The Journal of Immunology Functional Characterization of the CCL25 Promoter in Small Intestinal Epithelial Cells Suggests a Regulatory Role for Caudal-Related Homeobox (Cdx) Transcription Factors1 Anna Ericsson,2* Knut Kotarsky,2,3* Marcus Svensson,* Mikael Sigvardsson,† and William Agace* The chemokine CCL25 is selectively and constitutively expressed in the small intestinal epithelium and plays an important role in mediating lymphocyte recruitment to this site. In this study, we demonstrate that CCL25 expression in murine small intestinal epithelial cells is independent of signaling through the lymphotoxin ␤ receptor and is not enhanced by inflammatory stimuli, pathways involved in driving the expression of most other chemokines. We define a transcriptional start site in the CCL25 gene and a region ؊141 to ؊5 proximal of exon 1 that is required for minimal promoter activity in the small intestinal epithelial cell lines, MODE-K and mICc12. These cell lines expressed far less CCL25 mRNA than freshly isolated small intestinal epithelial cells indicating that they are missing important factors driving CCL25 expression. The CCL25 promoter contained putative binding sites for the intestinal epithelial-associated Caudal-related homeobox (Cdx) transcription factors Cdx-1 and Cdx-2, and small intestinal epithelial cells but not MODE-K and mICc12 cells expressed Cdx-1 and Cdx-2. EMSA analysis demonstrated that Cdx proteins were present in nuclear extracts from freshly isolated small intestinal epithelial cells but not in MODE-K or mICcl2 cells, and bound to putative Cdx sites within the CCL25 promoter. Finally, cotransfection of MODE-K cells with Cdx transcription factors significantly increased CCL25 promoter activity as well as endogenous CCL25 mRNA levels. Together these results demonstrate a unique pattern of regulation for CCL25 and suggest a role for Cdx proteins in regulating CCL25 transcription. The Journal of Immunology, 2006, 176: 3642–3651. hemokines are a large family of low m.w. proteins initiation of immune responses, and immune surveillance of primarily recognized for their role as leukocyte che- healthy peripheral tissues (2). The expression of homeostatic C moattractants and in regulating leukocyte trafficking. chemokines in lymphoid organs and the intestine is largely They function through seven transmembrane G protein-coupled dependent on lymphotoxin (LT)4 ␤ receptor signaling (3–5). receptors to induce directed cellular migration and enhanced The division of chemokines into inflammatory and homeostatic integrin-mediated adhesion, which are processes critical for chemokines is, however, not absolute because many homeo- leukocyte extravasation (1). Chemokines can be divided into static chemokines can be up-regulated in response to inflam- two groups, inflammatory and homeostatic chemokines, based matory stimuli (6, 7), and inflammatory chemokines can target on their regulation and function (2). Inflammatory chemokines noneffector leukocytes at sites of leukocyte development (8). control the recruitment of effector leukocytes, including cells The chemokine CCL25 is selectively and constitutively ex- from both the innate and adaptive immune response, to sites of pressed in the small intestine and thymus, primarily by resident infection or inflammation, and can be induced in a wide variety epithelial cells (9–12). Its sole functional receptor, CCR9, is of cells upon exposure to host or pathogen-derived inflamma- expressed on small intestinal lymphocytes, a subset of circulat- tory stimuli (2). Homeostatic chemokines, by contrast, are ing gut tropic lymphocytes, and thymocytes (13, 14). Analysis constitutively expressed in primary and secondary lymphoid Ϫ Ϫ of CCR9 / mice, and in vivo studies using neutralizing organs and in tertiary tissues, such as the skin and intestine, Ϫ Ϫ anti-CCL25 Ab, or CCR9 / TCR transgenic T cells have where they control lymphocyte migration during hemopoiesis, demonstrated a central role for CCL25/CCR9 in the generation of the small intestinal lymphocyte compartment (14–21). De- spite the importance of CCL25/CCR9 in small intestinal immu- *Immunology Section, and †Hematopoietic Stem Cell Laboratory, Stem Cell Center, Lund University, Lund, Sweden nity, the mechanisms underlying the selective and constitutive Received for publication July 22, 2005. Accepted for publication January 6, 2006. expression of CCL25 in the small intestine are not understood. The costs of publication of this article were defrayed in part by the payment of page In the current study, we have examined expression and charges. This article must therefore be hereby marked advertisement in accordance regulation of CCL25 in small intestinal epithelial cells. Our with 18 U.S.C. Section 1734 solely to indicate this fact. results demonstrate that CCL25 displays a unique pattern of 1 This work was supported by grants from the Swedish Medical Research Council; the regulation compared with other inflammatory or homeostatic Crafoordska, O¨ sterlund, Åke Wiberg, Richard and Ruth Julins, Nanna Svartz and Kocks Foundations; the Swedish Medical Society; the Royal Physiographic Society; chemokines and suggest a role for the Caudal-related homeobox the Swedish Foundation for Strategic Research “Microbes and Man” program and transcription factors in enhancing CCL25 promoter activity in INGVAR II program; and a Crohns and Colitis Foundation of America project grant (to W.A.). K.K. is supported by a SWEGENE Postdoctoral Fellowship. the small intestine. 2 A.E. and K.K. contributed equally to this work. 3 Address correspondence and reprint requests to Dr. Knut Kotarsky, Immunology Section, Lund University, Biomedical Centre I-13, S-22184 Lund, Sweden. E-mail address: [email protected] 4 Abbreviations used in this paper: LT, lymphotoxin; IEL, intraepithelial lymphocyte. Copyright © 2006 by The American Association of Immunologists, Inc. 0022-1767/06/$02.00 The Journal of Immunology 3643 Materials and Methods 5Ј-GCGGAATTCACCATGTATGTGGGCTATGTGCTG-3Ј and anti- Ј Ј Mice sense 5 -GCGGAATTCTATGGCAGAAACTCCTCTTTCACA-3 , and for Cdx-2 were sense 5Ј-GCGGAATTCACCATGTACGTGAGCTAC Germfree or conventional Swiss Webster mice were from Taconic Farms, CTTCTG-3Ј and antisense 5Ј-GCGGAATTCACTGGGTGACAGTG and C57BL/6 mice were from the Microbiology, Immunology, and Gly- GAGTTTAAAACC-3Ј. Primers for ␤-actin were sense 5Ј-GGTGGGAAT cobiology animal facility and the Biomedical Centre animal facility (Lund GGGTCAGAAGGACT-3Ј and antisense 5Ј-CCACGCTCGGTCAG University, Lund, Sweden). Small intestinal tissue from athymic mice with GATCTTCAT-3Ј. Primers for CCL25 were sense 5Ј-ATAGGCAATA a truncated common cytokine receptor ␥-chain (CR␥Ϫ/Y nu/nu) and CR␥ϩ CACGCTACAAGC-3Ј and antisense 5Ј-GCGGAATTCGTCTTCAAAG nu/ϩ mice was provided by Dr. H. Ishikawa (Keio University School of GCACCTTGGGCATGG-3Ј. Primers for cytokeratin 18 were sense Medicine, Tokyo, Japan), tissue from TNFR1Ϫ/Ϫ mice was provided by 5Ј-AGATCGACAATGCCCGCCTTG-3Ј and antisense 5Ј-AGACTTG Dr. N. Lycke (University of Gothenburg, Gothenburg, Sweden), and tissue GTGGTGACAACTGT-3Ј. Primers for Madcam-1 were sense 5Ј- from LT␣Ϫ/Ϫ and LT␤Ϫ/Ϫ mice was provided by Dr. D. Finke (University CCTGAGTCTGAGGTAGCTGTGG-3Ј and 5Ј-GAGTGCCTGTGT of Lausanne, Lausanne, Switzerland). All animal studies were approved by GTCTGACAGCAT-3Ј antisense and for intestinal alkaline phosphatase the local ethical committee. sense 5Ј-GCCGTGAAAGTGCTAAGCAGG-3Ј and antisense 5Ј- GGTCAGAGTGTCGCGTTCACTA-3Ј. CCL25 mRNA levels were deter- Epithelial cell isolation and cell line culture mined by real-time RT-PCR as previously described (14). Epithelial cells were removed from the small intestine with EDTA. Briefly, the small intestine was rinsed with ice-cold PBS, inverted, and cut into Rapid amplification of cDNA ends 5-cm fragments. Intestinal fragments were then incubated in PBS contain- Mouse small intestinal cells were isolated by EDTA treatment. Total RNA ing 30 mM EDTA for 30 min at 37°C on a rotating platform and EDTA was prepared using Stratagene RNA Miniprep kit. For 5Ј RACE total RNA was changed every 5 min. Murine small intestinal epithelial crypts were was reverse transcribed onto magnetic beads (Dynal Biotech) using Su- isolated and cultured as described (22). RT-PCR for cytokeratin 18 ex- perscript III (Invitrogen Life Technologies) according to manufacturer’s pression was performed to confirm the epithelial identity of cultured cells instructions. The single-stranded cDNA was tailed with dATP on the 3Ј (data not shown). The murine small intestinal epithelial cell lines MODE-K end using terminal transferase EC 2.7.7.31 (Roche). The second strand was provided by Dr. P. Ernst (University of Texas Medical Branch, Galveston, synthesized with Pfx-polymerase (Invitrogen Life Technologies) and the TX) and S1-H10 provided by Dr. J. I. Gordon (Washington University following adaptor primer: 5Ј-GTCCGCGGCCGCGTAATACGACTCAC School of Medicine, St. Louis, MO), and the murine fibroblast cell line TATAGGGCGTTTTTTTTTTTTTTTTTTT-3Ј. The cDNA was removed BALB/3T3 provided by Dr. C. Owman (Lund University, Lund, Sweden) from the magnetic beads and subjected to PCR using the first gene-specific were maintained in DMEM (Invitrogen Life Technologies) supplemented primer 5Ј-GCGGAATTCTTTGATCCTGTGCTGGTAACCCAGG-3Ј and with 10% FCS (Sigma-Aldrich), nonessential amino acids (Invitrogen Life the adaptor primer. The product was used in a consecutive PCR with a Technologies), 1 mM sodium pyruvate (Invitrogen Life Technologies), and second gene-specific primer 5Ј-GCGGAATTCGTCTTCAAAGGCACCT