Stat5 and Sp1 Regulate Transcription of the Cyclin D2 Gene in Response to IL-2 Anthony Martino, James H. Holmes IV, James D. Lord, James J. Moon and Brad H. Nelson This information is current as of September 27, 2021. J Immunol 2001; 166:1723-1729; ; doi: 10.4049/jimmunol.166.3.1723 http://www.jimmunol.org/content/166/3/1723 Downloaded from References This article cites 51 articles, 28 of which you can access for free at: http://www.jimmunol.org/content/166/3/1723.full#ref-list-1 Why The JI? Submit online. http://www.jimmunol.org/ • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average by guest on September 27, 2021 Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2001 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Stat5 and Sp1 Regulate Transcription of the Cyclin D2 Gene in Response to IL-21 Anthony Martino,2 James H. Holmes IV, James D. Lord, James J. Moon, and Brad H. Nelson The IL-2R promotes rapid expansion of activated T cells through signals mediated by the adaptor protein Shc and the transcription factor Stat5. The mechanisms that engage the cell cycle are not well defined. We report on the transcriptional regulation of the cell cycle gene cyclin D2 by the IL-2R. IL-2-responsive induction of a luciferase reporter gene containing 1624 bp of the cyclin D2 promoter/ enhancer was studied in the murine CD8؉ T cell line CTLL2. Reporter gene deletional analysis and EMSAs indicate an IL-2-regulated enhancer element flanks nucleotide ؊1204 and binds a complex of at least three proteins. The enhancer element is bound constitutively by Sp1 and an unknown factor(s) and inducibly by Stat5 in response to IL-2. The Stat5 binding site was essential for IL-2-mediated reporter gene activity, and maximum induction required the adjacent Sp1 binding site. Receptor mutagenesis studies in the pro-B cell line BA/FG (a derivative of the BA/F3 cell line) demonstrated a correlation between Stat5 activity and cyclin D2 mRNA levels when the Downloaded from Stat5 signal was isolated, disrupted, and then rescued. Further, a dominant-negative form of Stat5 lacking the trans-activation domain inhibited induction of cyclin D2 mRNA. We propose that the IL-2R regulates the cyclin D2 gene in part through formation of an enhancer complex containing Stat5 and Sp1. The Journal of Immunology, 2001, 166: 1723–1729. he cytokine IL-2 contributes to immune responses in part and activates two downstream pathways. The Grb2/Sos complex is by promoting rapid proliferation of activated T cells. Sev- recruited to Shc (9–11), and Sos activates the Ras-mitogen-acti- http://www.jimmunol.org/ T eral components of the IL-2R complex transduce the pro- vated protein kinase pathway up-regulating genes such as c-fos and liferative signal from the cell surface (1, 2), but the mechanisms c-jun (12, 13). Additionally, the phosphatidylinositol-3 kinase that control expression of cell cycle-regulatory genes remain (PI3K) signaling pathway is activated through recruitment of the poorly defined. In this report we investigate the molecular mech- adaptor protein GAB2 (14–17). While no genes have been linked anisms by which the IL-2R regulates transcription of the cell cycle directly to the PI3K pathway, regulation of cyclin D3 and p27kip1 gene cyclin D2. expression, pRB phosphorylation, and E2F activity is attributed to The high affinity IL-2R complex includes three proteins (3). PI3K activity in response to IL-2 in T cells (18, 19). IL-2R␣ regulates ligand receptor affinity, and IL-2R␤ and the A second proliferative signal activated by the IL-2R involves ␥ ␥ 3 by guest on September 27, 2021 common -chain ( c) initiate intracellular signaling. The tyrosine the transcription factor Stat5 (20, 21). Stat5 is recruited to phos- kinases Janus kinase 1 (Jak1) and Jak3 associate with the mem- phorylated Y510 (7), becomes tyrosine phosphorylated, homo- ␤ ␥ brane proximal regions of IL-2R and c, respectively, and un- and/or heterodimerizes with other Stat molecules, and directly dergo catalytic activation upon ligand-induced heterodimerization translocates to the nucleus (22). Expression of Stat5 mutants that ␤ ␥ of IL-2R and c (4–6). Activation of Jak1 and Jak3 leads to lack trans-activation potential impairs the IL-2-activated prolifer- phosphorylation of at least three tyrosine residues on IL-2R␤ 338 392 510 ative response in lymphocytes (23, 24), and Stat5-deficient mice (Y ,Y , and Y ) (7). Primed by the phosphorylation events, have compromised T cell proliferative responses (25). Growth- the receptor complex generates two major proliferative signals. related target genes of Stat5 in the context of the IL-2R include One signal is mediated by the adaptor protein Shc (7, 8), which c-myc, bcl-x, bcl2 (23), IL-2R␣ (26, 27), and pim-1 (28). Other binds to phosphorylated Y338, undergoes tyrosine phosphorylation, target genes of Stat5 that have been identified downstream of other cytokine receptors include cyclin D1 (29) and p21cip1 (30). The D-type cyclins (D1, D2, and D3) are among the first reg- Virginia Mason Research Center, Seattle, WA 98101; and Department of Immuno- ulatory proteins to appear in G in response to mitogens. Cyclin D2 logy, University of Washington, Seattle, WA 98195 1 and D3 mRNA levels increase in early G in primary T cells stim- Received for publication July 10, 2000. Accepted for publication November 8, 2000. 1 ulated by IL-2 (31) or stimulated by PHA and 12-O-tetradecanoyl- The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance phorbol-13-acetate (32). Cyclin D2 and D3 protein expression is with 18 U.S.C. Section 1734 solely to indicate this fact. up-regulated in primary T cells stimulated by anti-CD3, but not in 1 This work was supported by Grant GM57931 from the National Institutes of Health T cells derived from Stat5-deficient mice (25). Several mecha- and by grants from The M. J. Murdock Charitable Trust and The William Randolph nisms for cyclin regulation that may be relevant to IL-2R signaling Hearst Foundation. A.M. was supported by a National Research Service Award Se- nior Fellowship from the National Cancer Institute. have been identified. In an erythroleukemia cell line, IL-3-activated 2 Address correspondence and reprint requests to Dr. Anthony Martino, Virginia Ma- Stat5 regulates the transcriptional activity of cyclin D1 at a specific son Research Center, Benaroya Research Institute, 1201 9th Avenue, Seattle, WA gene enhancer region (29). In colon carcinoma and breast cancer cell 98101-6907. E-mail address: [email protected] lines, serum-activated PI3K controls mRNA translation of D-type cy- 3 ␥ ␥ Abbreviations used in this paper: c, common -chain; Jak, Janus kinase; PI3K, clins (33). Finally, c-Myc has been implicated in the transcriptional phosphatidylinositol 3-kinase; SOE, splice overlap extension; buffer H, 20 mM HEPES (pH 7.9), 1 mM EDTA, 0.1 mM EGTA, 2 mM magnesium chloride, 1 mM induction of cyclin D2 in Rat1 and NIH-3T3 cells (34). sodium o-vanadate, 20 mM sodium fluoride, 1 mM DTT, 0.1 mM 4-(2-aminoethyl)- We have studied the transcriptional regulation of the cyclin D2 benzenesulfonyl fluoride, and 1 ␮g/ml leupeptin; TTBS, 0.1 M, pH 7.5; Tris base, 0.9% sodium chloride, and 0.05% Tween 20; TAD, trans-activation domain; CREB, gene in T cells. We report evidence of an enhancer element in the cAMP responsive element binding; CBP, CREB binding protein. cyclin D2 promoter/enhancer that binds Stat5, specificity protein Copyright © 2001 by The American Association of Immunologists 0022-1767/01/$02.00 1724 CYCLIN D2 GENE REGULATION IN RESPONSE TO IL-2 BY Stat5 AND Sp1 (Sp)1, and an unknown factor(s). Maximal enhancer activity re- and 0.15 M sodium citrate, pH 7.0) and UV cross-linked. Blots were pre- quires both the Stat5 and Sp1 binding sites, suggesting functional hybridized at 43°C in hybridization buffer (1 M sodium phosphate (pH cooperation between these factors. Additionally, receptor mu- 7.1), 2 mM EDTA, 2% BSA, 10% SDS, 50% formamide, and 0.16 mg/ml yeast transfer RNA or herring sperm DNA). To generate nucleic acid tagenesis studies indicate the endogenous cyclin D2 gene is reg- probes, a 1.2-kb EcoRI fragment of the cyclin D2 gene and a 1.2-kb PstI ulated by the Stat5 pathway downstream of IL-2 in the absence of fragment of murine GAPDH cDNA were radiolabeled with [␣-32P]dCTP Shc-mediated signaling. Thus, this work reveals a direct pathway using a random primed labeling kit (Roche, Indianapolis, IN) and were from the IL-2R to a key cell cycle regulatory gene via a mecha- purified with Centri-Sep spin columns (Princeton Separations, Adelphia, NJ). Probes were boiled for 5 min and added to blots in hybridization nism involving the transcription factors Stat5 and Sp1. buffer. After overnight incubation at 43°C, blots were washed two or three times with 2ϫ SSC/0.1% SDS at 55°C before autoradiography.