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• W2068192226 abstract "LIGHT (TNFSF14) is a newly identified tumor necrosis factor superfamily member involved in the regulation of immune responses by control of activation, maturation, and survival of immune effector cells. Despite the immunological relevance of the LIGHT protein, little knowledge is available as to how light gene expression is regulated. In T-lymphocytes, most LIGHT surface expression and transcript accumulation occurs after T cell activation. In this study, we have shown that these events are blocked at the transcriptional level by cyclosporin A, an immuno-suppressive drug. Besides, we identified a role for Ca2+-signaling pathways and NFAT transcription factors in T cell activation-induced LIGHT expression. To further investigate this process, we have identified, cloned, and characterized a 2.1-kilobase 5′-flanking DNA genomic fragment from the human light gene. We have shown the transcriptional activity of the herein-identified minimal 5′ regulatory region of human light gene parallels the endogenous expression of light in T cells. Moreover, we demonstrated that induced LIGHT promoter activity can be equally blocked by cyclosporin A treatment or dominant negative NFAT overexpression and further identified by site-directed mutagenesis and electrophoretic mobility supershift analysis of a NFAT transcription factor binding site within the human light minimal promoter. Finally, Sp1 and Ets1 binding sites were identified and shown to regulate light basal promoter activity. Thus, the present study establishes a molecular basis to further understand the mechanisms governing human light gene expression and, consequently, could potentially lead to novel therapeutic manipulations that control the signaling cascade, resulting in LIGHT production in conditions characterized by immunopathologic activation of T cells. LIGHT (TNFSF14) is a newly identified tumor necrosis factor superfamily member involved in the regulation of immune responses by control of activation, maturation, and survival of immune effector cells. Despite the immunological relevance of the LIGHT protein, little knowledge is available as to how light gene expression is regulated. In T-lymphocytes, most LIGHT surface expression and transcript accumulation occurs after T cell activation. In this study, we have shown that these events are blocked at the transcriptional level by cyclosporin A, an immuno-suppressive drug. Besides, we identified a role for Ca2+-signaling pathways and NFAT transcription factors in T cell activation-induced LIGHT expression. To further investigate this process, we have identified, cloned, and characterized a 2.1-kilobase 5′-flanking DNA genomic fragment from the human light gene. We have shown the transcriptional activity of the herein-identified minimal 5′ regulatory region of human light gene parallels the endogenous expression of light in T cells. Moreover, we demonstrated that induced LIGHT promoter activity can be equally blocked by cyclosporin A treatment or dominant negative NFAT overexpression and further identified by site-directed mutagenesis and electrophoretic mobility supershift analysis of a NFAT transcription factor binding site within the human light minimal promoter. Finally, Sp1 and Ets1 binding sites were identified and shown to regulate light basal promoter activity. Thus, the present study establishes a molecular basis to further understand the mechanisms governing human light gene expression and, consequently, could potentially lead to novel therapeutic manipulations that control the signaling cascade, resulting in LIGHT production in conditions characterized by immunopathologic activation of T cells. tumor necrosis factor TNF superfamily nuclear factor of activated T cells reverse transcription thymidine kinase T cell receptor dendritic cell untranslated region cyclosporin A phorbol 12-myristate 13-acetate monoclonal antibody rapid amplification of cDNA ends calcineurin phosphatase dominant negative electrophoretic mobility shift assay nucleotides The TNF1 superfamily (TNFSF) is formed of type II transmembrane glycoproteins that can be cleaved and secreted as active trimeric cytokines. Most TNFSFs are expressed in the immune system, where they play important roles in lymphocyte activation, regulation of the immune response, and the development of lymphoid tissues (1Locksley R.M. Killeen N. Lenardo M.J. Cell. 2001; 104: 487-501Abstract Full Text Full Text PDF PubMed Scopus (3018) Google Scholar). Hence, expression of TNFSF is highly regulated, whereas its dysregulation can lead to severe pathologies (2Kontoyiannis D. Pasparakis M. Pizarro T.T. Cominelli F. Kollias G. Immunity. 1999; 10: 387-398Abstract Full Text Full Text PDF PubMed Scopus (1101) Google Scholar, 3Matsumoto M. J. Med. Invest. 1999; 46: 141-150PubMed Google Scholar, 4Sharma 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. Shi Y.F. Pharmacol. Ther. 2000; 88: 333-347Crossref PubMed Scopus (185) Google Scholar). LIGHT protein (TNFSF14) is a newly identified TNFSF member (5Mauri 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. Ware C.F. Immunity. 1998; 8: 21-30Abstract Full Text Full Text PDF PubMed Scopus (651) Google Scholar) expressed by activated T-lymphocytes (6Morel 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. Costello R.T. J. Immunol. 2000; 165: 4397-4404Crossref PubMed Scopus (152) Google Scholar) but also monocytes, granulocytes, and immature dendritic cells (DCs) (7Tamada K. Shimozaki K. Chapoval A.I. Zhai Y., Su, J. Chen S.F. Hsieh S.L. Nagata S., Ni, J. Chen L. J. Immunol. 2000; 164: 4105-4110Crossref PubMed Scopus (325) Google Scholar, 8Zhai 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. Yang D. J. Clin. Invest. 1998; 102: 1142-1151Crossref PubMed Scopus (235) Google Scholar). LIGHT is a ligand for three TNFSF receptors: herpes virus entry mediator, mainly expressed by T cells (5Mauri 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. Ware C.F. Immunity. 1998; 8: 21-30Abstract Full Text Full Text PDF PubMed Scopus (651) Google Scholar, 6Morel 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. Costello R.T. J. Immunol. 2000; 165: 4397-4404Crossref PubMed Scopus (152) Google Scholar, 9Morel Y. Truneh A. Sweet R.W. Olive D. Costello R.T. J. Immunol. 2001; 167: 2479-2486Crossref PubMed Scopus (159) Google Scholar); lymphotoxin β receptor, expressed by stromal and non-lymphoid hematopoietic cells (8Zhai 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. Yang D. J. Clin. Invest. 1998; 102: 1142-1151Crossref PubMed Scopus (235) Google Scholar); and the decoy receptor 3 (DcR3/TR6), which is predominantly expressed in lung tissue and the colon carcinoma cell line SW480 and might modulate LIGHT function in vivo (10Yu K.Y. Kwon B., Ni, J. Zhai Y. Ebner R. Kwon B.S. J. Biol. Chem. 1999; 274: 13733-13736Abstract Full Text Full Text PDF PubMed Scopus (343) Google Scholar). Functionally, LIGHT regulates apoptosis (8Zhai 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. Yang D. J. Clin. Invest. 1998; 102: 1142-1151Crossref PubMed Scopus (235) Google Scholar, 11Wang J., Lo, J.C. Foster A., Yu, P. Chen H.M. Wang Y. Tamada K. Chen L. Fu Y.X. J. Clin. Invest. 2001; 108: 1771-1780Crossref PubMed Scopus (216) Google Scholar) but is also involved in the control of the immune response by enhancing T cell proliferation and cytokine secretion (7Tamada K. Shimozaki K. Chapoval A.I. Zhai Y., Su, J. Chen S.F. Hsieh S.L. Nagata S., Ni, J. Chen L. J. Immunol. 2000; 164: 4105-4110Crossref PubMed Scopus (325) Google Scholar, 12Harrop 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. Young P.R. J. Biol. Chem. 1998; 273: 27548-27556Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar, 13Tamada K. Shimozaki K. Chapoval A.I. Zhu G. Sica G. Flies D. Boone T. Hsu H., Fu, Y.X. Nagata S., Ni, J. Chen L. Nat. Med. 2000; 6: 283-289Crossref PubMed Scopus (275) Google Scholar) as well as by inducting DC maturation (9Morel Y. Truneh A. Sweet R.W. Olive D. Costello R.T. J. Immunol. 2001; 167: 2479-2486Crossref PubMed Scopus (159) Google Scholar). Thus, LIGHT was shown both in vitro andin vivo to regulate cell death and survival (7Tamada K. Shimozaki K. Chapoval A.I. Zhai Y., Su, J. Chen S.F. Hsieh S.L. Nagata S., Ni, J. Chen L. J. Immunol. 2000; 164: 4105-4110Crossref PubMed Scopus (325) Google Scholar, 11Wang J., Lo, J.C. Foster A., Yu, P. Chen H.M. Wang Y. Tamada K. Chen L. Fu Y.X. J. Clin. Invest. 2001; 108: 1771-1780Crossref PubMed Scopus (216) Google Scholar), to play a role in antitumor activity (7Tamada K. Shimozaki K. Chapoval A.I. Zhai Y., Su, J. Chen S.F. Hsieh S.L. Nagata S., Ni, J. Chen L. J. Immunol. 2000; 164: 4105-4110Crossref PubMed Scopus (325) Google Scholar, 13Tamada K. Shimozaki K. Chapoval A.I. Zhu G. Sica G. Flies D. Boone T. Hsu H., Fu, Y.X. Nagata S., Ni, J. Chen L. Nat. Med. 2000; 6: 283-289Crossref PubMed Scopus (275) Google Scholar), and to interfere with virus infection (5Mauri 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. Ware C.F. Immunity. 1998; 8: 21-30Abstract Full Text Full Text PDF PubMed Scopus (651) Google Scholar, 14Benedict C.A. Banks T.A. Senderowicz L., Ko, M. Britt W.J. Angulo A. Ghazal P. Ware C.F. Immunity. 2001; 15: 617-626Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Moreover, different experimental models have demonstrated that transgenic expression of light in the T cell compartment leads to hyperactivated peripheral T cell population, altered T cell homeostasis, and severe autoimmune diseases (15Wang J. Chun T., Lo, J.C., Wu, Q. Wang Y. Foster A. Roca K. Chen M. Tamada K. Chen L. Wang C.R. Fu Y.X. J. Immunol. 2001; 167: 5099-5105Crossref PubMed Scopus (71) Google Scholar,16Shaikh R.B. Santee S. Granger S.W. Butrovich K. Cheung T. Kronenberg M. Cheroutre H. Ware C.F. J. Immunol. 2001; 167: 6330-6337Crossref PubMed Scopus (208) Google Scholar). Despite the immunological relevance of the LIGHT protein and its specific cell and tissue distribution, little knowledge is available as to how light gene expression is regulated. lightis up-regulated in T cells, whereas constitutive expression of the gene in immature DCs is down-regulated in mature DCs (6Morel 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. Costello R.T. J. Immunol. 2000; 165: 4397-4404Crossref PubMed Scopus (152) Google Scholar, 7Tamada K. Shimozaki K. Chapoval A.I. Zhai Y., Su, J. Chen S.F. Hsieh S.L. Nagata S., Ni, J. Chen L. J. Immunol. 2000; 164: 4105-4110Crossref PubMed Scopus (325) Google Scholar). Interestingly, the activation-induced expression of light in T cell lymphocytes is correlated with a decrease of herpes virus entry mediator membrane expression, suggesting a regulatory feedback loop controlling LIGHT functions (6Morel 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. Costello R.T. J. Immunol. 2000; 165: 4397-4404Crossref PubMed Scopus (152) Google Scholar). Post-translational modification of LIGHT by processing from a membrane into a soluble form by matrix metalloproteinases (6Morel 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. Costello R.T. J. Immunol. 2000; 165: 4397-4404Crossref PubMed Scopus (152) Google Scholar) or an alternative splicing of lightmRNA (17Granger S.W. Butrovich K.D. Houshmand P. Edwards W.R. Ware C.F. J. Immunol. 2001; 167: 5122-5128Crossref PubMed Scopus (74) Google Scholar) has been identified. However detailed molecular mechanisms governing its transcription are lacking. Recently, the mouselight cDNA (18Misawa K. Nosaka T. Kojima T. Hirai M. Kitamura T. Cytogenet. Cell Genet. 2000; 89: 89-91Crossref PubMed Scopus (12) Google Scholar) and the genomic organization of the human light gene (17Granger S.W. Butrovich K.D. Houshmand P. Edwards W.R. Ware C.F. J. Immunol. 2001; 167: 5122-5128Crossref PubMed Scopus (74) Google Scholar) have been partially elucidated, but the 5′-untranslated regions (UTRs) were not characterized. In T-lymphocyte, most LIGHT surface expression and transcript accumulation occurs after T cell activation (6Morel 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. Costello R.T. J. Immunol. 2000; 165: 4397-4404Crossref PubMed Scopus (152) Google Scholar). In this report, we show that these events are blocked at the transcriptional level by cyclosporin A (CsA), an immuno-suppressive drug acting on the phosphatase calcineurin. We identified a role for Ca2+-signaling pathways and NFAT transcription factors in T cell activation-induced light transcription and a role for both Ets and Sp1 families of transcription factors in basal constitutive expression of light in T cells. The Jurkat T cell line (JA16 clone (19Bagnasco M. Nunes J. Lopez M. Cerdan C. Pierres A. Mawas C. Olive D. Eur. J. Immunol. 1989; 19: 823-827Crossref PubMed Scopus (33) Google Scholar)) was maintained in RPMI 1640 medium supplemented with 10% fetal calf serum. Peripheral blood mononuclear cells from healthy donors were isolated on Ficoll-Hypaque gradients (20Costello R. Cerdan C. Pavon C. Brailly H. Hurpin C. Mawas C. Olive D. Eur. J. Immunol. 1993; 23: 608-613Crossref PubMed Scopus (45) Google Scholar). T-lymphocytes were isolated as the CD2-positive peripheral blood mononuclear cell population, corresponding to cells that adhere to sheep erythrocytes in the E-rosetting technique but fail to adhere to plastic dishes after overnight incubation in medium and 30% fetal calf serum. T-lymphocytes and the Jurkat T cell line were stimulated with 1 ng/ml and 20 ng/ml PMA (Sigma), respectively, and/or 1 μg/ml and 2 μg/ml ionomycin (Calbiochem), respectively, either in the presence or absence of CsA at 5–100 ng/ml (Calbiochem). The monoclonal antibody (mAb) 289 (kindly gift by Alessandro Moretta) recognizes the CD3ε chain of the T cell receptor complex and was immobilized on plastic culture dishes for TCR stimulation. The expression of light mRNA and protein was analyzed by RT-PCR and cyto-fluorometry, respectively, using the previously described primers pair and anti-LIGHT 2C8 monoclonal antibody (6Morel 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. Costello R.T. J. Immunol. 2000; 165: 4397-4404Crossref PubMed Scopus (152) Google Scholar). The transcription start site of the human lightgene was mapped in T-lymphocytes by using the 5′-RACE system for rapid amplification of cDNA ends, version 2.0 (Invitrogen) according to the manufacturer's protocol. In brief, total RNA from T-lymphocytes stimulated with PMA and ionomycin for 16 h was reversed-transcribed using the primer GSP1. PCR was performed on dC tailed with the 5′-RACE-abridged anchor primer associated with GSP2. The nested amplification was performed with the 5′-RACE-abridged universal amplification and GSP3 primers. The three GSP1, -2, and -3 primer sequences are shown in Fig.1. The amplified products were cloned using the pGEM-T Easy Vector System (Promega Corp, Madison, WI) and sequenced. The genomic sequence (−2186pb/+1) lying upstream to the human lightgene was isolated by PCR using the following primers: light/sense 5′-GAGTCAAGACGCAGATGAGCAGGGGGAGCC-3′ and light/antisense 5′-CCGAAGCTTGCCCAAGGTGTCTGGAGCAGGGCTGACACGC-3′. The PCR product was cloned in pGEM-T Easy Vector. The fragments generated by enzymatic restrictions of pGEM-T/pLIGHT(2148) with BglII,NdeI, and KpnI, respectively, for the 5′ site andHindIII for the 3′ site were cloned into the reporter vector pGL2-Basic (Promega); these reporter constructs are called pLIGHT(2148)-luc, pLIGHT(1033)-luc, and pLIGHT(175)-luc, respectively. The primers used for the other light promoter constructs were: LIGHT(441)-luc, 5′-CTACTCGAGCTTGTCTCTCTGGCTCCACCAG-3′; LIGHT(124)-luc, 5′-CTACTCGAGCTCTAAAGGCGGCCCACGGGTG-3′, and LIGHT(91)-luc, 5′-CTACTCGAGCGTGCACAGCCCAGGAGTGTTGAG-3′ and the 3′ primer described above. The XhoI and HindIII sites in these primers are underlined. The amplified cDNA fragments were cloned into the XhoI and HindIII sites in pGL2Basic. Whole nucleotide sequences from these constructs were confirmed by sequencing. pLIGHT(441)-luc and pLIGHT(175)-luc were used as templates for mutagenesis performed by the QuikChange site-directed mutagenesis method (Stratagene). The primers used are: M1, 5′-GGCTCCACCAGAAGACTCGCAGGGACCCTTCTTGC-3′; M2, 5′-GAAGCATCCAAGAAGCTCAAGCTGGGGGCTCCC-3′; M3, 5′-CATTTTCAGAAGCCTCTCTCAAGTGTGAGAGTCTG-3′; M4, 5′-GCGGCGGGTACCGGACTCAGAGGAGGGTGAGTG-3′; M5, 5′-GAGCAATTTCGGTTGAGTCTGAGGTTGAAGGACCC-3′; Sp1mut, 5′-GAGGAGGGTGAGTGGTTGAAGTTGTGTTGCTGAACCCCAGCTC-3′. The mutations were confirmed by sequencing. The pDN-NFAT expression construct was previously described (21Chow C.W. Rincon M. Davis R.J. Mol. Cell. Biol. 1999; 19: 2300-2307Crossref PubMed Scopus (160) Google Scholar). The calcineurin phosphatase (CN) cDNA (22Clipstone N.A. Crabtree G.R. Nature. 1992; 357: 695-697Crossref PubMed Scopus (1476) Google Scholar) was subcloned from pBJ5 into the BamHI-digested BDNA4 expression vector (23Yang W.C. Ghiotto M. Castellano R. Collette Y. Auphan N. Nunes J.A. Olive D. Int. Immunol. 2000; 12: 1547-1552Crossref PubMed Scopus (15) Google Scholar). 107Jurkat T cells were electroporated at 960 microfarads and 250 V using Bio-Rad Gene Pulser with 25 μg of pGL2/basic or the variouslight promoter-driven luciferase firefly constructs (pLIGHT-luc) together with 5 μg of thymidine kinase-luciferaseRenilla plasmid. The cells were incubated for 2 h, then left unstimulated or stimulated for 16 h as previously described (23Yang W.C. Ghiotto M. Castellano R. Collette Y. Auphan N. Nunes J.A. Olive D. Int. Immunol. 2000; 12: 1547-1552Crossref PubMed Scopus (15) Google Scholar). The cells were collected, washed in phosphate-buffered saline, lysed to determine the luciferase activity by using the dual luciferase reporter assay according to manufacturer's instructions (Promega), and read using a luminometer (Dynex). The transfection efficiency was normalized to luciferase Renilla activity and corrected for protein content as determined by the Bradford protein assay (Bio-Rad). The reported values represent the average of three independent transfections, with standard deviation as error bars. Nuclear extracts from Jurkat cells were prepared as previously described (24Van Lint C. Emiliani S. Ott M. Verdin E. EMBO J. 1996; 15: 1112-1120Crossref PubMed Scopus (484) Google Scholar). Electrophoretic mobility shift assay were performed with 10 μg of nuclear protein extract or 0.25–1 ng of recombinant protein in a 20-μl reaction mixture containing 2 μg of DNase-free bovine serum albumin (Amersham Biosciences), 1 μg of poly(dI-dC) (AmershamBiosciences) as nonspecific DNA competitor, 1 mmdithiothreitol, 20 mm Hepes buffer, pH 7.3, and 10% (V/V) glycerol. The binding reactions were incubated for 20 min at room temperature with 10,000–24,000 cpm of double-stranded oligonucleotide 5′ end-labeled with [γ-32P]ATP using the T4 polynucleotide kinase. The reaction mixture was loaded directly onto a 6% non-denaturing polyacrylamide 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 the wild-type NFAT binding site in the light promoter (M3wt), 5′-CAGAAGCCTCTGGAAAGTGTGAGAGTC-3′, and its mutated homologue (M3mut), 5′-CAGAAGCCTCTCTCAAGTGTGAGAGTC-3′; for the control NFAT binding site in the FasL promoter (FasLwt), 5′-TAGCTATGGAAACTCTATA-3′; for the oligonucleotide containing the Ets binding site (M4wt), 5′-GCGGGTACCGGAAGAAGAGGAGGGTG-3′ and its mutated version (M4mut), 5′-GCGGGTACCGGACTCAGAGGAGGGTG-3′; finally, for the oligonucleotide containing the Sp1 binding site (Sp1wt), 5′-GAGTGGGGGAAGGGGTGGGGCTGAA-3′, and its mutated version (M4mut), 5′-GAGTGGTTGAAGTTGTGTTGCTGAA-3′, and the Sp1 consensus 5′-ATTCGATCGGGGCGGGGCGAGC-3′ (Promega). For supershift assays, 1 μl of monoclonal antibodies against NFATc (sc-7294), NFATp (sc-7295), or CREB (cAMP-response element-binding protein; sc-271) as a negative control or 1 μl of polyclonal antibodies against Sp1 (sc-059x), Sp2 (sc-643x), Sp3 (sc-644x), or Sp4 (sc-645x) (Santa Cruz Biotechnology) were added to the binding reaction mixture at the end of the binding reaction for an additional 30 min of incubation at room temperature before electrophoresis. For blocking assays, antibodies against Ets-1 (sc-350) and Ets-2 (sc-351) or MEF-2 (sc-313x) as a negative control (Santa Cruz Biotechnology) were added at the beginning of the binding reaction before the nuclear extracts. When indicated, a 150-fold molar excess of unlabeled homologous oligonucleotide (Ets, 5′-tcgggctcgagataaacaggaagtggtctcgg-3′) or heterologous oligonucleotide (Ebox, 5′-AGCTTCAGACCACGTGGTCGGG-3′ or Oct, 5′-TGTCGAATGCAAATCACTAGAA-3′) oligonucleotide was used as competitor. The induction oflight in T cells requires cellular activation by the TCR/CD3 (8Zhai 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. Yang D. J. Clin. Invest. 1998; 102: 1142-1151Crossref PubMed Scopus (235) Google Scholar) or by treatment with PMA and calcium ionophore (6Morel 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. Costello R.T. J. Immunol. 2000; 165: 4397-4404Crossref PubMed Scopus (152) Google Scholar). A common feature to ionomycin- and CD3-initiated signal transduction is the increase of [Ca2+]i followed by the nuclear translocation of the NFAT transcription factors. The CsA immuno-suppressive drug blocks induced transcription of several cytokines (for review, see Ref. 25Kiani A. Rao A. Aramburu J. Immunity. 2000; 12: 359-372Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar) by inhibiting the phosphatase CN, a second messenger protein activated by [Ca2+]i and controlling the nuclear translocation of NFAT. Moreover, several TNS SF members such as TNFα, FasL, TRAIL (tumor necrosis factor-related apoptosis-inducing ligand), and CD40L are regulated by NFAT and inhibited by CsA (26Goldfeld A.E. Tsai E. Kincaid R. Belshaw P.J. Schrieber S.L. Strominger J.L. Rao A. J. Exp. Med. 1994; 180: 763-768Crossref PubMed Scopus (84) Google Scholar, 27Latinis K.M. Norian L.A. Eliason S.L. Koretzky G.A. J. Biol. Chem. 1997; 272: 31427-31434Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 28Mariani S.M. Krammer P.H. Eur. J. Immunol. 1998; 28: 1492-1498Crossref PubMed Scopus (155) Google Scholar, 29Schubert L.A. King G. Cron R.Q. Lewis D.B. Aruffo A. Hollenbaugh D. J. Biol. Chem. 1995; 270: 29624-29627Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). To get insight into the mechanisms of light gene expression, we performed experiments with CsA. As shown in Fig.1, panels A and B, LIGHT was absent from the cell surface of activated lymphocytes incubated in the presence of CsA. To specify whether the effect of CsA on expression was transcriptional, we also analyzed thelight RNA expression. As shown in panel C, a faint signal was detected in unstimulated T-lymphocytes at 30 and 33 PCR cycles. In contrast, light messenger was detected at 27 PCR cycles in PMA plus ionomycin-stimulated lymphocytes with a quantitatively more significant signal. In contrast, the presence of CsA during T cell stimulation significantly decreased lightRNA expression, which remained undetectable at 27 PCR cycles and produced a faint signal at 30 PCR cycles. To normalize both quantity and quality of the RT products, we used the β-actin housekeeping gene as a control. Results from these experiments demonstrate that the induction of light transcription by PMA plus ionomycin is prevented by CsA and hint to the likely involvement of the NFAT family of transcription factors. To investigate the regulation by CsA of the LIGHT transcription, we identified the promoter region in the human light gene by multiple BLAST analysis between human light cDNA sequence (accession number NM_003807) compared with a genomic sequence data base. Wholelight cDNA sequences matched the chromosome 19p13.3 (accession number NT_0111458.12) at nt 366054–371642 (including intron sequences) with 100% identity. These results were confirmed by Grangeret al. (17Granger S.W. Butrovich K.D. Houshmand P. Edwards W.R. Ware C.F. J. Immunol. 2001; 167: 5122-5128Crossref PubMed Scopus (74) Google Scholar) during the preparation of this manuscript. To identify the transcription initiation site of humanlight, we used a 5′-RACE approach. Using downstream oligonucleotide sequences present in the first exon, a single PCR product was amplified. Sequence analysis revealed that the major start site maps 91 bp upstream to the ATG. To confirm this result, RT-PCR reactions were performed with two 5′ primers located near the identified transcription start site (Primers A andB, see Fig. 2 A) and the same 3′ primer within exon 1 (primer GSP2). Using T cell-derived reverse-transcribed mRNA as a template, the B/GSP2 but not the A/GSP2 primer pairs allowed amplification of the expected 128-bp fragments (Fig. 2 B), thereby confirming the preferential usage of the transcription initiation start site located 91 bp upstream to the ATG. Next, homologies to known cis-regulatory elements were determined using the MatInspector (Genomatix Software, Munich, Germany) and TESS (Transcription Element Search System, CBIL, US) programs. A TATA motif (nt −116 to −121) and GC-rich domains were identified close to the transcription initiation site (Fig. 3 A). Furthermore, the proximal promoter region of light resembles other known cytokine gene promoters, particularly promoters controlling TNFSF gene products, in that it possesses several potential binding sites for TCR-inducible transcription factors such as NFAT (Fig.3 A).Figure 3light promoter/reporter deletional analysis. A, nucleotide sequence of the potential promoter and of the first exon of the human light gene (AF542509). The ATG site is boldface (was assigned the position nucleotide +1). Three gene-specific primers (GSP1, -2, and -3) used in 5′-RACE procedure and mapping of the 5′ end of lightmRNA are underlined. Two primers (A and B) used in PCR to map the transcription start site are underlined (see “Results”). The first nucleotide of the 5′-RACE product is indicated by an arrow. Putative cis-acting motifs areunderlined or boxed. The constructs were generated by cloning progressively 5′-truncated human lightpromoter fragments into the pGL2/basic luciferase vector.Negative numbers denote bp distances from translational start codon (panel B). Jurkat cells were transiently cotransfected with these reporter constructs and pRL-TK to control for transfection efficiency. Cells were either left unstimulated (panel C) or stimulated with PMA plus ionomycin (panel D) for 16 h. RLU, relative light units.View Large Image Figure ViewerDownload (PPT) To evaluate promoter activity of the 5′-UTR regions of the humanlight gene, the 2148 bp of genomic DNA immediately 5′ to the translational start site were cloned in pGL2/Basic. Promoter activity of the construct pLIGHT(2148)-luc was assayed by measuring firefly luciferase activity after transient transfection into the JA16 Jurkat T cell line, which expresses light mRNA (Fig.2 C) and protein (data not shown) after T-cell activation. Similarly, treatment of Jurkat T cells with PMA plus ionomycin or CD3-immobilized antibody significantly enhanced the exogenous luciferase activity by ∼200- and 20-fold for PMA plus ionomycin and CD3, respectively (Fig. 2 D). To map the minimal promoter region in the light gene required for initiation and induction of gene transcription, luciferase reporter constructs containing progressive deletions of the 2148-bp genomic DNA fragment were generated (Fig. 3 B). Each construct as well as the control vector pGL2/Basic were transiently transfected into Jurkat T cells and assayed for reporter activity. Our results show that the 2148-bp fragment induces a large increase in basal luciferase activity and that deletions of DNA regions upstream to nt −175 (relative to the ATG) did not significantly decrease basal luciferase expression as compared with the full-length 2148-bp fragment (Fig. 3 C). However, deletion of an additional 53 bp (from nt −175 to −122) reduced reporter activity to a level similar to that obtained with the promoterless luciferase construct (pGL2/Basic) (Fig. 3 C). These results suggest that the cis-regulatory elements required for the basal transcription of the light gene are located in a 175-bp region upstream to the ATG and containing the transcription initiatio" @default.
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• W2068192226 date "2002-11-01" @default.
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• W2068192226 title "Mechanisms Regulating Expression of the Tumor Necrosis Factor-related light Gene" @default.
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• W2068192226 doi "https://doi.org/10.1074/jbc.m207689200" @default.
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