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

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

Activation of β-catenin signaling increases <i>RHBG</i> expression.

<p>A) HEK293T cells were treated with LiCL (20mM) for 24 hours. β-catenin localization was determined by immunofluorescence using β-catenin antibody. Nuclei were stained with DAPI. B) HEK293T cells were treated with LiCL (10 or 20mM) for 24 hours. β-catenin protein level was determined in total cell lysates by immunoblotting using β-catenin antibody. C) Same culture conditions as in B. The <i>RHBG</i> mRNA level was determined by qRT-PCR.</p

β-catenin knockdown decreases <i>RHBG</i> expression in SW480 colon cancer cells.

<p>A) Representative results of immunofluorescence of β-catenin localization in SW480 cells using β-catenin antibody. Nuclei were stained with DAPI. B-G) SW480 cells were reverse transfected with β-catenin or control (scramble) siRNAs. 72 hours after transfection, expression levels of <i>β-catenin</i> mRNA (B), β-catenin protein (C), <i>RHBG</i> mRNA (D), <i>Axin2</i> mRNA (E), <i>Cyclin D1</i> mRNA (F) and <i>RHCG</i> mRNA (G) were determined.</p

β-catenin knockdown decreases <i>RHBG</i> expression in HepG2 hepatoma cells.

<p>A-F) HepG2 cells were reverse transfected with β-catenin or control (scramble) siRNAs. 72 hours after transfection, expression levels of <i>β-catenin</i> mRNA (A), β-catenin protein (B), <i>RHBG</i> mRNA (C), <i>Axin2</i> mRNA (D), <i>Cyclin D1</i> mRNA (E) and <i>RHCG</i> mRNA (F) were determined.</p

<i>RHBG</i> expression is dependent on TCF4.

<p>A-B) HepG2 cells were treated with PKF118-310 (0,2 or 0,4 μM) for 24 hours. The <i>RHBG</i> (A) and <i>GLUL</i> (B) mRNA levels were determined by qRT-PCR. C-G) HepG2 cells were reverse transfected with TCF4 or control (scramble) siRNAs. 72 hours after transfection, levels of <i>TCF4</i> mRNA (C), TCF4 protein (D), <i>RHBG</i> mRNA (E), <i>Axin2</i> mRNA (F), and <i>Cylcin D1</i> mRNA (G), were determined.</p

<i>RHBG</i> and <i>GLUL</i> are highly expressed in HepG2 hepatoma cells.

<p>A) The level of mRNA expression of <i>RHBG</i> in HEK293T, HepG2 and Hep3B cells was determined by qRT-PCR and normalized to β-actin. B) The mRNA expression of <i>GLUL</i> in HepG2 and Hep3B was determined by qRT-PCR. C) Western Blot analysis of GS protein from total cell lysates of HepG2 and Hep3B cells. D) The level of mRNA expression of <i>RHCG</i> in HEK293T, HepG2 and Hep3B cells was determined by qRT-PCR and normalized to β-actin. E) Western Blot analysis of β-catenin protein from total cell lysates of HEK293T, HepG2 and Hep3B cells.</p

Deletion analysis of <i>RHBG</i> promoter sequence.

<p>HepG2 cells were transfected with the empty plasmid (pGL3) or <i>RHBG</i> promoter (pGL3-RHBG) together with Renilla plasmid. 48 hours after transfection, <i>RHBG</i> promoter activity in total cell lysates was determined by luciferase assay. B) HepG2 cells were transfected with <i>RHBG</i> promoter (pGL3-RHBG) or the indicated construct together with Renilla plasmid. 48 hours after transfection, <i>RHBG</i> promoter activity was determined by measuring luciferase activity in total cell lysates. Data are expressed as mean of triplicate determinations ± S.E.M of the pGL3-RHBG construct relative to pGL3-Basic. C) HepG2 cells were transfected with the indicated construct together with Renilla plasmid. 48 hours after transfection, <i>RHBG</i> promoter activity was determined by measuring luciferase activity in total cell lysates.</p

Promoter region of <i>RHBG</i> gene.

<p>The potential human <i>RHBG</i> promoter sequence was obtained from eukaryotic promoter database (<a href="http://epd.vital-it.ch/" target="_blank">http://epd.vital-it.ch/</a>). Black arrow (↓) indicates the predicted transcription start site (TSS) which is designated nucleotide 0. The GC boxes are shadowed. A selection of potential binding sites (with 0 or less than 5% dissimilarity) of transcription factors identified using PROMO [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128683#pone.0128683.ref054" target="_blank">54</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128683#pone.0128683.ref055" target="_blank">55</a>] is underlined. Potential TCF4 binding sites are indicated with empty boxes. Horizontal arrows (→) indicate the starting residue position of each promoter construct analyzed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128683#pone.0128683.g008" target="_blank">Fig 8</a>.</p

TCF4 knockdown decreases <i>RHBG</i> expression in SW480 cells.

<p>A-E) SW480 cells were reverse transfected with TCF4 or control (scramble) siRNAs. 72 hours after transfection, levels of <i>TCF4</i> mRNA (A), TCF4 protein (B) <i>RHBG</i> mRNA (C), <i>Axin2</i> mRNA (D), and <i>Cyclin D1</i> mRNA (E), were determined.</p

TCF4/β-catenin binds to the <i>RHBG</i> promoter.

<p>HepG2 cells were cross-linked with formaldehyde followed by chromatin digestion. Chromatin immunoprecipitations were performed using antibodies targeting either β-catenin, TCF4 or IgG, as a control. Purified DNA was analyzed by qPCR using the indicated primers. The amount of immunoprecipitated DNA with each antibody is represented as signal relative to IgG (equivalent to 1) (n = 2).</p