Mechanisms Regulating Expression of the Tumor NecrosisFactor-related light GeneROLE OF CALCIUM-SIGNALING PATHWAY IN THE TRANSCRIPTIONAL CONTROL*

Received for publication, July 30, 2002Published, JBC Papers in Press, September 4, 2002, DOI 10.1074/jbc.M207689200

Remy Castellano‡§, Carine Van Lint¶�, Valentine Peri‡, Emmanuelle Veithen¶, Yannis Morel**,Regis Costello**, Daniel Olive‡**, and Yves Collette‡ ‡‡From the ‡Institut de Cancerologie et d’Immunologie de Marseille, Universite de la Mediterranee, INSERM, Unite 119,13009 Marseille, France, ¶Universite Libre de Bruxelles, Institut de Biologie et de Medecine Moleculaire, Laboratoire deChimie Biologique, 6041 Gosselies, Belgique, and **Institut Paoli-Calmette, Laboratoire d’Immunologie des Tumeurs,13009 Marseille, France

LIGHT (TNFSF14) is a newly identified tumor necro-sis factor superfamily member involved in the regula-tion of immune responses by control of activation, mat-uration, and survival of immune effector cells. Despitethe immunological relevance of the LIGHT protein, littleknowledge is available as to how light gene expressionis regulated. In T-lymphocytes, most LIGHT surface ex-pression and transcript accumulation occurs after T cellactivation. In this study, we have shown that theseevents are blocked at the transcriptional level by cyclo-sporin A, an immuno-suppressive drug. Besides, weidentified a role for Ca2�-signaling pathways and NFATtranscription factors in T cell activation-induced LIGHTexpression. To further investigate this process, we haveidentified, cloned, and characterized a 2.1-kilobase 5�-flanking DNA genomic fragment from the human lightgene. We have shown the transcriptional activity of theherein-identified minimal 5� regulatory region of humanlight gene parallels the endogenous expression of lightin T cells. Moreover, we demonstrated that inducedLIGHT promoter activity can be equally blocked by cy-closporin A treatment or dominant negative NFAT over-expression and further identified by site-directed mu-tagenesis and electrophoretic mobility supershiftanalysis of a NFAT transcription factor binding sitewithin the human light minimal promoter. Finally, Sp1and Ets1 binding sites were identified and shown toregulate light basal promoter activity. Thus, the presentstudy establishes a molecular basis to further under-stand the mechanisms governing human light gene ex-pression and, consequently, could potentially lead tonovel therapeutic manipulations that control the signal-ing cascade, resulting in LIGHT production in condi-tions characterized by immunopathologic activation ofT cells.

The TNF1 superfamily (TNFSF) is formed of type II trans-membrane glycoproteins that can be cleaved and secreted asactive trimeric cytokines. Most TNFSFs are expressed in theimmune system, where they play important roles in lympho-cyte activation, regulation of the immune response, and thedevelopment of lymphoid tissues (1). Hence, expression ofTNFSF is highly regulated, whereas its dysregulation can leadto severe pathologies (2–4).

LIGHT protein (TNFSF14) is a newly identified TNFSFmember (5) expressed by activated T-lymphocytes (6) but alsomonocytes, granulocytes, and immature dendritic cells (DCs)(7, 8). LIGHT is a ligand for three TNFSF receptors: herpesvirus entry mediator, mainly expressed by T cells (5, 6, 9);lymphotoxin � receptor, expressed by stromal and non-lym-phoid hematopoietic cells (8); and the decoy receptor 3 (DcR3/TR6), which is predominantly expressed in lung tissue and thecolon carcinoma cell line SW480 and might modulate LIGHTfunction in vivo (10).

Functionally, LIGHT regulates apoptosis (8, 11) but is alsoinvolved in the control of the immune response by enhancing Tcell proliferation and cytokine secretion (7, 12, 13) as well as byinducting DC maturation (9). Thus, LIGHT was shown both invitro and in vivo to regulate cell death and survival (7, 11), toplay a role in antitumor activity (7, 13), and to interfere withvirus infection (5, 14). Moreover, different experimental modelshave demonstrated that transgenic expression of light in the Tcell compartment leads to hyperactivated peripheral T cellpopulation, altered T cell homeostasis, and severe autoimmunediseases (15, 16).

Despite the immunological relevance of the LIGHT proteinand its specific cell and tissue distribution, little knowledge isavailable as to how light gene expression is regulated. light isup-regulated in T cells, whereas constitutive expression of thegene in immature DCs is down-regulated in mature DCs (6, 7).Interestingly, the activation-induced expression of light in Tcell lymphocytes is correlated with a decrease of herpes virusentry mediator membrane expression, suggesting a regulatoryfeedback loop controlling LIGHT functions (6). Post-transla-tional modification of LIGHT by processing from a membraneinto a soluble form by matrix metalloproteinases (6) or analternative splicing of light mRNA (17) has been identified.However detailed molecular mechanisms governing its tran-

* This work was supported by INSERM, the Universite Libre deBruxelles, and by grants from the Ligue Nationale Francaise Contre leCancer, Fonds National de la Recherche Scientifique (Belgium),Televie-program, International Brachet Stiftung, Commissariat gen-eral aux Relations Internationales/INSERM cooperation, region Wal-lonne-Commission Europeenne FEDER, and the Theyskens-MineurFoundation. The costs of publication of this article were defrayed in partby the payment of page charges. This article must therefore be herebymarked “advertisement” in accordance with 18 U.S.C. Section 1734solely to indicate this fact.

§ Fellow of the Association Nationale de Recherche contre le Syn-drome d’Immuno-Def ıcience Acquise.

� Maıtre de Recherches of the Fonds National de la RechercheScientifique.

‡‡ To whom correspondence should be addressed: Institut de Cancer-ologie et d’Immunologie de Marseille, U119 INSERM, 27 Boulevard LeiRoure, 13009 Marseille, France. Tel.: 33-491-75-84-13; Fax: 33-491-26-03-64; E-mail: collette@marseille.inserm.fr.

1 The abbreviations used are: TNF, tumor necrosis factor; TNFSF,TNF superfamily; NFAT, nuclear factor of activated T cells; RT, reversetranscription; TK, thymidine kinase; TCR, T cell receptor; DC, dendriticcell; UTR, untranslated region; CsA, cyclosporin A; PMA, phorbol 12-myristate 13-acetate; mAb, monoclonal antibody; RACE, rapid ampli-fication of cDNA ends; CN, calcineurin phosphatase; DN, dominantnegative; EMSA, electrophoretic mobility shift assay; nt, nucleotides.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 277, No. 45, Issue of November 8, pp. 42841–42851, 2002© 2002 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

This paper is available on line at http://www.jbc.org 42841

by guest on September 23, 2020

http://ww

w.jbc.org/

Dow

nloaded from

scription are lacking. Recently, the mouse light cDNA (18) andthe genomic organization of the human light gene (17) havebeen partially elucidated, but the 5�-untranslated regions(UTRs) were not characterized.

In T-lymphocyte, most LIGHT surface expression and tran-script accumulation occurs after T cell activation (6). In thisreport, we show that these events are blocked at the transcrip-tional level by cyclosporin A (CsA), an immuno-suppressivedrug acting on the phosphatase calcineurin. We identified arole for Ca2�-signaling pathways and NFAT transcription fac-tors in T cell activation-induced light transcription and a rolefor both Ets and Sp1 families of transcription factors in basalconstitutive expression of light in T cells.

MATERIALS AND METHODS

Cells, Culture Conditions, and T Cell Stimulation—The Jurkat T cellline (JA16 clone (19)) was maintained in RPMI 1640 medium supple-mented with 10% fetal calf serum. Peripheral blood mononuclear cellsfrom healthy donors were isolated on Ficoll-Hypaque gradients (20).T-lymphocytes were isolated as the CD2-positive peripheral bloodmononuclear cell population, corresponding to cells that adhere tosheep erythrocytes in the E-rosetting technique but fail to adhere toplastic dishes after overnight incubation in medium and 30% fetal calfserum. T-lymphocytes and the Jurkat T cell line were stimulated with1 ng/ml and 20 ng/ml PMA (Sigma), respectively, and/or 1 �g/ml and 2�g/ml ionomycin (Calbiochem), respectively, either in the presence orabsence of CsA at 5–100 ng/ml (Calbiochem). The monoclonal antibody(mAb) 289 (kindly gift by Alessandro Moretta) recognizes the CD3�chain of the T cell receptor complex and was immobilized on plasticculture dishes for TCR stimulation.

LIGHT Endogenous Quantification; RT-PCR and Flow Cytometry—The expression of light mRNA and protein was analyzed by RT-PCRand cyto-fluorometry, respectively, using the previously described prim-ers pair and anti-LIGHT 2C8 monoclonal antibody (6).

Mapping of the Transcription Start Site in the light Gene—The tran-scription start site of the human light gene was mapped inT-lymphocytes by using the 5�-RACE system for rapid amplification ofcDNA ends, version 2.0 (Invitrogen) according to the manufacturer’sprotocol. In brief, total RNA from T-lymphocytes stimulated with PMAand ionomycin for 16 h was reversed-transcribed using the primerGSP1. PCR was performed on dC tailed with the 5�-RACE-abridgedanchor primer associated with GSP2. The nested amplification wasperformed with the 5�-RACE-abridged universal amplification andGSP3 primers. The three GSP1, -2, and -3 primer sequences are shownin Fig.1. The amplified products were cloned using the pGEM-T EasyVector System (Promega Corp, Madison, WI) and sequenced.

Cloning of the Human light Gene Promoter—The genomic sequence(�2186pb/�1) lying upstream to the human light gene was isolated byPCR using the following primers: light/sense 5�-GAGTCAAGACGCAG-ATGAGCAGGGGGAGCC-3� and light/antisense 5�-CCGAAGCTTGCC-CAAGGTGTCTGGAGCAGGGCTGACACGC-3�. The PCR product wascloned in pGEM-T Easy Vector. The fragments generated by enzymaticrestrictions of pGEM-T/pLIGHT(2148) with BglII, NdeI, and KpnI,respectively, for the 5� site and HindIII for the 3� site were cloned intothe reporter vector pGL2-Basic (Promega); these reporter constructsare called pLIGHT(2148)-luc, pLIGHT(1033)-luc, and pLIGHT(175)-luc, respectively. The primers used for the other light promoter con-structs were: LIGHT(441)-luc, 5�-CTACTCGAGCTTGTCTCTCTGGCT-CCACCAG-3�; LIGHT(124)-luc, 5�-CTACTCGAGCTCTAAAGGCGGC-CCACGGGTG-3�, and LIGHT(91)-luc, 5�-CTACTCGAGCGTGCACAG-CCCAGGAGTGTTGAG-3� and the 3� primer described above. The XhoIand HindIII sites in these primers are underlined. The amplified cDNAfragments were cloned into the XhoI and HindIII sites in pGL2Basic.Whole nucleotide sequences from these constructs were confirmed bysequencing.

Plasmid Constructs—pLIGHT(441)-luc and pLIGHT(175)-luc wereused as templates for mutagenesis performed by the QuikChange site-directed mutagenesis method (Stratagene). The primers used are: M1,5�-GGCTCCACCAGAAGACTCGCAGGGACCCTTCTTGC-3�; M2, 5�-G-AAGCATCCAAGAAGCTCAAGCTGGGGGCTCCC-3�; M3, 5�-CATTTT-CAGAAGCCTCTCTCAAGTGTGAGAGTCTG-3�; M4, 5�-GCGGCGGG-TACCGGACTCAGAGGAGGGTGAGTG-3�; M5, 5�-GAGCAATTTCGG-TTGAGTCTGAGGTTGAAGGACCC-3�; Sp1mut, 5�-GAGGAGGGTGA-GTGGTTGAAGTTGTGTTGCTGAACCCCAGCTC-3�. The mutationswere confirmed by sequencing. The pDN-NFAT expression constructwas previously described (21). The calcineurin phosphatase (CN) cDNA

(22) was subcloned from pBJ5 into the BamHI-digested BDNA4 expres-sion vector (23).

Electroporation and Luciferase Assays—107 Jurkat T cells were elec-troporated at 960 microfarads and 250 V using Bio-Rad Gene Pulserwith 25 �g of pGL2/basic or the various light promoter-driven luciferasefirefly constructs (pLIGHT-luc) together with 5 �g of thymidine kinase-luciferase Renilla plasmid. The cells were incubated for 2 h, then leftunstimulated or stimulated for 16 h as previously described (23). Thecells were collected, washed in phosphate-buffered saline, lysed to de-termine the luciferase activity by using the dual luciferase reporterassay according to manufacturer’s instructions (Promega), and readusing a luminometer (Dynex). The transfection efficiency was normal-ized to luciferase Renilla activity and corrected for protein content asdetermined by the Bradford protein assay (Bio-Rad). The reportedvalues represent the average of three independent transfections, withstandard deviation as error bars.

Electrophoretic Mobility Shift Assay—Nuclear extracts from Jurkatcells were prepared as previously described (24). Electrophoretic mobil-ity shift assay were performed with 10 �g of nuclear protein extract or0.25–1 ng of recombinant protein in a 20-�l reaction mixture containing2 �g of DNase-free bovine serum albumin (Amersham Biosciences), 1�g of poly(dI-dC) (Amersham Biosciences) as nonspecific DNA compet-itor, 1 mM dithiothreitol, 20 mM Hepes buffer, pH 7.3, and 10% (V/V)glycerol. The binding reactions were incubated for 20 min at roomtemperature with 10,000–24,000 cpm of double-stranded oligonucleo-tide 5� end-labeled with [�-32P]ATP using the T4 polynucleotide kinase.The reaction mixture was loaded directly onto a 6% non-denaturingpolyacrylamide gel and electrophoresed at room temperature in 1�TGE buffer (25 mM Tris acetate, pH 8.3, 190 mM glycine, 1 mM EDTA).The double-stranded oligonucleotides used were as follows: for thewild-type NFAT binding site in the light promoter (M3wt), 5�-CAGAA-GCCTCTGGAAAGTGTGAGAGTC-3�, and its mutated homologue(M3mut), 5�-CAGAAGCCTCTCTCAAGTGTGAGAGTC-3�; for the con-trol NFAT binding site in the FasL promoter (FasLwt), 5�-TAGCTAT-GGAAACTCTATA-3�; for the oligonucleotide containing the Ets bind-ing site (M4wt), 5�-GCGGGTACCGGAAGAAGAGGAGGGTG-3� and itsmutated version (M4mut), 5�-GCGGGTACCGGACTCAGAGGAGGGT-G-3�; finally, for the oligonucleotide containing the Sp1 binding site(Sp1wt), 5�-GAGTGGGGGAAGGGGTGGGGCTGAA-3�, and its muta-ted version (M4mut), 5�-GAGTGGTTGAAGTTGTGTTGCTGAA-3�, andthe Sp1 consensus 5�-ATTCGATCGGGGCGGGGCGAGC-3� (Promega).

For supershift assays, 1 �l of monoclonal antibodies against NFATc(sc-7294), NFATp (sc-7295), or CREB (cAMP-response element-bindingprotein; sc-271) as a negative control or 1 �l of polyclonal antibodiesagainst Sp1 (sc-059x), Sp2 (sc-643x), Sp3 (sc-644x), or Sp4 (sc-645x)(Santa Cruz Biotechnology) were added to the binding reaction mixtureat the end of the binding reaction for an additional 30 min of incubationat room temperature before electrophoresis. For blocking assays, anti-bodies against Ets-1 (sc-350) and Ets-2 (sc-351) or MEF-2 (sc-313x) as anegative control (Santa Cruz Biotechnology) were added at the begin-ning of the binding reaction before the nuclear extracts. When indi-cated, a 150-fold molar excess of unlabeled homologous oligonucleotide(Ets, 5�-tcgggctcgagataaacaggaagtggtctcgg-3�) or heterologous oligonu-cleotide (Ebox, 5�-AGCTTCAGACCACGTGGTCGGG-3� or Oct, 5�-TG-TCGAATGCAAATCACTAGAA-3�) oligonucleotide was used ascompetitor.

RESULTS

light Transcription Is CsA-sensitive—The induction of lightin T cells requires cellular activation by the TCR/CD3 (8) or bytreatment with PMA and calcium ionophore (6). A commonfeature to ionomycin- and CD3-initiated signal transduction isthe increase of [Ca2�]i followed by the nuclear translocation ofthe NFAT transcription factors. The CsA immuno-suppressivedrug blocks induced transcription of several cytokines (for re-view, see Ref. 25) by inhibiting the phosphatase CN, a secondmessenger protein activated by [Ca2�]i and controlling thenuclear translocation of NFAT. Moreover, several TNS SFmembers such as TNF�, FasL, TRAIL (tumor necrosis factor-related apoptosis-inducing ligand), and CD40L are regulatedby NFAT and inhibited by CsA (26–29).

To get insight into the mechanisms of light gene expression,we performed experiments with CsA. As shown in Fig. 1, panelsA and B, LIGHT was absent from the cell surface of activatedlymphocytes incubated in the presence of CsA. To specify

LIGHT Transcriptional Regulation in T-lymphocytes42842

by guest on September 23, 2020

http://ww

w.jbc.org/

Dow

nloaded from

whether the effect of CsA on expression was transcriptional, wealso analyzed the light RNA expression. As shown in panel C,a faint signal was detected in unstimulated T-lymphocytes at30 and 33 PCR cycles. In contrast, light messenger wasdetected at 27 PCR cycles in PMA plus ionomycin-stimulatedlymphocytes with a quantitatively more significant signal. Incontrast, the presence of CsA during T cell stimulation signif-icantly decreased light RNA expression, which remained unde-tectable at 27 PCR cycles and produced a faint signal at 30 PCRcycles. To normalize both quantity and quality of the RT prod-ucts, we used the �-actin housekeeping gene as a control.Results from these experiments demonstrate that the induc-tion of light transcription by PMA plus ionomycin is preventedby CsA and hint to the likely involvement of the NFAT familyof transcription factors.

Characterization of Cis-acting Regions Involved in the LightPromoter-mediated Transcription—To investigate the regula-tion by CsA of the LIGHT transcription, we identified thepromoter region in the human light gene by multiple BLASTanalysis between human light cDNA sequence (accession num-ber NM_003807) compared with a genomic sequence data base.Whole light cDNA sequences matched the chromosome 19p13.3(accession number NT_0111458.12) at nt 366054–371642 (in-cluding intron sequences) with 100% identity. These resultswere confirmed by Granger et al. (17) during the preparation ofthis manuscript. To identify the transcription initiation site ofhuman light, we used a 5�-RACE approach. Using downstreamoligonucleotide sequences present in the first exon, a singlePCR product was amplified. Sequence analysis revealed thatthe major start site maps 91 bp upstream to the ATG. Toconfirm this result, RT-PCR reactions were performed with two5� primers located near the identified transcription start site(Primers A and B, see Fig. 2A) and the same 3� primer withinexon 1 (primer GSP2). Using T cell-derived reverse-transcribedmRNA as a template, the B/GSP2 but not the A/GSP2 primerpairs allowed amplification of the expected 128-bp fragments(Fig. 2B), thereby confirming the preferential usage of thetranscription initiation start site located 91 bp upstream to theATG. Next, homologies to known cis-regulatory elements weredetermined using the MatInspector (Genomatix Software,Munich, Germany) and TESS (Transcription Element SearchSystem, CBIL, US) programs. A TATA motif (nt �116 to �121)and GC-rich domains were identified close to the transcriptioninitiation site (Fig. 3A). Furthermore, the proximal promoterregion of light resembles other known cytokine gene promoters,particularly promoters controlling TNFSF gene products, inthat it possesses several potential binding sites for TCR-induc-ible transcription factors such as NFAT (Fig. 3A).

To evaluate promoter activity of the 5�-UTR regions of thehuman light gene, the 2148 bp of genomic DNA immediately 5�to the translational start site were cloned in pGL2/Basic. Pro-moter activity of the construct pLIGHT(2148)-luc was assayedby measuring firefly luciferase activity after transient trans-fection into the JA16 Jurkat T cell line, which expresses lightmRNA (Fig. 2C) and protein (data not shown) after T-cellactivation. Similarly, treatment of Jurkat T cells with PMAplus ionomycin or CD3-immobilized antibody significantly en-hanced the exogenous luciferase activity by �200- and 20-foldfor PMA plus ionomycin and CD3, respectively (Fig. 2D).

To map the minimal promoter region in the light generequired for initiation and induction of gene transcription,luciferase reporter constructs containing progressive deletionsof the 2148-bp genomic DNA fragment were generated (Fig.3B). Each construct as well as the control vector pGL2/Basicwere transiently transfected into Jurkat T cells and assayed forreporter activity. Our results show that the 2148-bp fragmentinduces a large increase in basal luciferase activity and thatdeletions of DNA regions upstream to nt �175 (relative to theATG) did not significantly decrease basal luciferase expressionas compared with the full-length 2148-bp fragment (Fig. 3C).However, deletion of an additional 53 bp (from nt �175 to�122) reduced reporter activity to a level similar to that ob-tained with the promoterless luciferase construct (pGL2/Basic)(Fig. 3C). These results suggest that the cis-regulatory ele-ments required for the basal transcription of the light gene arelocated in a 175-bp region upstream to the ATG and containingthe transcription initiation site, the putative TATA box, GC-rich domains, and one SP1 site (Fig. 3A).

Because light transcription is optimal upon T cell activation,the different deletion constructs were assayed for luciferase

FIG. 1. Effects of CsA on light expression in primary humanT-lymphocytes. Purified T-lymphocytes were incubated either withmedium alone or PMA plus ionomycin (P � I) in the presence or absenceof cyclosporin A (70 ng/ml), and the expression of light was assessed byflow cytometry (panels A and B) or RT-PCR (panel C). Data are repre-sentative from three independent experiments performed with samplesfrom different healthy blood donors. A, empty histograms correspond tothe isotypic control mAb, and filled histograms correspond to anti-LIGHT mAb (the data presented correspond to % and mean fluores-cence values (MFI) of positive cells after subtraction of the backgrounddetermined by the isotypic control mAb). B, a kinetic flow cytometryanalysis was performed from base-line conditions to 96 h. Results arepresented as mean fluorescence values, but similar results were ob-tained using the % of positive cells. The absence of direct cytotoxicity ofCsA was assessed by trypan blue exclusion (data not shown). C, semi-quantitative RT-PCR was performed using light (27, 30, and 33 cycles)and �-actin (25 cycles) primer pairs in non-saturating conditions.

LIGHT Transcriptional Regulation in T-lymphocytes 42843

by guest on September 23, 2020

http://ww

w.jbc.org/

Dow

nloaded from

activity upon treatment with PMA plus ionomycin. ThepLIGHT(441)-luc construct displayed a reporter gene activitysimilar to that obtained with the full-length pLIGHT(2148)-lucconstruct. In contrast, cells transfected with pLIGHT(175)-luc,pLIGHT (122)-luc, and pLIGHT (89)-luc showed markedly re-duced luciferase activity in response to the T cell activation, ascompared with less deleted constructs (Fig. 3D). These dataindicate that the region located between nt �441 and �175 isrequired for light promoter induction, whereas the nt �175 to�122 region is essential for light basal transcription. Moreover,these results are in good agreement with the wealth of putativebinding sites for inducible transcription factors present in theformer region (Fig. 3A).

NFAT Family Members Participate in the Induction of lightTranscription in Jurkat T Cells—To determine whether theherein described promoter regulatory cis-acting regions ofhuman light gene are regulated by CsA, we transfected thepLIGHT(441)-luc construct into Jurkat T cells. Treatment ofcells with CsA before activation almost completely (90%) abro-gated the increase in reporter gene activity of pLIGHT(441)-lucobserved after PMA and ionomycin treatment (Fig. 4A). Toinvestigate further the role of CN, an expression vector codingfor a constitutively active CN was cotransfected withpLIGHT(441)-luc into Jurkat T cells. CN significantly inducedthe pLIGHT(441)-luc-mediated transcription (Fig. 6B). ThusCN and CsA can regulate the LIGHT promoter activity. Thissuggested that NFAT binding transcriptional elements mightcontribute to the light gene transcriptional regulation con-ferred by the immediate 5�-flanking region, as previously de-scribed for TNF�, FasL, or CD40L (26, 27, 29).

To address directly a role of NFAT in light transcription, aNFAT dominant negative expression construct (pDN-NFAT)was cotransfected into Jurkat T cells. DN-NFAT overexpres-sion decreased both the transcription of the pLIGHT(441)-lucconstruct and membrane expression of the endogenous LIGHTprotein (Fig. 4, A–C). Together, these results strongly support arole for NFAT family members in light transcription. Moreover,the activation signal requirement and sensitivity to the CsA orNFAT inhibition observed with the light promoter reporterconstructs faithfully paralleled expression of the endogenouslight gene.

Identification of a Functional NFAT Binding Site in Minimallight Promoter—To further explore the role of NFAT in lighttranscription, we searched for NFAT binding sites within theminimal light promoter using MatInspector and TESS pro-grams with deliberately reduced identity standards. Fiveputative NFAT binding sites, termed NFAT M1 to M5, wereidentified (Fig. 3A). To test the functional significance of theseputative NFAT binding sites for light promoter activity, thecorresponding residues were mutated independently in thecontext of the pLIGHT(441)-luc promoter construct. The tran-scriptional activity of the mutated constructs was comparedwith that of the native pLIGHT construct in transfection ex-periments using PMA and ionomycin as the stimuli. As shownin Fig. 5, mutation of the M3 and M4 sites reduced activation-induced luciferase activity by more than 80 and 50%, respec-tively, whereas M1-, M2-, and M5-mutated pLIGHT promoterconstructs displayed similar fold induction as compared withthe wild-type construct. The double M3- plus M4-mutated con-struct presented an almost completely reduced induction. Themutation of M4 also reduced the basal promoter activity. Theseobservations suggest the M3 and M4 binding sites are func-tional in vivo in transfected cells. These results are in agree-ment with the fact that only M3, M4, and M5 (not M1 and M2)sites are conserved between mouse and human (data notshown).

FIG. 2. light promoter/reporter activity mirrors endogenousregulation of light expression in Jurkat T cells. A and B, pref-erential transcription start site used in primary T lymphocytes. A,schematic representation of the primers used in 5�-RACE procedure(GSP1 and GSP2) and in control RT-PCR (primers A and B). B, totalRNA extracted from PMA plus ionomycin-activated T cells was re-verse-transcribed (RT) and used in a PCR reaction (30 cycles) usingthe indicated primers pair. Genomic DNA from the same cells wasused as a positive control (DNA), and H2O was used as a negativecontrol of the PCR reaction. The PCR products were analyzed byagarose gel electrophoresis. C, light and �-actin transcripts weredetermined by semi-quantitative RT-PCR performed on total mRNAsamples from unstimulated (UN), PMA plus ionomycin (P � I, 14 h),or immobilized anti-CD3Ab (3coat, 14 h)-activated Jurkat T cells. Thenumbers correspond to PCR cycles performed. D, the pLIGHT(2148)-luc construct was transiently transfected into Jurkat T cells. Trans-fected cells were either left untreated or stimulated overnight byPMA, ionomycin (IONO), PMA plus ionomycin (P � I), or immobilizedanti-CD3Ab (3coat). Data are the average of three independent ex-periments � S.D., presented as fold induction calculated as the ratioof values obtained for stimulated cells over unstimulated cells andcorrected for transfection efficiency by normalization to pRL-TKactivity.

LIGHT Transcriptional Regulation in T-lymphocytes42844

by guest on September 23, 2020

http://ww

w.jbc.org/

Dow

nloaded from

FIG. 3. light promoter/reporter deletional analysis. A, nucleotide sequence of the potential promoter and of the first exon of the human lightgene (AF542509). The ATG site is boldface (was assigned the position nucleotide �1). Three gene-specific primers (GSP1, -2, and -3) used in5�-RACE procedure and mapping of the 5� end of light mRNA are underlined. Two primers (A and B) used in PCR to map the transcription startsite are underlined (see “Results”). The first nucleotide of the 5�-RACE product is indicated by an arrow. Putative cis-acting motifs are underlinedor boxed. The constructs were generated by cloning progressively 5�-truncated human light promoter fragments into the pGL2/basic luciferasevector. Negative numbers denote bp distances from translational start codon (panel B). Jurkat cells were transiently cotransfected with thesereporter constructs and pRL-TK to control for transfection efficiency. Cells were either left unstimulated (panel C) or stimulated with PMA plusionomycin (panel D) for 16 h. RLU, relative light units.

LIGHT Transcriptional Regulation in T-lymphocytes 42845

by guest on September 23, 2020

http://ww

w.jbc.org/

Dow

nloaded from

light M3 Site Binds NFATc and NFATp—EMSA experi-ments were next performed to verify the binding of NFATproteins to M3 and M4 sites. First, radiolabeled light M3 andM4 oligonucleotides were used as probes and incubated withrecombinant NFAT (DNA binding domain) (30). As shown inFig. 8A, the wild-type M3 but not the mutated probe formed acomplex with recombinant NFAT protein, although the bindingby M3 was less important than that by the control probe de-rived from FasL 5� regulatory regions (27). In contrast, the M4probe formed no detectable complex with recombinant NFAT(Fig. 6A).

Next, to confirm that NFAT transcription factors participatein light transcription in a M3 but not M4 site-dependent man-

ner, we compared the effect of the CN overexpression onpLIGHT(441)-luc reporter constructs. As shown Fig. 6B, muta-tion in the M3 site reduced CN-induced luciferase activity topGL2/Basic level, whereas M4-mutated LIGHT promoter con-struct displayed a higher fold induction as compared with thewild-type construct. The higher fold induction by mutated M4over wild-type construct could be explained by a reduced basalpromoter activity. These results suggested that M3 but not M4site participates to NFAT-induced light transcription.

Finally, to address whether NFAT proteins from activatedT-lymphocytes can bind the M3 site, the M3 probe was used inEMSA experiments with nuclear extracts prepared fromJurkat T cells either left unstimulated or activated with immo-bilized anti-CD3 mAb or PMA plus ionomycin. In addition,nuclear extracts were prepared from cells stimulated withPMA plus ionomycin in the presence of CsA to block signalingevents leading to NFAT nuclear translocation. As shown in Fig.7, both activations via the TCR/CD3 and by PMA plus ionomy-cin induced formation of a retarded complex (lanes 2 and 3).The mobility of the complex was indistinguishable from thatobtained with the FasL-derived NFAT probe (Fig. 7, lane 18). A150-fold molar excess of unlabeled oligonucleotides containingeither the M3 site or the FasL-derived NFAT site specificallycompeted for binding of this complex (lanes 6 and 8). In con-trast, a heterologous Ebox competitor did not compete for bind-ing to the same probe (lane 5). Oligonucleotides containing amutated M3 site or a mutated FasL-derived NFAT site did notcompete for binding of nuclear extract by the wild-type light M3probe (lanes 7 and 9). This suggests that the proteins fromactivated nuclear extracts that form complexes with eitherprobes are likely to have similar binding specificities.

Moreover, treatment with CsA totally inhibited formation ofthe complex (Fig. 7, lane 4), suggesting that NFAT is impli-cated. To further investigate whether the light M3 site bindsNFAT proteins from activated Jurkat T cells nuclear extracts,supershift experiments were performed using NFAT-specificantibodies raised against NFATp and NFATc (which constitutethe T cell-specific isoforms of NFAT). In these experiments thespecific complex was supershifted (Fig. 7), indicating that thelight M3 probe binds NFAT proteins from activated nuclearextracts (lanes 11–13). A nonspecific isotypic control antibodydid not alter the retarded complex (Fig. 9, lane 10). Based onthese in vitro binding studies together with the transfectionresults described above, we conclude that NFAT regulates lighttranscription at least in part through binding to the M3 sitelocated within the minimal light promoter.

Sp1 and Ets Family Members Control Basal Transcription ofthe light Gene—Deletion analysis suggested that cis-regulatoryelements required for basal activity of the light gene are locatedbetween the nt �175 and �122, upstream to the ATG (Fig. 3C).Moreover, the above experiments have demonstrated that theM4 site located at nt �169/�160 is necessary for induced butalso for maximal basal transcription of light (Fig. 5). Besidessequence similarity with previously described NFAT bindingsites, the M4 site also presents homology to the Ets consensusmotif (GGAA), especially Ets1, which is another lymphoid-specific transcription factor. Consequently, we performed EM-SAs using radiolabeled M4 oligonucleotides corresponding tothe putative Ets binding site. As shown in the Fig. 8A, threeindependent-stimulated DNA complexes were detected (C1–C3). As expected, these complexes did not co-migrate withNFAT-FasL complexes. These complexes were easily competedby increasing amounts of unlabeled Ets oligonucleotides butnot by the same amounts of an heterologous Oct oligonucleotide(Fig. 8B). Furthermore, formation of the C3 complex was pre-vented by preincubation of the nuclear extracts with anti-Ets-1

FIG. 4. Role of NFAT transcription factors in light expressionin Jurkat T cells. A, Jurkat T cells were transfected with either 10 �gof control vector DNA or 10 �g of NFAT negative dominant formexpression construct (pDN-NFAT) along with 15 �g of thepLIGHT(441)-luc reporter construct. Cells were left unstimulated orstimulated with PMA plus ionomycin in the presence or absence of 5 or50 ng/ml CsA and assayed for luciferase activity. Luciferase reportergene activity is expressed as the percentage of maximal activity ob-tained with PMA and ionomycin stimulation. B, the pLIGHT(441)-lucconstruct was cotransfected with 20 �g either DNA control vector or thecalcineurin dominant positive expression construct (CN) with or with-out 3 �g of the pDN-NFAT construct. Transfected cells were stimulatedwith PMA for 16 h. C, cell surface LIGHT protein was determined byflow cytometry performed on pDN-NFAT- and pCDNA3-transfectedJurkat T cells after PMA plus ionomycin stimulation.

LIGHT Transcriptional Regulation in T-lymphocytes42846

by guest on September 23, 2020

http://ww

w.jbc.org/

Dow

nloaded from

and -2 antibodies but not with a control antibody or with theanti-Ets-related Elk1 (Fig. 8C) and anti-GABP � and � (datanot shown) antibodies. Thus, Ets-1 and Ets-2 bind to the M4site implicated in light promoter basal activity.

In addition, based on sequence analysis, one potential Sp1(nt �144/�131) binding site was also identified (Fig. 3A). More-over, FasL is regulated by the Sp1 transcription factor family,which is usually associated with basal promoter activity (31).To investigate the binding of Sp1 family members to the po-tential Sp1 site, EMSAs were performed. As shown in Fig. 9A,nuclear extracts from Jurkat T cells formed three complexeswith the consensus Sp1 probe and four complexes with thelight-derived Sp1 probe (C1–C4). These four complexes werenot altered by activation. Mutation in light Sp1 site eliminatedor dramatically reduced the formation of these complexes. Toresolve the composition of the C1, C2, C3, and C4 complexes,supershifts were conducted in the presence of control antibodyor of anti-Sp1, -Sp2, -Sp3, and -Sp4 antibodies. Both Sp1 andSp3 antibodies induced the supershift of the C1 and C2 com-plexes, respectively (Fig. 9A, sixth and eighth lanes, suggestingthat the Sp1 and Sp3 nuclear factors were present in thesecomplexes. The identity of other nuclear factors implicated inC3 and C4 remains to be determined. The specificity of Sp1 andSp3 binding to the Sp1 sites in the light promoter was assayedby competition. As shown in Fig. 9A, the wild-type light-derived unlabeled Sp1 probe was as efficient as the consensusSp1 probe to compete for binding to the light Sp1 probe (secondand third lanes). In contrast, the unlabeled Oct oligonucleotidedid not compete the light Sp1 probe (first lane). Mutation in theSp1 probe abolished competition for binding to the wild-typelight Sp1 probe (fourth lane). Thus, these results show that Sp1and Sp3 were able to bind in an activation-independent man-ner to the herein-identified light Sp1 binding site.

To investigate the functional role of the Sp1 binding site inlight promoter activity, we introduced Sp1 mutations inpLIGHT(175)-luc and pLIGHT(441)-luc constructs. The mu-tated reporter constructs were transiently transfected intoJurkat T cells and analyzed for basal luciferase activity.Mutation in the Sp1 binding site in these promoter constructssignificantly reduced luciferase activity (Fig. 9B). Moreover,

the double-mutated construct pLIGHT(M4�Sp1mut)-lucshowed no detectable activity as compared with the pGL2Basicvector (Fig. 9B). Thus, we conclude that both the Sp1 bindingsite (nt �144/�132) and the Ets binding site (nt �168/165) areimportant for light basal transcription.

DISCUSSION

The biological importance of LIGHT has been highlightedrecently by work demonstrating its critical role in both co-stimulation of T-lymphocytes and the lymphoid organogenesis(32). However, relatively little is known about factors thatcontrol expression of LIGHT in T-cells. Here, we described forthe first time a signaling pathway that controls the humanlight gene expression in T lymphocytes. We demonstrated thatinduced light gene transcription can be blocked by CsA treat-ment or DN-NFAT overexpression and further identified bysite-directed mutagenesis and electrophoretic mobility super-shift analysis of a NFATc/NFATp transcription factor bindingsite within the human light minimal promoter.

The studies presented here provide an initial characteriza-tion of the transcriptional regulation of the human light en-hancer/promoter region in human T cells. The 2.1-kilobase5�-flanking DNA of the human light gene was cloned and ex-pressed as a functional promoter in T cells. Deletion analysisdemonstrate that the �441 bp of the immediate 5� flank issufficient for substantial activity in reporter gene assays, sug-gesting that cis-acting elements minimally required for thehuman light gene transcription are contained in this region.The importance of this region is further supported by its greatsimilarity with mouse sequences (56.6% of identity for the 500nt of genomic DNA immediately 5� to the translational lightstart site, data not shown). Results with reporter gene con-structs containing 441 bp of light flank appeared to accuratelyreflect the endogenous gene requirements for the [Ca2�]i-reg-ulated signaling pathway, including NFAT-dependent tran-scription, inhibition by CsA, and induced expression in T-lym-phocytes. In addition, cis-acting elements contained within aminimal �175-bp fragment shown to bind Sp1 and Ets tran-scription factors were evidenced to control the constitutivetranscriptional activity of light expression.

FIG. 5. Transcriptional activity of light promoter mutants. The different pLIGHT(441)-luc mutated constructs were generated as describedunder “Materials and Methods.” The filled boxes represent putative NFAT sites (M1 to M5). The mutations in these sites are symbolized by hatchedboxes. Jurkat T cells were transiently cotransfected with the indicated reporter construct and the pRL-TK to control for transfection efficiency.Cells were left unstimulated (hatched bars) or were stimulated by PMA plus ionomycin for 16 h (filled bars). RLU, relative light units

LIGHT Transcriptional Regulation in T-lymphocytes 42847

by guest on September 23, 2020

http://ww

w.jbc.org/

Dow

nloaded from

The cloning and characterization of the 5�-untranslatedgenomic DNA sequence (2.1 kilobase) allowed us to identify atranscription initiation site preferentially used in T cells. Thissite maps 91 nucleotides upstream to the translation start siteand is further supported by the presence of a proximal TATAconsensus sequence (�115 nt). However, this transcription ini-tiation site is located 43 bp upstream of the 5� end cDNAidentified in an activated T cell cDNA library but downstreamof the 5� end of the cDNA clone identified in an other study (12),suggesting that alternative transcription start sites could beused. Because previous Northern blot analysis identified tran-scripts of 2.5–3.5 kilobases (5, 12) for the human light gene, the3�-UTR regions is expected to contain additional regulatoryelements and, likewise, other TNFSF family members (33).Alternatively, spliced isoforms of light mRNA remains to beidentified (17).

Transient transfection of Jurkat T cells with the pLIGHT-(-2148bp)-luc construct shows inducible activity after stimula-tion with PMA plus ionomycin or TCR ligation but not witheither PMA or ionomycin alone (Fig. 2D), suggesting the coop-eration of several transcription factors or the integration ofsignaling at light regulatory elements. We have explored a rolefor NF�B since this family of transcription factors is induced byPMA. However, in agreement with the lack of induction of lightreporter gene transcription by PMA, neither the overexpres-sion of NF�B family members nor that of I�B� and -� kinases,known to regulate the nuclear translocation of NF�B, couldinduce light reporter gene transcription despite efficient regu-lation of an NF�B reporter construct (data not shown). Con-versely, the role of NFAT was examined, and a signaling path-way driving light transcription was described in this study. TheNFAT transcription factor family was identified as significantand necessary for the transactivation of the light gene tran-scription and light minimal promoter activity in lymphocyte Tcells after activation by stimuli that trigger the calcineurinphosphatase via the [Ca2�]i. Moreover, we mapped a functionalNFAT binding site within the minimal human light promoter(M3) that binds NFATc and NFATp in vitro as evidenced byEMSA experiments. NFATc and NFATp are the predominantNFAT family isoforms expressed in mature T-lymphocytes. Theproximity of the AP1 sites to the NFAT M3 site in the minimallight promoter (Fig. 3A) suggests a potential cooperation be-tween NFAT and AP1 family members as has been reported forother cytokine regulatory regions (for review, see Ref. 34).Thus, the activation-induced transcription of interleukin-2 andTNF� but not FasL is highly dependent on the cooperativeinteraction of NFAT proteins with Fos/Jun or ATF proteins

FIG. 6. Transcriptional activity of the putative NFAT bindingsites. A, recombinant NFAT protein (0.25 or 1 ng) was incubated withwild-type (wt) or mutated (mut) M3 and M4 probes and a FasL promot-er-derived NFAT probe (28). Protein-DNA complexes were resoled on a6% nondenaturing polyacrylamide gel. The NFAT complexes are indi-cated by an arrow. B, different pLIGHT(441)-luc mutant reporter con-structs were cotransfected with either the DNA control vector or thepCN construct into Jurkat T cells stimulated with PMA for 16 h.Results are representative of three independent experiments and arepresented as the fold induction obtained by the ratio of values deter-mined in CN-transfected cells over mock-transfected cells.

FIG. 7. The light M3 probe binds NFAT from activated Jurkatnuclear extracts. Nuclear extracts were prepared from unstimulated(UN) Jurkat cells or from Jurkat cells stimulated with immobilizedanti-CD3mAb (TCR) or with PMA and ionomycin in the absence (P � I)or in the presence of CsA (P � I � CsA) for 5 h. The double-strandedcompetitor oligonucleotides were mixed at a 150-fold molar excess withthe probe. Twenty minutes after the addition of the probe, nuclearextract were mixed with affinity-purified mAbs directed against theindicated transcription factors. Arrows identify specific retarded com-plexes corresponding to NFATp (p) and NFATc (c).

LIGHT Transcriptional Regulation in T-lymphocytes42848

by guest on September 23, 2020

http://ww

w.jbc.org/

Dow

nloaded from

(34). Future studies will further assess the contribution ofthese sites to light gene transcription.

The role of NFAT in the transcriptional regulation of light isin agreement with the fact that NFAT proteins have beenimplicated in the CsA-sensitive transcriptional activation of anumber of cytokines, including TNFSF members, expressed byactivated T cells such as TNF� (26), Fas-L (27), TRAIL (tumornecrosis factor-related apoptosis-inducing ligand (28)), andCD40L (29). However, in our EMSA experiments, both therecombinant and the endogenous NFAT proteins from Jurkat Tcells bound the fasL proximal NFAT site with higher affinitythan the light M3 site. In our study, the integrity of the NFATbinding site named M3 was required for the transcriptionalinduction of the pLIGHT(441)-luc reporter construct by over-expression of calcineurin plus PMA stimulation. Because thistranscriptional induction is blocked by co-expression of a dom-inant negative NFAT-mutant, we conclude that M3 accountson its own for the NFAT-dependent transcriptional regulationof the pLIGHT(441)-luc construct. Although light, fasL,CD40L, and tnf� share a common NFAT-dependent transcrip-tional regulatory pathway, light clearly differs by the numberand the avidity of its identified NFAT binding site. However,we cannot rule out the existence of additional unidentifiedNFAT regulatory binding sites.

Another potential role in unstimulated T cells of the 5�-UTRgenomic DNA region is its participation in driving the consti-tutive level of light transcription (Fig. 3D). Our results demon-strate that the basal transcription of human light in T-lympho-cytes is regulated by the sequence contained betweennucleotide �175 and the translational start site of the lightpromoter region. A Sp1 binding site was identified within thisregion. Sp1 transcription factors are ubiquitously expressedproteins that contribute to the constitutive expression of sev-eral cytokine genes including fasL (31, 35). Our EMSA analyses

have shown the Sp1 and Sp3 bind in vitro to light promoterelements. Sp1 has been described as a positive regulator oftranscription, whereas Sp3 has been shown to either activateor repress transcription in different cell types. In addition, therole of Sp3 is promoter- and cell type-specific, and the ratio ofSp1 to Sp3 may be important for the transcriptional regulationand the control of tissue-specific expression. Interestingly, ithas been demonstrated that Sp1 sites could play a critical rolein activation of fasL and tnf� gene transcription by functionalsynergism with the PMA plus ionomycin-induced pathway andNFAT/Jun/ATF2, respectively (36–38). Therefore, Sp1 proteinsmay play a role in both basal and inducible light expression,although the mutation in the light Sp1 binding site did notaffect the induction of the light reporter activity (data notshown).

Moreover, we found that in Jurkat T cells, an Ets bindingsite (nt �165 to �169) must be intact for efficient basal tran-scription mediated by the light promoter region. The Ets familyof transcription factors is defined by a highly conserved DNAbinding domain called the ETS domain, which recognizes a5�-GGA(A/T)-3� core motif. In our experiments, Ets1 proteincan bind light promoter elements mapped to the M4 site (Fig.8). This result is in agreement with the regulation of otherhuman TNFSF promoters by the transcription factor Ets (39–41). Preferential expression of Ets1 occurs in B, T, and NK cellsof adult mice, with high levels in the peripheral T cells (42).Knockout of the Ets1 gene demonstrates an essential role forEts1 protein in survival, maturation and activation of the T celllineage (43, 44). These data parallel the function of LIGHTprotein in negative selection (15) and T cell costimulation (12),suggesting an eventual defect of light expression in Ets1 knock-out model. In addition, because Ets1 is mainly expressed in Tcells, further studies may examine the role of other Ets familymembers, such as PU1 expressed in immature DCs.

FIG. 8. The light M4 probe binds Ets1and Ets2 from Jurkat nuclear extracts. A, EMSA was performed with 10 �g of cellular lysates usingthe labeled wild-type (wt) or mutated (mut) light M4 probes or a fasL-derived probe (28) as a control. Nuclear extracts were prepared fromunstimulated (UN) and stimulated Jurkat T cells as described in Fig. 9. Three specific complexes were identified with the M4 probe (C1 to C3) andone with the fasL-derived NFAT probe (NFAT) and are indicated by arrows. P � I, PMA and ionomycin. B, the double-stranded competitoroligonucleotides were added up to a 150-fold molar excess over the probes. C, 20 min after the addition of the M4 probe, unstimulated nuclearextracts were mixed with affinity-purified monoclonal antibodies directed against the indicated transcription factors. Arrows identify specificretarded complexes.

LIGHT Transcriptional Regulation in T-lymphocytes 42849

by guest on September 23, 2020

http://ww

w.jbc.org/

Dow

nloaded from

Although most of Ets proteins bind to DNA as monomer,DNA binding activity is enhanced or modulated by other fac-tors. Thus, it has been proposed that the ability of the Etsfamily members to mediate trans-activation depends on inter-

actions with other DNA-binding proteins. For example, Ets1has been demonstrated to functionally interact with NF�B,cJun, AP1, Sp1, CBP/p300, Ets2 (for review, see Ref. 45). Dis-ruption of both the Ets and the Sp1 binding site identified inour study resulted in a marked reduction of light promoteractivity (Fig 9B), suggesting that these two binding sites coop-erate to regulate light promoter transcriptional activity. Aphysical interaction between Ets1 and Sp1 and/or Sp3 to act onlight promoter could be envisaged.

Hence, this study begins to address the regulation of LIGHTexpression. Our data identify LIGHT as an additional target ofCsA and evidence the critical role for NFAT transcription fac-tors in activation-induced LIGHT expression in T cells. Conse-quently, the modulation of LIGHT expression might be instru-mental in the immunosuppressive activity of CsA undertransplantation circumstances (13, 46).

Acknowledgments—We thank S. Just for technical assistance andA. Vialle, V. Goffin, and members from Van Lint and Olive laboratoriesfor helpful advice and support.

REFERENCES

1. Locksley, R. M., Killeen, N., and Lenardo, M. J. (2001) Cell 104, 487–5012. Kontoyiannis, D., Pasparakis, M., Pizarro, T. T., Cominelli, F., and Kollias, G.

(1999) Immunity 10, 387–3983. Matsumoto, M. (1999) J. Med. Invest. 46, 141–1504. Sharma, K., Wang, R. X., Zhang, L. Y., Yin, D. L., Luo, X. Y., Solomon, J. C.,

Jiang, R. F., Markos, K., Davidson, W., Scott, D. W., and Shi, Y. F. (2000)Pharmacol. Ther. 88, 333–347

5. Mauri, D. N., Ebner, R., Montgomery, R. I., Kochel, K. D., Cheung, T. C., Yu,G. L., Ruben, S., Murphy, M., Eisenberg, R. J., Cohen, G. H., Spear, P. G.,and Ware, C. F. (1998) Immunity 8, 21–30

6. Morel, Y., Schiano, D. C. J., Harrop, J., Deen, K. C., Holmes, S. D., Wattam,T. A., Khandekar, S. S., Truneh, A., Sweet, R. W., Gastaut, J. A., Olive, D.,and Costello, R. T. (2000) J. Immunol. 165, 4397–4404

7. Tamada, K., Shimozaki, K., Chapoval, A. I., Zhai, Y., Su, J., Chen, S. F., Hsieh,S. L., Nagata, S., Ni, J., and Chen, L. (2000) J. Immunol. 164, 4105–4110

8. Zhai, Y., Guo, R., Hsu, T. L., Yu, G. L., Ni, J., Kwon, B. S., Jiang, G. W., Lu, J.,Tan, J., Ugustus, M., Carter, K., Rojas, L., Zhu, F., Lincoln, C., Endress, G.,Xing, L., Wang, S., Oh, K. O., Gentz, R., Ruben, S., Lippman, M. E., Hsieh,S. L., and Yang, D. (1998) J. Clin. Invest. 102, 1142–1151

9. Morel, Y., Truneh, A., Sweet, R. W., Olive, D., and Costello, R. T. (2001)J. Immunol. 167, 2479–2486

10. Yu, K. Y., Kwon, B., Ni, J., Zhai, Y., Ebner, R., and Kwon, B. S. (1999) J. Biol.Chem. 274, 13733–13736

11. Wang, J., Lo, J. C., Foster, A., Yu, P., Chen, H. M., Wang, Y., Tamada, K.,Chen, L., and Fu, Y. X. (2001) J. Clin. Invest. 108, 1771–1780

12. Harrop, J. A., McDonnell, P. C., Brigham-Burke, M., Lyn, S. D., Minton, J.,Tan, K. B., Dede, K., Spampanato, J., Silverman, C., Hensley, P., DiPrinzio,R., Emery, J. G., Deen, K., Eichman, C., Chabot-Fletcher, M., Truneh, A.,and Young, P. R. (1998) J. Biol. Chem. 273, 27548–27556

13. Tamada, K., Shimozaki, K., Chapoval, A. I., Zhu, G., Sica, G., Flies, D., Boone,T., Hsu, H., Fu, Y. X., Nagata, S., Ni, J., and Chen, L. (2000) Nat. Med. 6,283–289

14. Benedict, C. A., Banks, T. A., Senderowicz, L., Ko, M., Britt, W. J., Angulo, A.,Ghazal, P., and Ware, C. F. (2001) Immunity 15, 617–626

15. Wang, J., Chun, T., Lo, J. C., Wu, Q., Wang, Y., Foster, A., Roca, K., Chen, M.,Tamada, K., Chen, L., Wang, C. R., and Fu, Y. X. (2001) J. Immunol. 167,5099–5105

16. Shaikh, R. B., Santee, S., Granger, S. W., Butrovich, K., Cheung, T.,Kronenberg, M., Cheroutre, H., and Ware, C. F. (2001) J. Immunol. 167,6330–6337

17. Granger, S. W., Butrovich, K. D., Houshmand, P., Edwards, W. R., and Ware,C. F. (2001) J. Immunol. 167, 5122–5128

18. Misawa, K., Nosaka, T., Kojima, T., Hirai, M., and Kitamura, T. (2000) Cyto-genet. Cell Genet. 89, 89–91

19. Bagnasco, M., Nunes, J., Lopez, M., Cerdan, C., Pierres, A., Mawas, C., andOlive, D. (1989) Eur. J. Immunol. 19, 823–827

20. Costello, R., Cerdan, C., Pavon, C., Brailly, H., Hurpin, C., Mawas, C., andOlive, D. (1993) Eur. J. Immunol. 23, 608–613

21. Chow, C. W., Rincon, M., and Davis, R. J. (1999) Mol. Cell. Biol. 19, 2300–230722. Clipstone, N. A., and Crabtree, G. R. (1992) Nature 357, 695–69723. Yang, W. C., Ghiotto, M., Castellano, R., Collette, Y., Auphan, N., Nunes, J. A.,

and Olive, D. (2000) Int. Immunol. 12, 1547–155224. Van Lint, C., Emiliani, S., Ott, M., and Verdin, E. (1996) EMBO J. 15,

1112–112025. Kiani, A., Rao, A., and Aramburu, J. (2000) Immunity 12, 359–37226. Goldfeld, A. E., Tsai, E., Kincaid, R., Belshaw, P. J., Schrieber, S. L.,

Strominger, J. L., and Rao, A. (1994) J. Exp. Med. 180, 763–76827. Latinis, K. M., Norian, L. A., Eliason, S. L., and Koretzky, G. A. (1997) J. Biol.

Chem. 272, 31427–3143428. Mariani, S. M., and Krammer, P. H. (1998) Eur. J. Immunol. 28, 1492–149829. Schubert, L. A., King, G., Cron, R. Q., Lewis, D. B., Aruffo, A., and

Hollenbaugh, D. (1995) J. Biol. Chem. 270, 29624–29627

FIG. 9. Transcriptional activity and binding of Sp1 to lightpromoter elements. A, EMSA was performed with 10 �g of nuclearextracts prepared from Jurkat T cells left unstimulated (UN) or stim-ulated with PMA plus ionomycin using a labeled light-derived wild-type(wt) or mutated (mut) putative Sp1 probes or a Sp1 consensus controlprobe. The double-stranded competitor oligonucleotides were mixed at a150-fold molar excess with the probes. Twenty minutes after the addi-tion of the probe, nuclear extracts were mixed with affinity-purifiedmonoclonal antibodies directed against the indicated transcription fac-tors. Arrows identify specific retarded complexes (C1 to C4). B, JurkatT cells were cotransfected with the different mutated reporter con-structs and pRL-TK to control for transfection efficiency. One repre-sentative experiment of at least three independent experiments isshown. RLU, relative light units.

LIGHT Transcriptional Regulation in T-lymphocytes42850

by guest on September 23, 2020

http://ww

w.jbc.org/

Dow

nloaded from

30. Luo, C., Burgeon, E., and Rao, A. (1996) J. Exp. Med. 184, 141–14731. Xiao, S., Marshak-Rothstein, A., and Ju, S. T. (2001) Eur. J. Immunol. 31,

3339–334832. Scheu, S., Alferink, J., Potzel, T., Barchet, W., Kalinke, U., and Pfeffer, K.

(2002) J. Exp. Med. 195, 1613–162433. Han, J., Huez, G., and Beutler, B. (1991) J. Immunol. 146, 1843–184834. Macian, F., Lopez-Rodriguez, C., and Rao, A. (2001) Oncogene 20, 2476–248935. McClure, R. F., Heppelmann, C. J., and Paya, C. V. (1999) J. Biol. Chem. 274,

7756–776236. Chou, C. F., Peng, H. W., Wang, C. Y., Yang, Y. T., and Han, S. H. (2000)

J. Biomed. Sci. 7, 136–14337. Xiao, S., Matsui, K., Fine, A., Zhu, B., Marshak-Rothstein, A., Widom, R. L.,

and Ju, S. T. (1999) Eur. J. Immunol. 29, 3456–346538. Tsai, E. Y., Yie, J., Thanos, D., and Goldfeld, A. E. (1996) Mol. Cell. Biol. 16,

5232–5244

39. Kramer, B., Wiegmann, K., and Kronke, M. (1995) J. Biol. Chem. 270,6577–6583

40. Tomaras, G. D., Foster, D. A., Burrer, C. M., and Taffet, S. M. (1999) J. Leu-kocyte Biol. 66, 183–193

41. Tsai, E. Y., Falvo, J. V., Tsytsykova, A. V., Barczak, A. K., Reimold, A. M.,Glimcher, L. H., Fenton, M. J., Gordon, D. C., Dunn, I. F., and Goldfeld,A. E. (2000) Mol. Cell. Biol. 20, 6084–6094

42. Bassuk, A. G., and Leiden, J. M. (1997) Adv. Immunol. 64, 65–10443. Bories, J. C., Willerford, D. M., Grevin, D., Davidson, L., Camus, A., Martin, P.,

Stehelin, D., and Alt, F. W. (1995) Nature 377, 635–63844. Muthusamy, N., Barton, K., and Leiden, J. M. (1995) Nature 377, 639–64245. Li, R., Pei, H., and Watson, D. K. (2000) Oncogene 19, 6514–652346. Ye, Q., Fraser, C. C., Gao, W., Wang, L., Busfield, S. J., Wang, C., Qiu, Y.,

Coyle, A. J., Gutierrez-Ramos, J. C., and Hancock, W. W. (2002) J. Exp.Med. 195, 795–800

LIGHT Transcriptional Regulation in T-lymphocytes 42851

by guest on September 23, 2020

http://ww

w.jbc.org/

Dow

nloaded from

Régis Costello, Daniel Olive and Yves ColletteRémy Castellano, Carine Van Lint, Valentine Péri, Emmanuelle Veithen, Yannis Morel,

CONTROLROLE OF CALCIUM-SIGNALING PATHWAY IN THE TRANSCRIPTIONAL

Gene:lightMechanisms Regulating Expression of the Tumor Necrosis Factor-related

doi: 10.1074/jbc.M207689200 originally published online September 4, 20022002, 277:42841-42851.J. Biol. Chem. 

  10.1074/jbc.M207689200Access the most updated version of this article at doi:

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

to choose from all of JBC's e-mail alertsClick here

  http://www.jbc.org/content/277/45/42841.full.html#ref-list-1

This article cites 46 references, 20 of which can be accessed free at

by guest on September 23, 2020

http://ww

w.jbc.org/

Dow

nloaded from