Krüppel-Like Factor 2, a Novel Immediate-Early Transcriptional Factor, Regulates IL-2 Expression in T Lymphocyte Activation This information is current as of September 25, 2021. Jinghai Wu and Jerry B. Lingrel J Immunol 2005; 175:3060-3066; ; doi: 10.4049/jimmunol.175.5.3060 http://www.jimmunol.org/content/175/5/3060 Downloaded from References This article cites 35 articles, 19 of which you can access for free at: http://www.jimmunol.org/content/175/5/3060.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 25, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2005 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Kru¨ppel-Like Factor 2, a Novel Immediate-Early Transcriptional Factor, Regulates IL-2 Expression in T Lymphocyte Activation1 Jinghai Wu and Jerry B. Lingrel2 Ag presentation to T lymphocytes and subsequent activation are characterized by a cascade of signaling events, some of which result in the transcriptional activation of a diverse set of genes. An important example is the induction of the IL-2 gene, which is a critical event in the escalation of T cell activation. Previous studies have found that expression of Kru¨ppel-like factor 2 (KLF2), a zinc finger transcription factor, is extinguished after T cell activation. However, the biological role of KLF2 during T cell activation is still unknown. In this study we found that KLF2 protein degradation is delayed, and KLF2 expression is up-regulated during the early stage of T cell activation in primary T cells. Within a few hours, this process is reversed, and KLF2 expression Downloaded from is turned off. Next, we found that the expression of KLF2 significantly increases IL-2 production 4-fold in activated T cells, resulting from activation of the IL-2 promoter. By narrowing down the 2.0-kb IL-2 promoter region, we found that the KLF2 responsive element in the IL-2 promoter is a CACCC element, the KLF consensus binding motif. Moreover, KLF2 binds to this promoter in vivo under different conditions. Our studies show that KLF2 regulates IL-2 promoter activity in the earliest stages of T cell activation, indicating that KLF2 may act as a novel immediate-early transcriptional factor to maximally prime T cell activation. The Journal of Immunology, 2005, 175: 3060–3066. http://www.jimmunol.org/ n essential feature of the cellular immune response is the expressed in fetal and adult lungs, heart, as well as several other activation of T lymphocytes. T cells are activated by organs (4, 5). It is absolutely required for normal embryogenesis A triggering the TCR/CD3 complex by a specific Ag, in and late stages of lung development (6–8). KLF2 homozygous collaboration with costimulatory and adhesion receptors. This is a null mice die in utero due to severe hemorrhage resulting from complex process that involves multiple enzymes, adapters, and defects of the mature blood vessel wall (6). KLF2 does not seem other transcriptional factors, which induces a cascade of metabolic to be a regulator of the initiation of blood vessel morphogenesis; events resulting in the transcriptional activation of a large number however, it is active in the late stages of blood vessel wall assem- of different genes (1). Of these genes, the best characterized is the bly and stabilization (7). KLF2 also appears to play an important by guest on September 25, 2021 IL-2 gene, whose induction is critical for T cell activation and role in endothelial cell activation (9, 10). differentiation. The quantity of IL-2 produced is a major determi- Additionally, KLF2 plays an important role in T cell function. nant of whether an effective response can be generated. Previous KLF2 is developmentally induced during the maturation of single- studies have indicated that several transcriptional factors, includ- positive CD4ϩ or CD8ϩ T cells and prevents mature circulating ing AP-1, NFAT, NF-␬B, and CD28RE/AP, are important positive cells from undergoing apoptosis (11, 12). Furthermore, it is re- regulators of IL-2 gene expression (2). Also, a CACCC binding quired to maintain CD4ϩ or CD8ϩ single-positive T cells in their site for specificity protein 1 and an early growth response gene-1 quiescent state (13). Also, KLF2 inhibits Jurkat T leukemia cell binding site have been recently identified immediately upstream of growth via direct up-regulation of the cyclin-dependent kinase in- the distal NFAT site (3). Because many of these transcriptional hibitor p21WAF1/CIP1 (14). Intriguingly, KLF2 is rapidly extin- factor binding sites vary from consensus sequences in other genes, guished after T cell activation and is re-expressed in CD8ϩ mem- it appears that these differences may in part account for the T ory T cells (11, 15), indicating that KLF2 might also play a crucial cell-specific expression of IL-2. role in regulating the activation of T cells and the survival of mem- Kru¨ppel-like factor 2 (KLF23; previously termed LKLF) is a ory T cells. However, the exact biological role of KLF2 in T cell member of the closely related family of the KLFs. KLF2 is highly activation is still unknown. In the studies presented in this paper, we found that KLF2 transcription and protein expression are up- Department of Molecular Genetics, Biochemistry, and Microbiology, University of regulated during the early stage of T cell activation. Moreover, the Cincinnati, College of Medicine, Cincinnati, OH 45267 expression of KLF2 significantly increases IL-2 production, up to Received for publication December 30, 2004. Accepted for publication June 22, 2005. 4-fold in activated T cells. Our studies also suggest that KLF2 The costs of publication of this article were defrayed in part by the payment of page maximally regulates IL-2 promoter activity directly via a CACCC charges. This article must therefore be hereby marked advertisement in accordance KLF2 binding site in the earlier stage of T cell activation. Our with 18 U.S.C. Section 1734 solely to indicate this fact. findings indicate that KLF2 may be required to maximally prime T 1 This work was supported by National Institutes of Health Grant RO1HL57281 (to cell activation as a novel immediate-early transcriptional factor. J.B.L.). 2 Address correspondence and reprint requests to Dr. Jerry B. Lingrel, Department of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati Col- Materials and Methods lege of Medicine, 231 Albert Sabin Way, Cincinnati, OH 45267. E-mail address: Plasmid constructs [email protected] 3 Abbreviations used in this paper: KLF, Kru¨ppel-like factor; ChIP, chromatin im- Full-length mouse KLF2 cDNA tagged at the C terminus with hemagglu- munoprecipitation; Dox, doxycycline; HA, hemagglutinin; mt, mutant; PPAR␥, per- tinin (HA) was generated by PCR and inserted into BamHI/HindIII restric- oxisomal proliferator-activated receptor ␥; wt, wild type. tion sites of vector pBK-CMV (Stratagene) as previously described (14). Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00 The Journal of Immunology 3061 Subsequently, the insert including full-length, wild-type KLF2 (KLF2-wt) Generation of promoter constructs and site-directed mutagenesis with the HA tag was subcloned into the pTRE2hyg vector (BD Clontech) to generate Tet/KLF2 as a response plasmid for doxycycline (Dox) A 2-kb fragment of IL-2 enhancer/promoter sequence, provided by Dr. V. induction. Boussiotis (Harvard Medical School, Boston, MA) (18), was cloned into the pT81-Luc reporter vector. The Ϫ585 IL-2 promoter construct, was Cell line and stable cell transfection inserted into the SalI/BglII site of pT81-luc. The empty vector of pT81-luc, NFAT, and NF-␬B luciferase reporter constructs were gifts from Dr. D. The human Jurkat tet-on CD4ϩ T cell line (BD Clontech) was maintained McKean (Mayo Clinic, Rochester, NY) (19, 20). 4xAP-1 and CD28RE/AP in RPMI 1640 medium supplemented with 10% Tet System Approved FBS luciferase reporters were provided by Dr. X.-F. Wang (Duke University (BD Clontech), 100 ␮g/ml G418, 100 U/ml penicillin, 100 ␮g/ml strepto- Medical Center, Durham, NC) (21) and Dr. A. Weiss (University of Cal- mycin, and 100 ␮g/ml gentamicin. The transfection of 30 ␮g of Tet/ ifornia, San Francisco, CA) (22), respectively. Three additional IL-2 lu- KLF2-wt plasmid was conducted by electroporation of 2 ϫ 107 cells with ciferase deletion mutants (pIL2–310, pIL2–290, and pIL2–206) were cre- settings at 950 ␮F and 240 V, respectively. Stably transfected cells were ated to narrow down the potential KLF2-responsive sites. PCR was used to selected by growth in 200 ␮g/ml hygromycin (BD Clontech) and 200 amply these deletion mutants and to introduce a KpnI restriction site at the ␮g/ml G418 (Invitrogen Life Technologies). Clones from single cells were 5Ј end, starting from IL-2 promoter region sites Ϫ310, Ϫ290, and Ϫ206, generated by limited dilution. Protein expression was induced by resus- respectively, and the BglII restriction site at the 3Ј end, ending in the IL-2 pending cells at a density of 105/ml in complete medium with Dox transcriptional start codon (forward, 5Ј-CGAGGTACCTTTGTGTCTC (1 ␮g/ml; Sigma-Aldrich), a derivative of tetracycline.