human OX40 genomic clones Immunology, University Hospital Benjamin Franklin, Free University Berlin, Hin- denburgdamm 30, D-12200 Berlin, Germany. E-mail: [email protected] The human placental cosmid library pWE15 (Stratagene, Heidelberg, Ger- 3 Abbreviations used in this paper: OX40L, OX40 ligand; ATL, adult T cell leukemia; many) was screened by standard molecular biology techniques using ran- HTLV-I, human T cell leukemia virus type I; Inr, initiator element. dom-primed radio-labeled human OX40 cDNA (3). The cosmid carrying Copyright © 2000 by The American Association of Immunologists 0022-1767/00/$02.00 264 Tax TRANSACTIVATES OX40 THROUGH NF-␬B the OX40 gene was sequenced from both strands of sample DNAs on an of each plasmid DNA. Cell extract preparation and luciferase assay were automated sequencer (Perkin-Elmer/ABI, Weiterstadt, Germany). The se- performed 48 h later according to the Luciferase assay system kit (Pro- quence was searched for possible transcription factor binding sites using mega). Enzyme activity was measured with a Lumat LB9501 (Berthold, the programs FACTOR (HUSAR, EMBL, Heidelberg, Germany) and Mat- Wildbad, Germany). Transfection efficiency was normalized by cotrans- Inspector (34). fection of a reporter plasmid, pCH101 (Pharmacia Biotech, Freiburg, Ger- many), containing the ␤-galactosidase gene under the control of the SV40 Cell lines and cell culture promoter. ␤-galactosidase activity was determined using the Galacto-light Ϫ kit (Tropix, Bedford, MA), following manufacturer’s instructions. For The OX40 lymphoid cell lines L363 and DG75 were obtained from the comparison of promoter activities in different cell lines, luciferase activities Deutsche Sammlung fu¨r Mikroorganismen und Zellkulturen (Braun- ϩ were referred to luciferase control vector, pGL2 control, containing the schweig, Germany). The OX40 but HTLV-I-negative lymphoid cell line SV40 promoter and enhancer. All measurements were conducted in dupli- Co (35) was a generous gift from Dr. D. B. Jones (Southhampton, U.K.). cates from three independent transfections. The OX40ϩ but HTLV-I-negative lymphoid cell line CCRF-CEM and the OX40ϩ and HTLV-I-positive lymphoid cell lines HUT-102 and MT-2 were obtained from the American Type Culture Collection (Manassas, Nuclear extracts and gel mobility shift assay VA). All cell lines were cultured in RPMI 1640, containing 10% FCS, 2 Nuclear extracts were prepared as previously described (37). Five micro- mM L-glutamine, 100 U/ml penicillin, and 100 ␮g/ml streptomycin and grams of extract were incubated with 1 ng of labeled DNA (20,000 cpm) incubated at 37°C and 5% CO2.
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