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      Wnt/β-Catenin Signaling Regulates the Expression of the Ammonium Permease Gene RHBG in Human Cancer Cells

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          Abstract

          Ammonium is a metabolic waste product mainly detoxified by the liver. Hepatic dysfunction can lead to cytotoxic accumulation of circulating ammonium and to subsequent encephalopathy. Transmembrane ammonium transport is a widely spread process ensured by the highly conserved proteins of the Mep-Amt-Rh superfamily, including the mammalian Rhesus (Rh) factors. The regulatory mechanisms involved in the control of RH genes expression remain poorly studied. Here we addressed the expression regulation of one of these factors, RHBG. We identify HepG2 hepatocellular carcinoma cells and SW480 colon adenocarcinoma cells as expressing RHBG and show that its expression relies on β-catenin signaling. siRNA-mediated β-catenin knockdown resulted in significant reduction of RHBG mRNA in both cell lines. Pharmaceutical inhibition of the TCF4/β-catenin interaction or knockdown of the transcription factor TCF4 also downregulated RHBG expression. We identify a minimal RHBG regulatory sequence displaying a promoter activity and show that β-catenin and TCF4 bind to this fragment in vivo. We finally characterize the role of potential TCF4 binding sites in RHBG regulation. Taken together, our results indicate RHBG expression as a direct target of β-catenin regulation, a pathway frequently deregulated in many cancers and associated with tumorigenesis.

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          Most cited references49

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          Wnt/β-Catenin/Tcf Signaling Induces the Transcription of Axin2, a Negative Regulator of the Signaling Pathway

          Axin2/Conductin/Axil and its ortholog Axin are negative regulators of the Wnt signaling pathway, which promote the phosphorylation and degradation of β-catenin. While Axin is expressed ubiquitously, Axin2 mRNA was seen in a restricted pattern during mouse embryogenesis and organogenesis. Because many sites of Axin2 expression overlapped with those of several Wnt genes, we tested whether Axin2 was induced by Wnt signaling. Endogenous Axin2 mRNA and protein expression could be rapidly induced by activation of the Wnt pathway, and Axin2 reporter constructs, containing a 5.6-kb DNA fragment including the promoter and first intron, were also induced. This genomic region contains eight Tcf/LEF consensus binding sites, five of which are located within longer, highly conserved noncoding sequences. The mutation or deletion of these Tcf/LEF sites greatly diminished induction by β-catenin, and mutation of the Tcf/LEF site T2 abolished protein binding in an electrophoretic mobility shift assay. These results strongly suggest that Axin2 is a direct target of the Wnt pathway, mediated through Tcf/LEF factors. The 5.6-kb genomic sequence was sufficient to direct the tissue-specific expression of d2EGFP in transgenic embryos, consistent with a role for the Tcf/LEF sites and surrounding conserved sequences in the in vivo expression pattern of Axin2 . Our results suggest that Axin2 participates in a negative feedback loop, which could serve to limit the duration or intensity of a Wnt-initiated signal.
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            PROMO: detection of known transcription regulatory elements using species-tailored searches.

            We have developed a set of tools to construct positional weight matrices from known transcription factor binding sites in a species or taxon-specific manner, and to search for matches in DNA sequences.
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              The cyclin D1 gene is a target of the beta-catenin/LEF-1 pathway.

              beta-Catenin plays a dual role in the cell: one in linking the cytoplasmic side of cadherin-mediated cell-cell contacts to the actin cytoskeleton and an additional role in signaling that involves transactivation in complex with transcription factors of the lymphoid enhancing factor (LEF-1) family. Elevated beta-catenin levels in colorectal cancer caused by mutations in beta-catenin or by the adenomatous polyposis coli molecule, which regulates beta-catenin degradation, result in the binding of beta-catenin to LEF-1 and increased transcriptional activation of mostly unknown target genes. Here, we show that the cyclin D1 gene is a direct target for transactivation by the beta-catenin/LEF-1 pathway through a LEF-1 binding site in the cyclin D1 promoter. Inhibitors of beta-catenin activation, wild-type adenomatous polyposis coli, axin, and the cytoplasmic tail of cadherin suppressed cyclin D1 promoter activity in colon cancer cells. Cyclin D1 protein levels were induced by beta-catenin overexpression and reduced in cells overexpressing the cadherin cytoplasmic domain. Increased beta-catenin levels may thus promote neoplastic conversion by triggering cyclin D1 gene expression and, consequently, uncontrolled progression into the cell cycle.
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                Author and article information

                Contributors
                Role: Academic Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                1 June 2015
                2015
                : 10
                : 6
                : e0128683
                Affiliations
                [001]Biology of Membrane Transport Laboratory, IBMM, Université Libre de Bruxelles, Gosselies, Belgium
                University of Kentucky, UNITED STATES
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Conceived and designed the experiments: AM AMM. Performed the experiments: AM CD RA JVA. Analyzed the data: AM CD RA JVA AMM. Wrote the paper: AM AMM.

                Article
                PONE-D-15-02088
                10.1371/journal.pone.0128683
                4452261
                26029888
                a8243655-f4c3-4489-adb8-fd677a2f0365
                Copyright @ 2015

                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

                History
                : 19 January 2015
                : 29 April 2015
                Page count
                Figures: 9, Tables: 2, Pages: 20
                Funding
                This work was supported by grants from the Belgian Fonds de la Recherche Scientifique F.R.S.-FNRS (F.R.S.M. 3.4633.09, M.I.S. F.4.521.10.F), the Fédération Wallonie-Bruxelles (Action de Recherche Concertée), the International Brachet Stiftung, the Jean Brachet and the Alice et David Van Buuren foundations. A.M. is a scientific research worker supported by a Télévie grant, C.D. was a scientific research worker of the F.R.S.-FNRS, JVA was supported by a da Vinci program, and A.M.M. is a senior research associate of the F.R.S.-FNRS. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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