Txk, a Member of Nonreceptor Tyrosine Kinase of Tec Family, Acts as a Th1 Cell-Specific Transcription Factor and Regulates IFN- γ Gene Transcription This information is current as of September 29, 2021. Yuko Takeba, Hiroko Nagafuchi, Mitsuhiro Takeno, Jun-ichi Kashiwakura and Noboru Suzuki J Immunol 2002; 168:2365-2370; ; doi: 10.4049/jimmunol.168.5.2365 http://www.jimmunol.org/content/168/5/2365 Downloaded from References This article cites 40 articles, 21 of which you can access for free at: http://www.jimmunol.org/content/168/5/2365.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! 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Txk, a Member of Nonreceptor Tyrosine Kinase of Tec Family, Acts as a Th1 Cell-Specific Transcription Factor and Regulates IFN-␥ Gene Transcription1 Yuko Takeba,2* Hiroko Nagafuchi,2† Mitsuhiro Takeno,* Jun-ichi Kashiwakura,* and Noboru Suzuki3* Precise mechanisms responsible for Th1 cell activation and differentiation are not fully elucidated. We have recently reported that Txk, a member of Tec family nonreceptor tyrosine kinase, is expressed on Th1/Th0 cells, and Txk regulates specifically IFN-␥ gene expression. In this study, we found that Txk bound to IFN-␥ promoter region. Txk transfection increased transcriptional activity of IFN-␥ promoter plus luciferase constructs severalfold, including IFN-␥ promoter ؊538, ؊208, and ؊53. IFN-␥ promoter ؊39 was refractory to the Txk transfection. The actual site to which Txk bound was the element consisting of ؊53 and ؊39 bp from Downloaded from the transcription start site of human IFN-␥ gene, a site distinct from several previously characterized binding sites. We found that the entire ؊53/؊39 region was necessary for the binding to and function of Txk, because mutant promoter oligoDNA that contained contiguous five base substitutions dispersed throughout the ؊53/؊39 inhibited the binding, and the mutant promoters did not respond to the Txk transfection. Similar sequences of this element are found within the 5؅ flanking regions of several Th1 cell-associated protein genes. Thus, Txk is expressed on Th1/Th0 cells with the IFN-␥ production and acts as a Th1 cell-specific transcription factor. The Journal of Immunology, 2002, 168: 2365–2370. http://www.jimmunol.org/ uch interest has focused on Th1 and Th2 subsets that presumed to function in vivo as important signaling mediators have been characterized on the basis of the discrete (26–31). Schneider et al. (26) suggested that TCR can utilize M cytokine production profiles; Th1 cells secrete IL-2, mouse Rlk (as well as ZAP-70) in the phosphorylation of key sites IFN-␥, and lymphotoxin and are important for the cell-mediated in the adaptor protein, SLP-76, leading to the up-regulation of response; Th2 cells produce IL-4, IL-5, IL-10, and IL-13 and pro- Th1-preferred cytokine IL-2. Similarly, Rajagopal et al. (27) iden- vide help for Ig production (1–4). Accumulating evidence sug- tified the T cell-specific adaptor protein, RIBP, which binds to gests that distinct signaling molecules and transcription factors mouse Rlk/Txk and modulates production of IL-2 and IFN-␥. mediate cytokine expression pattern in Th1 and Th2 cells (5–13). However, information concerning roles of Txk in human T lym- by guest on September 29, 2021 However, to date, precise mechanisms responsible for the differ- phocyte function is limited. We have recently reported that Txk entiation and development of polarized Th1 responses are not fully expression is restricted to Th1/Th0 cells with IFN-␥-producing clarified in humans. Especially, intracellular signaling pathway potential, and that Txk transfection resulted in severalfold increase specific for Th1 cells remains elucidated. of IFN-␥ mRNA expression and protein production by up-regu- The Tec family has emerged recently as a subfamily of nonre- lating IFN-␥ enhancer activity specifically (32). This finding ceptor tyrosine kinases, consisting of Tec, Btk, Itk/Tsk/Emt, Bmx, prompted us to study a mechanism of Txk to provoke IFN-␥ pro- and Txk/Rlk, all of which are importantly involved in the lym- duction in humans. phocyte signaling pathways (14–24). Recently, Itk, the T cell-as- sociated Tec family kinase, has been suggested for the involve- Materials and Methods ment of Th2 cell development (5, 25). Txk/Rlk has been shown to Plasmid vectors be involved in signaling pathways of lymphocyte activation and is Human Txk cDNA in ␭ phage was provided by G. W. Litman (University of South Florida, St. Petersburg, FL) (16). Full-length Txk cDNA was ligated into a mammalian expression vector, pME18S (SR-␣ promoter; † *Departments of Immunology and Medicine, and Institute of Medical Science, St. provided by K. Maruyama, Tokyo Medical and Dental University, Tokyo, Marianna University School of Medicine, Kawasaki, Kanagawa, Japan Japan), as described (32). Received for publication May 8, 2001. Accepted for publication December 21, 2001. IFN-␥ promoter plus luciferase plasmids were kindly provided by C. B. The costs of publication of this article were defrayed in part by the payment of page Wilson (University of Washington, Seattle, WA) and H. A. Young (Na- charges. This article must therefore be hereby marked advertisement in accordance tional Cancer Institute, Frederick, MD) (33, 34). with 18 U.S.C. Section 1734 solely to indicate this fact. The IFN-␥ promoter mutant plus luciferase plasmids were created using 1 QuickChange site-directed mutagenesis kit (Stratagene, La Jolla, CA.). This work was supported, in part, by a grant for the Promotion of the Advancement ␥ Ϫ of Education and Research in Graduate Schools from the Promotion and Mutual Aid Briefly, pIFN- promoter 208 plus luciferase was used as a template. The Corporation for Private Schools of Japan; a grant-in-aid for Scientific Research primers containing the desired mutation were extended during PCR cycling Project 13037033 and special coordination funds from the Ministry of Education, by PfuTurbo DNA polymerase (Stratagene). The amplification cycle con- Culture, Sports and Technology of Japan; Comprehensive Research on Aging and sisted of one cycle of denaturation (95°C) for 1 min, followed by 18 cycles Health research grants from the Ministry of Health, Labor and Welfare of Japan; and of denaturation (95°C) for 30 s, annealing for 1 min (55°C), and polymer- a grant from the SRF Foundation. ization for 10 min (68°C). After PCR cycling, the PCR product was treated 2 Y.T. and H.N. contributed equally to this study. with Dpn I that is specific for methylated and hemimethylated DNA, and 3 Address correspondence and reprint requests to Dr. Noboru Suzuki, Departments of the synthesized nonmethylated DNA containing the mutation was recov- Immunology and Medicine, St. Marianna University School of Medicine, 2-16-1, ered. The resultant mutant vector was used for transformation of Esche- Sugao, Miyamae-ku, Kawasaki, Kanagawa 216-8511, Japan. E-mail address: richia coli, DH5␣. Their sequences have been verified by DNA sequenc- [email protected] ing. c-Jun expression vector has been reported previously (35). Copyright © 2002 by The American Association of Immunologists 0022-1767/02/$02.00 2366 Txk AS A HUMAN Th1 LYMPHOCYTE-SPECIFIC TRANSCRIPTION FACTOR Transfection into Jurkat cells and luciferase assay dialyzed against buffer consisting of 20 mM HEPES, 20% glycerol, 0.2 mM EDTA, 0.5 mM PMSF, and 0.5 mM DTT. The protein concentration Purified plasmids were transfected into Jurkat cells by electroporation, as in the nuclear extracts was determined by Bio-Rad protein assay kit (Bio- ␮ ␥ ␮ described (30). In brief, 5 g of pIFN- promoter plus luciferase, 5 gof Rad, Richmond, CA). In some experiments, human Th1 cell lines were 4 ␮ pRSV-chloramphenicol acetyltransferase (CAT), and 10 g of pME18S- established, as described previously (32), and nuclear proteins of the cells Txk (Txk transfection) or pME18S (empty vector; mock transfection) were were recovered. cotransfected. Forty-eight hours after transfection, the Jurkat cells were stimulated with 1 ␮g/ml PHA and cultured for various periods. Thereafter, DNA-protein-binding assay protein assay, luciferase assay, and CAT-ELISA (Roche Diagnostics, To- kyo, Japan) of the cell lysates were conducted (32). IL-2 promoter plus A gel shift assay was performed using digoxigenin gel shift kit (Boehringer luciferase vector was also included as a control promoter vector. In some Mannheim Biochemica, Mannheim, Germany). In brief, digoxigenin-la- experiments, c-Jun expression vector was used to transfect Jurkat cells to beled DNA fragments were incubated at room temperature for 15 min with obtain control nuclear proteins (35). 5–10 ␮g of nuclear proteins. Protein-DNA complexes were separated from free probe on a polyacrylamide gel. Thereafter, the gels were electrically Immunoblotting analysis transferred to nylon membrane and detected by chemiluminescence. We verified that a 20-fold excess of specific cold oligonucleotide competed the The cells were lysed with buffer containing 50 mM Tris, pH 8, 1% Nonidet binding of the protein to the digoxigenin-labeled probe, whereas a similar P-40, 150 mM NaCl, and the protease inhibitors, as described (35). Equiv- excess from another site would not compete (see Figs.