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• W2091518548 abstract "The thyroid hormone receptor TRα1 regulates intestinal development and homeostasis by controlling epithelial proliferation in the crypts. This involves positive control of the Wnt/β-catenin pathway. To further investigate the effect of thyroid hormone-TRα1 signaling on the intestinal epithelium proliferating compartment, we performed a comparative transcription profile analysis on laser microdissected crypt cells recovered from wild type animals with normal or perturbed hormonal status, as well as from TR knock-out mice. Statistical analysis and an in silico approach allowed us to identify 179 differentially regulated genes and to group them into organized functional networks. We focused on the “cell cycle/cell proliferation” network and, in particular, on the Frizzled-related protein sFRP2, whose expression was greatly increased in response to thyroid hormones. In vitro and in vivo analyses showed that the expression of sFRP2 is directly regulated by TRα1 and that it activates β-catenin signaling via Frizzled receptors. Indeed, sFRP2 stabilizes β-catenin, activates its target genes, and enhances cell proliferation. In conclusion, these new data, in conjunction with our previous results, indicate a complex interplay between TRα1 and components of the Wnt/β-catenin pathway. Moreover, we describe in this study a novel mechanism of action of sFRP2, responsible for the activation of β-catenin signaling. The thyroid hormone receptor TRα1 regulates intestinal development and homeostasis by controlling epithelial proliferation in the crypts. This involves positive control of the Wnt/β-catenin pathway. To further investigate the effect of thyroid hormone-TRα1 signaling on the intestinal epithelium proliferating compartment, we performed a comparative transcription profile analysis on laser microdissected crypt cells recovered from wild type animals with normal or perturbed hormonal status, as well as from TR knock-out mice. Statistical analysis and an in silico approach allowed us to identify 179 differentially regulated genes and to group them into organized functional networks. We focused on the “cell cycle/cell proliferation” network and, in particular, on the Frizzled-related protein sFRP2, whose expression was greatly increased in response to thyroid hormones. In vitro and in vivo analyses showed that the expression of sFRP2 is directly regulated by TRα1 and that it activates β-catenin signaling via Frizzled receptors. Indeed, sFRP2 stabilizes β-catenin, activates its target genes, and enhances cell proliferation. In conclusion, these new data, in conjunction with our previous results, indicate a complex interplay between TRα1 and components of the Wnt/β-catenin pathway. Moreover, we describe in this study a novel mechanism of action of sFRP2, responsible for the activation of β-catenin signaling. The thyroid hormones (TH), 3The abbreviations used are: TH, thyroid hormone; WT, wild type; TRE, thyroid hormone-responsive element; RT-QPCR, reverse transcription-quantitative PCR; TR, thyroid hormone receptor; BrdUrd, bromodeoxyuridine; GFP, green fluorescent protein; PTU, propylthiouracil; IP, immunoprecipitation; T3, triiodothyronine; T4, thyroxine.3The abbreviations used are: TH, thyroid hormone; WT, wild type; TRE, thyroid hormone-responsive element; RT-QPCR, reverse transcription-quantitative PCR; TR, thyroid hormone receptor; BrdUrd, bromodeoxyuridine; GFP, green fluorescent protein; PTU, propylthiouracil; IP, immunoprecipitation; T3, triiodothyronine; T4, thyroxine. T3 and T4, control cell proliferation, cell differentiation, and apoptosis depending on tissue targets (1Yen P.M. Ando S. Feng X. Liu Y. Maruvada P. Xia X. Mol. Cell. Endocrinol. 2006; 246: 121-127Crossref PubMed Scopus (180) Google Scholar). This is well illustrated during TH-dependent amphibian metamorphosis (2Tata J.R. Mol. Cell. Endocrinol. 2006; 246: 10-20Crossref PubMed Scopus (184) Google Scholar). The action of TH is mediated by T3 binding to nuclear receptors (TRs), encoded by the TRα and TRβ genes (3Laudet V. Hanni C. Coll J. Catzeflis F. Stehelin D. EMBO J. 1992; 11: 1003-1013Crossref PubMed Scopus (567) Google Scholar). The TRs activate or repress transcription of target genes by binding to specific DNA sequences named thyroid hormone-responsive elements (TRE) (1Yen P.M. Ando S. Feng X. Liu Y. Maruvada P. Xia X. Mol. Cell. Endocrinol. 2006; 246: 121-127Crossref PubMed Scopus (180) Google Scholar). In some organs of the gastrointestinal tract, TH-TRs stimulate cell proliferation (4Bockhorn M. Frilling A. Benko T. Best J. Sheu S.Y. Trippler M. Schlaak J.F. Broelsch C.E. Eur. Surg. Res. 2007; 39: 58-63Crossref PubMed Scopus (26) Google Scholar, 5Columbano A. Pibiri M. Deidda M. Cossu C. Scanlan T.S. Chiellini G. Muntoni S. Ledda-Columbano G.M. Endocrinology. 2006; 147: 3211-3218Crossref PubMed Scopus (37) Google Scholar, 6Kress E. Rezza A. Nadjar J. Samarut J. Plateroti M. Mol. Endocrinol. 2008; 22: 47-55Crossref PubMed Scopus (27) Google Scholar, 7Plateroti M. Gauthier K. Domon-Dell C. Freund J.N. Samarut J. Chassande O. Mol. Cell. Biol. 2001; 21: 4761-4772Crossref PubMed Scopus (114) Google Scholar, 8Plateroti M. Kress E. Mori J.I. Samarut J. Mol. Cell. Biol. 2006; 26: 3204-3214Crossref PubMed Scopus (103) Google Scholar).The intestinal epithelium is characterized by continuous and rapid cell renewal, fuelled by adult stem cells located in the crypts of Lieberkühn (9Stappenbeck T.S. Wong M.H. Saam J.R. Mysorekar I.U. Gordon J.I. Curr. Opin. Cell Biol. 1998; 10: 702-709Crossref PubMed Scopus (127) Google Scholar). Epithelial cells acquire differentiated phenotypes during their migration up to the villi, where they eventually die and are shed into the lumen. In the mouse, the whole process lasts 3-4 days (9Stappenbeck T.S. Wong M.H. Saam J.R. Mysorekar I.U. Gordon J.I. Curr. Opin. Cell Biol. 1998; 10: 702-709Crossref PubMed Scopus (127) Google Scholar, 10Karam S.M. Front. Biosci. 1999; 4: D286-D298Crossref PubMed Google Scholar). This continuous cell renewal is regulated by several intrinsic (i.e. transcription factors) and extrinsic (i.e. growth factors and hormones) components. A complex interplay between different signaling pathways maintains epithelial homeostasis (11He X.C. Zhang J. Tong W.G. Tawfik O. Ross J. Scoville D.H. Tian Q. Zeng X. He X. Wiedemann L.M. Mishina Y. Li L. Nat. Genet. 2004; 36: 1117-1121Crossref PubMed Scopus (850) Google Scholar, 12Taipale J. Beachy P.A. Nature. 2001; 411: 349-354Crossref PubMed Scopus (1168) Google Scholar). We recently showed that the TRα1 receptor controls the proliferation of intestinal epithelium progenitors (8Plateroti M. Kress E. Mori J.I. Samarut J. Mol. Cell. Biol. 2006; 26: 3204-3214Crossref PubMed Scopus (103) Google Scholar). The mechanism behind this effect involves direct transcriptional regulation of the Ctnnb1 gene, which encodes β-catenin, the intracellular mediator of the canonical Wnt pathway (13Clevers H. Cell. 2006; 127: 469-480Abstract Full Text Full Text PDF PubMed Scopus (4427) Google Scholar).Canonical Wnt is activated when Wnt proteins bind to the Frizzled receptors, allowing stabilization and nuclear translocation of β-catenin. β-Catenin then binds to the TCF/LEF family of transcription factors to regulate the expression of Wnt target genes (13Clevers H. Cell. 2006; 127: 469-480Abstract Full Text Full Text PDF PubMed Scopus (4427) Google Scholar). Because some of these factors control intestinal progenitor cell proliferation (12Taipale J. Beachy P.A. Nature. 2001; 411: 349-354Crossref PubMed Scopus (1168) Google Scholar, 13Clevers H. Cell. 2006; 127: 469-480Abstract Full Text Full Text PDF PubMed Scopus (4427) Google Scholar), this pathway is thought to be a key regulator of intestinal homeostasis.To analyze the TH-responsive genes in the intestinal epithelium, we used global comparative transcription profiling of laser microdissected intestinal crypt cells. This approach allowed us to define in detail the cross-talk between genes and signaling pathways involved in the control of epithelial cell homeostasis. Moreover, we were able to extend our previous results regarding the control of β-catenin expression by TH. In fact, we identified a new target of TRα1, the secreted Frizzled-related protein sFRP2, which in these cells behaves as a positive regulator of β-catenin stabilization and signaling.EXPERIMENTAL PROCEDURESAnimal Treatment and Tissue Preparation—For microdissection, we used TRα0/0 (14Gauthier K. Plateroti M. Harvey C.B. Williams G.R. Weiss R.E. Refetoff S. Willott J.F. Sundin V. Roux J.P. Malaval L. Hara M. Samarut J. Chassande O. Mol. Cell. Biol. 2001; 21: 4748-4760Crossref PubMed Scopus (219) Google Scholar), TRβ-/- (15Gauthier K. Chassande O. Plateroti M. Roux J.P. Legrand C. Pain B. Rousset B. Weiss R. Trouillas J. Samarut J. EMBO J. 1999; 18: 623-631Crossref PubMed Scopus (333) Google Scholar), and wild type animals, housed and maintained with approval from the animal experimental committee of the Ecole Normale Superieure de Lyon (Lyon, France), and in accordance with European legislation on animal care and experimentation. TH deficiency in pups was induced by 0.15% propylthiouracil (PTU) in the diet (Harlan/Teklad) as described previously (8Plateroti M. Kress E. Mori J.I. Samarut J. Mol. Cell. Biol. 2006; 26: 3204-3214Crossref PubMed Scopus (103) Google Scholar). A group of PTU-treated animals received a single IP injection of TH (2.5 mg/kg T4 and 0.25 mg/kg T3 in 100 μl of phosphate-buffered saline) 24 h before sacrifice. Control animals were fed with standard mouse chow. Animals were euthanized at 14 days, and segments of proximal, median, and distal small intestine were embedded together in Tissue-Tek mounting medium and frozen at -80 °C for cryosectioning. For RNA and protein analysis in the whole mucosa, animals were sacrificed at 14 days, and the intestine was quickly removed and fixed in 4% paraformaldehyde for immunohistochemistry or frozen in liquid nitrogen for RNA and/or protein extraction.Isolation of Villus-Crypt Epithelial Fractions—The sequential isolation of mouse small intestinal epithelial cells along the villus-crypt axis has been described previously and validated (16Weiser M.M. J. Biol. Chem. 1973; 248: 2536-2541Abstract Full Text PDF PubMed Google Scholar). 1-Month-old mice were maintained under a standard chow diet and either untreated or TH-injected (a single IP injection). They were euthanized 24 h after the injection. TH status was confirmed by measuring the circulating levels of free T3 and T4 (Biomerieux).Laser Capture Microdissection and GeneChip Analysis—Tissue-Tek-embedded intestine fragments were prepared for LCM by using the protocol described previously (17Stappenbeck T.S. Hooper L.V. Manchester J.K. Wong M.H. Gordon J.I. Methods Enzymol. 2002; 356: 167-196Crossref PubMed Scopus (50) Google Scholar). Details for RNA extraction, labeling, and hybridization can be found in the supplemental Materials and Methods.Statistics—For pairwise comparisons (WT-PTU-treated versus WT-Control; WT-TH-injected versus WT-Control; WT-TH versus WT-PTU; TRα0/0 versus WT-Control; TRβ-/- versus WT-Control), we used Affymetrix Microarray Suite software 5.0 and two-tailed Student's T-Test (Zoe software). We also applied the analysis of variance for multiple comparisons. More details can be found in the supplemental “Materials and Methods.Ingenuity Pathway Analysis—The associations between the genes were further evaluated using the Ingenuity Pathways Analysis software (Ingenuity Systems). See also the supplemental Materials and Methods.Primary Culture of Intestine Epithelial Cells—Intestinal epithelial primary cultures were derived from 4- to 6-day-old neonatal mice, using the protocol and culture conditions described previously (8Plateroti M. Kress E. Mori J.I. Samarut J. Mol. Cell. Biol. 2006; 26: 3204-3214Crossref PubMed Scopus (103) Google Scholar). Either 2 × 10-7 m T3 or the vehicle alone was added to the culture medium for the indicated length of time. For proliferation studies, 10 μm BrdUrd was added to the culture medium during an overnight incubation. Recombinant sFRP2, Wnt3a, chimeric Fz4, and chimeric Fz7 (R & D Systems) were added to the culture medium for 24 h, at the indicated concentrations. In blocking experiments, 1 μg/ml of anti-sFRP2 (Santa Cruz Biotechnology) or anti-GFP (Roche Applied Science) antibodies were added to the culture medium for 24 h. After 4 days in culture, cells were washed twice with phosphate-buffered saline and frozen at -80 °C prior to being used for RNA or protein extraction, or they were fixed in 2% paraformaldehyde for immunofluorescence.RNA Preparation and Analysis—RNA was extracted from primary cultures with Absolutely RNA nanoprep kit (Stratagene), and from epithelial fractions with total RNA and protein isolation kit (Macherey-Nagel). Reverse transcription was performed using 1 μg of RNA and the Sprint PowerScript Pre-Primed SingleShots with random hexamer primers (Clontech). For RNA recovered by laser microdissection, 1 ng of RNA was retro-transcribed. See the supplemental Materials and Methods for details of quantitative PCR.Immunostaining and Western Blot—Immunolabeling for β-catenin (Santa Cruz Biotechnology) and BrdUrd (Roche Applied Science) was performed on 2% paraformaldehyde-fixed cell cultures. Staining for β-catenin was also performed on 5-μm paraffin sections. Secondary fluorescent antibodies were obtained from The Jackson Laboratories. For fluorescence (Zeiss Axioplan) or confocal microscopy (Zeiss Axiophot), nuclei were stained by Hoechst or by propidium iodide, respectively.Whole proteins from the intestine or from fractionated epithelial cells were extracted using the total RNA and protein isolation kit (Macherey-Nagel). Proteins from cell cultures were obtained by adding the SDS-loading buffer directly to the culture dish. The culture medium was concentrated with centrifugal filter devices (Centricon, Millipore). Proteins were separated and analyzed as described previously (8Plateroti M. Kress E. Mori J.I. Samarut J. Mol. Cell. Biol. 2006; 26: 3204-3214Crossref PubMed Scopus (103) Google Scholar). We used the following primary antibodies: anti-sFRP2 (Santa Cruz Biotechnology), anti-β-catenin (Santa Cruz Biotechnology), anti-activated β-catenin (Upstate), anti-actin (Sigma), anti-Dishevelled 1 (Santa Cruz Biotechnology), and anti-phospho-GSK3β (Ser-9; Cell Signaling).Chromatin Immunoprecipitation—The chromatin immunoprecipitation study was performed on collagenase-dispase separated epithelial fragments from 3- to 6-day-old mouse intestine as described previously (8Plateroti M. Kress E. Mori J.I. Samarut J. Mol. Cell. Biol. 2006; 26: 3204-3214Crossref PubMed Scopus (103) Google Scholar). For conventional PCR, we used the Euroblue Taq (Eurobio). Oligonucleotides are listed in supplemental Fig. S5B. All amplicons were sequenced.RESULTSCrypt Cell Isolation and Microarray Data—Crypt cells were from wild type (WT) animals with normal or perturbed TH status, as well as from TRα and TRβ knock-out mice (14Gauthier K. Plateroti M. Harvey C.B. Williams G.R. Weiss R.E. Refetoff S. Willott J.F. Sundin V. Roux J.P. Malaval L. Hara M. Samarut J. Chassande O. Mol. Cell. Biol. 2001; 21: 4748-4760Crossref PubMed Scopus (219) Google Scholar, 15Gauthier K. Chassande O. Plateroti M. Roux J.P. Legrand C. Pain B. Rousset B. Weiss R. Trouillas J. Samarut J. EMBO J. 1999; 18: 623-631Crossref PubMed Scopus (333) Google Scholar). The supplemental Fig. S1 summarizes the approach of laser microdissection. Statistic methods were used to identify the differentially expressed genes illustrated in supplemental Table S1.Fig. 1 shows examples of validation by RT-QPCR on RNA recovered from microdissected cells. Fig. 1, A and B, displays Ccnb1 (cyclin B1) and Mad2L1 (mitotic arrest deficient homolog-like 1) genes, which encode regulators of cell cycle progression and mitotic checkpoint, respectively (18Hardwick K.G. Curr. Biol. 2005; 15: R122-R124Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 19Lindqvist A. van Zon W. Karlsson Rosenthal C. Wolthuis R.M. PLoS Biol. 2007; 5: e123-e127Crossref PubMed Scopus (136) Google Scholar). Similar results obtained by microarray and by RT-QPCR approaches are illustrated in supplemental Fig. S2.For a finer in silico study, we used the software Ingenuity Pathway Analysis, and defined groups of functions and canonical pathways represented in the set of input genes (supplemental Fig. S3). Finally, we used the gene network tool, which links genes and/or proteins based on a knowledge base. The highest score corresponded to the network “cell cycle/cell proliferation,” which contained 35 input genes (Fig. 1C). This network gives good evidence of the global response to the TH-mediated proliferative stimulus in crypt cells. It is composed of two major nodes. One is Fos, which is up-regulated by TH treatment, coherent with its positive control of cell proliferation (20Hess J. Angel P. Schorpp-Kistner M. J. Cell Sci. 2004; 117: 5965-5973Crossref PubMed Scopus (933) Google Scholar). The second is Ctnnb1, encoding β-catenin, already described as a TH-TRα1 target (8Plateroti M. Kress E. Mori J.I. Samarut J. Mol. Cell. Biol. 2006; 26: 3204-3214Crossref PubMed Scopus (103) Google Scholar). An interesting result was the positive relationship between the Frizzled-related protein sFRP2 and β-catenin, in agreement with work described previously (21Lee J.L. Chang C.J. Wu S.Y. Sargan D.R. Lin C.T. Breast Cancer Res. Treat. 2004; 84: 139-149Crossref PubMed Scopus (48) Google Scholar).Sfrp2 Is a Target Gene of TH in Vivo and in Vitro—We focused on the Sfrp2 gene because its expression was highly stimulated upon treatment with TH, and it participates in the Wnt/β-catenin pathway. Moreover, the action of sFRP2 on canonical Wnt has been described in contradictory terms, either repression or activation (21Lee J.L. Chang C.J. Wu S.Y. Sargan D.R. Lin C.T. Breast Cancer Res. Treat. 2004; 84: 139-149Crossref PubMed Scopus (48) Google Scholar, 22Jones S.E. Jomary C. BioEssays. 2002; 24: 811-820Crossref PubMed Scopus (348) Google Scholar, 23Kawano Y. Kypta R. J. Cell Sci. 2003; 116: 2627-2634Crossref PubMed Scopus (1337) Google Scholar, 24Lee J.L. Chang C.J. Chueh L.L. Lin C.T. In Vitro Cell Dev. Biol. Anim. 2003; 39: 221-227Crossref PubMed Google Scholar, 25Mirotsou M. Zhang Z. Deb A. Zhang L. Gnecchi M. Noiseux N. Mu H. Pachori A. Dzau V. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 1643-1648Crossref PubMed Scopus (444) Google Scholar). Our goal was to clarify its role in intestinal epithelial progenitors.First, we validated sFRP2 differential expression depending on TH status or on TR genotype in vivo. Fig. 2A shows that sFRP2 mRNA levels in WT crypts were lower in hypothyroid conditions and were stimulated in hyperthyroid conditions compared with the control condition. Moreover, they decreased in TRα0/0 and increased in TRβ-/- compared with WT control crypts. It is worth noting that TRβ-/- mice are congenitally hyperthyroid (15Gauthier K. Chassande O. Plateroti M. Roux J.P. Legrand C. Pain B. Rousset B. Weiss R. Trouillas J. Samarut J. EMBO J. 1999; 18: 623-631Crossref PubMed Scopus (333) Google Scholar). We also quantified sFRP2 mRNA and protein in the intestines of TRα and TRβ mutant mice with altered TH status to evaluate whether TH responsiveness depended on a specific TR subtype (supplemental Fig. S4, A and B). WT and TRβ-/- animals both showed decreased sFRP2 mRNA and protein expression under hypothyroid conditions and an induction of sFRP2 after TH treatment. In TRα0/0 mice, sFRP2 expression was unchanged by alteration of TH levels. Slight differences in the results reported in Fig. 2A and supplemental Fig. S4A are probably because of the use of isolated crypt cells in the first case and the whole mucosa in the second.FIGURE 2In vivo and in vitro regulation of the Sfrp2 gene. A, validation of microarray data by RT-QPCR on RNA from intestinal crypts. Histograms illustrate mean ± S.D., n = 3. *, p < 0.05, and **, p < 0.01, compared with WT-C condition; $, p < 0.05, and $$, p < 0.01, compared with WT-PTU and TRα0/0 conditions by the Student's t test. B, representative Western blot analysis of sFRP2 in epithelial cells fractionated from the villus-crypt axis. The picture is representative of four independent experiments. 1-4, fractions from WT control animals; 1′-4′, fractions from WT animals injected with TH. 50 μg of protein per lane were separated on gel; 20 ng of sFRP2 (lane Ctr) was included as positive control. Histograms in the lower panel (mean ± S.D., n = 4) summarize densitometry analyses (by ImageQuant) from four independent experiments. Data are normalized to the amount of actin in each sample. Statistical analysis was conducted using the Student's t test. **, p < 0.01, compared with the preceding fractions of the same experimental condition. $$, p < 0.01, compared with the corresponding untreated fraction. C and D, Sfrp2 gene regulation by T3 in primary cultures by RT-QPCR analysis. Cells were treated with T3 during 24 h (C) or 6 h (D). Histograms illustrate mean ± S.D. (n = 6) from three independent experiments, each conducted in duplicate. **, p < 0.01 by the Student's t test; $, p < 0.05 compared with WT control by the Student's t test.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Finally, we quantified by Western blot sFRP2 protein levels in fractionated villus-enriched or crypt-enriched cells from the villus-crypt axis (16Weiser M.M. J. Biol. Chem. 1973; 248: 2536-2541Abstract Full Text PDF PubMed Google Scholar) (supplemental scheme Fig. S5). sFRP2 protein was present at comparable levels along the villus-crypt axis in control animals (Fig. 2B, lanes 1-4). However, TH injection increased the protein specifically in the two last fractions, corresponding to crypt-associated progenitors (Fig. 2B, lanes 3′ and 4′ versus lanes 3 and 4). It is worth pointing out that TRα1 is the only TR receptor expressed and exclusively present in crypt cells. This observation was made both in this study and previously (8Plateroti M. Kress E. Mori J.I. Samarut J. Mol. Cell. Biol. 2006; 26: 3204-3214Crossref PubMed Scopus (103) Google Scholar). Altogether, these data demonstrated that the regulation of sFRP2 expression by TH depended specifically on the TRα1 receptor.To analyze whether the regulation of sFRP2 by TH was epithelial cell-autonomous, we used intestinal epithelium primary cultures maintained in the absence or presence of T3 for 24 h (Fig. 2C). In WT cells, sFRP2 mRNA levels were clearly and significantly increased by T3 treatment. We obtained a similar result by short term T3 treatment (6 h, Fig. 2D), strongly suggesting that this gene might be a direct target of TRα1. The mRNA levels of sFRP2 in TRα0/0 cells maintained in control condition were significantly decreased compared with WT control cells. As expected, the up-regulation of sFRP2 mRNA by T3 was abolished in cells from TRα0/0 mutant mice (Fig. 2C).Characterization of a TRE in the Promoter of the sFRP2 Gene—Using an in silico approach, we found a putative TRE located at -924 bp from the transcription start site (supplemental Fig. S6). To check whether this TRE is a binding site for TRα1 in vivo, we carried out chromatin IP assays on freshly isolated intestinal epithelium. Sonicated chromatin was incubated with anti-TRα1, anti-TRβ1, or preimmune serum. The DNA precipitated and in starting inputs was analyzed by PCR (Fig. 3). Using primers that amplified a fragment of DNA comprising the sFRP2-TRE, we showed the presence of a band only in samples incubated with the anti-TRα1 antibody. A similar result was obtained for the positive control Ctnnb1-TRE (8Plateroti M. Kress E. Mori J.I. Samarut J. Mol. Cell. Biol. 2006; 26: 3204-3214Crossref PubMed Scopus (103) Google Scholar). We also quantified by QPCR the DNA precipitated by the TRα1 antibody, expressed as a percentage of the starting input. We obtained a value of 6.4 ± 0.3% (n = 4) for sFRP2-TRE and 5.8 ± 0.4% (n = 4) for Ctnnb1-TRE. Negative controls included Sfrp2 promoter regions located 1 and 3 kb from the TRE, and the TH-insensitive 36B4 gene. The binding of TRα1 to sFRP2-TRE was also demonstrated in vitro by gel shift analysis (supplemental Fig. S7). Altogether, these data clearly demonstrated that TRα1 binds sFRP2-TRE in intestinal epithelial cells both in vitro and in vivo.FIGURE 3Molecular analysis of the thyroid hormone-responsive element present in the Sfrp2 gene, by in vivo chromatin immunoprecipitation. Study by PCR of the DNA purified from the different samples before and after chromatin IP. The picture is representative of two independent experiments. Indicated on the right part of each panel is the fragment amplified on the Sfrp2, Ctnnb1, and 36B4 genes. C, preimmune serum; TRα1, anti-TRα1; TRβ1, anti-TRβ1; SI, starting input; Ctrl PCR, negative control for PCR mix.View Large Image Figure ViewerDownload Hi-res image Download (PPT)TH-stimulated sFRP2 Stabilizes β-Catenin through Frizzled and Promotes Cell Proliferation—sFRP2 action on Wnt/β-catenin appears different depending on the cellular/tissue system. Our results showing strong positive regulation by TH-TRα1 in vivo and in vitro were intriguing. We took advantage of the intestinal epithelium primary culture model to study in detail the action of sFRP2 on β-catenin as well as its function on epithelial progenitor cell proliferation.We treated WT or TRα0/0 cells with recombinant sFRP2 and monitored the mRNA levels of c-Myc (Fig. 4A) and cyclin D1 (not shown), which are established targets of β-catenin (26He T.C. Sparks A.B. Rago C. Hermeking H. Zawel L. da Costa L.T. Morin P.J. Vogelstein B. Kinzler K.W. Science. 1998; 281: 1509-1512Crossref PubMed Scopus (4047) Google Scholar, 27Shtutman M. Zhurinsky J. Simcha I. Albanese C. D'Amico M. Pestell R. Ben-Ze'ev A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5522-5527Crossref PubMed Scopus (1906) Google Scholar). A concentration of 50 ng/ml in the culture medium significantly stimulated the expression of both targets. This stimulation was similar to that obtained by treating the cells with the canonical Wnt3a ligand (Fig. 4A). The simultaneous addition of sFRP2 and Wnt3a resulted in similar expression levels of c-Myc mRNA as in each single treatment. We obtained similar results in primary cultures from both WT and TRα0/0 intestine, indicating that stimulation of β-catenin targets by exogenous sFRP2 was independent of TRα1 signaling.FIGURE 4sFRP2 stabilizes β-catenin in vitro. A, analysis of c-Myc mRNA expression by RT-QPCR in intestinal epithelium primary cultures maintained in different culture conditions: sFRP2 50 ng/ml, Wnt3a 10 ng/ml, and a combination of both sFRP2 and Wnt3a. Histograms illustrate mean ± S.D. (n = 4) from two independent experiments each conducted in duplicate. **, p < 0.01, by Student's t test compared with the control condition. B, analysis of β-catenin mRNA expression by RT-QPCR in intestinal epithelium primary cultures maintained in different culture conditions: sFRP2 50 ng/ml or T3. For inhibition experiments, we co-treated the cells with T3 and anti-sFRP2 or anti-GFP antibodies, as indicated. Histograms illustrate mean ± S.D. (n = 6) from two independent experiments each conducted in triplicate. **, p < 0.01 by Student's t test compared with the control condition. C, representative Western blot analysis of total and activated nonphosphorylated β-catenin in primary cultures treated either with T3 or with 50 ng/ml sFRP2 for 24 h. The picture is representative of three independent experiments, each conducted in duplicate. After densitometry analysis (ImageQuant), the levels of total or activated β-catenin in each condition were normalized to that of actin. The values were then normalized to the control (Ctrl) condition. **, p < 0.01 n = 3, compared with control by the Student's t test. D, representative Western blot analysis of sFRP2 in culture medium of cells treated or untreated with T3 during 24 h. 500 μg of concentrated proteins from the medium were loaded into the gel. 20 ng of recombinant protein (lane Ctr) was used as the positive control. The picture is representative of three independent experiments. E, analysis of c-Myc mRNA expression by RT-QPCR in primary cultures treated or untreated (C, control) with T3 for 24 h. For inhibition experiments we co-treated the cells with T3 and anti-sFRP2 or anti-GFP antibodies. Histograms illustrate mean ± S.D. (n = 6) from two independent experiments each conducted in triplicate. *, p < 0.05 and **, p < 0.01 by Student's t test.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Increased levels of β-catenin targets depend on increased availability of β-catenin (13Clevers H. Cell. 2006; 127: 469-480Abstract Full Text Full Text PDF PubMed Scopus (4427) Google Scholar). Because the levels of β-catenin mRNA were not changed by sFRP2 treatment in vitro (Fig. 4B), we focused on protein analysis and in particular on the amount of total as well as nonphosphorylated stabilized β-catenin. For this we used a specific antibody recognizing the N-terminal part of the protein only when not phosphorylated at residues Ser-37 and Thr-41. Wnt signals specifically increase the levels of dephosphorylated β-catenin as detected with this antibody (28van Noort M. Meeljik J. van der Zee R. Destree O." @default.
• W2091518548 created "2016-06-24" @default.
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• W2091518548 creator A5025041008 @default.
• W2091518548 creator A5038888136 @default.
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• W2091518548 date "2009-01-01" @default.
• W2091518548 modified "2023-10-15" @default.
• W2091518548 title "The Frizzled-related sFRP2 Gene Is a Target of Thyroid Hormone Receptor α1 and Activates β-Catenin Signaling in Mouse Intestine" @default.
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