Multi-Level Interactions between the Nuclear Receptor TRa1 and the WNT Effectors b-Catenin/Tcf4 in the Intestinal Epithelium Maria Sirakov 1¤a , Seham Skah 1¤b , Imtiaz Nisar Lone 2 , Julien Nadjar 1¤b , Dimitar Angelov 2 , Michelina Plateroti 1 * ¤b 1 Institut de Ge ´nomique Fonctionnelle de Lyon, Lyon, France, 2 Laboratoire de Biologie Mole ´culaire de la Cellule, Universite ´ de Lyon, Universite ´ Lyon 1, Ecole Normale Supe ´rieure de Lyon, Lyon, France Abstract Intestinal homeostasis results from complex cross-regulation of signaling pathways; their alteration induces intestinal tumorigenesis. Previously, we found that the thyroid hormone nuclear receptor TRa1 activates and synergizes with the WNT pathway, inducing crypt cell proliferation and promoting tumorigenesis. Here, we investigated the mechanisms and implications of the cross-regulation between these two pathways in gut tumorigenesis in vivo and in vitro. We analyzed TRa1 and WNT target gene expression in healthy mucosae and tumors from mice overexpressing TRa1 in the intestinal epithelium in a WNT-activated genetic background (vil-TRa1/Apc mice). Interestingly, increased levels of b-catenin/Tcf4 complex in tumors from vil-TRa1/Apc mice blocked TRa1 transcriptional activity. This observation was confirmed in Caco2 cells, in which TRa1 functionality on a luciferase reporter-assay was reduced by the overexpression of b-catenin/Tcf4. Moreover, TRa1 physically interacted with b-catenin/Tcf4 in the nuclei of these cells. Using molecular approaches, we demonstrated that the binding of TRa1 to its DNA target sequences within the tumors was impaired, while it was newly recruited to WNT target genes. In conclusion, our observations strongly suggest that increased b-catenin/Tcf4 levels i) correlated with reduced TRa1 transcriptional activity on its target genes and, ii) were likely responsible for the shift of TRa1 binding on WNT targets. Together, these data suggest a novel mechanism for the tumor-promoting activity of the TRa1 nuclear receptor. Citation: Sirakov M, Skah S, Lone IN, Nadjar J, Angelov D, et al. (2012) Multi-Level Interactions between the Nuclear Receptor TRa1 and the WNT Effectors b- Catenin/Tcf4 in the Intestinal Epithelium. PLoS ONE 7(4): e34162. doi:10.1371/journal.pone.0034162 Editor: Masaru Katoh, National Cancer Center, Japan Received November 16, 2011; Accepted February 23, 2012; Published April 3, 2012 Copyright: ß 2012 Sirakov et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the Institut National pour le Cancer (grant INCA-2009-175) and the Ligue contre le Cancer Department du Rhone. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: michela.plateroti@ens-lyon.fr ¤a Current address: Institut de Biologie et de Me ´decine Mole ´ culaires, Universite ´ libre de Bruxelles, Bruxelles, Belgium ¤b Current address: Centre de Ge ´ne ´tique et de Physiologie Mole ´culaire et Cellulaire, Universite ´ Lyon 1, Lyon, France Introduction The intestinal epithelium is a dynamic tissue that is continuously renewed through stem cells and committed progenitors, located in the crypts of Liberku ¨n [1,2]. The balance between proliferation and differentiation in the crypts is maintained by the fine cross- regulation among several pathways, including WNT, Hedgehog, Notch, BMP, and thyroid hormones (THs) [3–6]. These pathways are key players of intestinal homeostasis, and their deregulation is correlated with the onset of colorectal cancer [7–9]. The molecular basis of their action is only partially understood, and the mechanism of cross-regulation that occurs between these signaling pathways is still a puzzle. We previously demonstrated that TH signaling in the mouse intestine is involved in development, homeostasis and cancer susceptibility [10]#. THs act through nuclear hormone receptors, the TRs, which are encoded by THRa and THRb loci [11]. These transcription factors activate or repress the transcription of target genes by binding to specific DNA sequences called thyroid hormone response elements (TREs) [12], and they are involved in several cellular responses, such as cell proliferation, cell differentiation and apoptosis [5,13]. TH signaling in the mouse intestine is mediated by TRa1, given that TRa 0/0 [14] mice as well as hypothyroid mice have reduced cell proliferation in the crypts during development and in adulthood [15]. Recently, we showed that the constitutive TRa1 overex- pression in the intestinal epithelium (vil-TRa1 mice) allows increased cell proliferation and adenoma development; this overexpression also enhances the intestinal tumorigenic process in an Apc mutated genetic background (vil-TRa1/Apc +/1638N mice) [9]. Interestingly, one of the molecular features of the vil- TRa1/Apc mice is the increased activity of the WNT pathway compared with that of the Apc mutants [9]. This is in agreement with our previous studies showing that TRa1 activates the WNT pathway via the transcriptional regulation of the genes Ctnnb1 and Sfrp2 [15,16]. b-Catenin, the protein encoded by Ctnnb1, was originally identified as a structural component of cell adhesion complexes [17]. Upon increased cellular levels and nuclear accumulation, b- catenin interacts with transcription factors of the T-cell factor (Tcf) and lymphocyte-enhancer factor (Lef) families [18]. b-catenin/ PLoS ONE | www.plosone.org 1 April 2012 | Volume 7 | Issue 4 | e34162