Thyroid Hormone Receptor {alpha}1 Directly Controls Transcription of the {beta}-Catenin Gene in Intestinal Epithelial Cells

Thyroid hormones, T3 and T4, are known regulators of intestinedevelopment. The best characterized example is the remodelingof the gastrointestinal tract during amphibian metamorphosis.Thyroid hormones act via nuclear receptors, the TRs, which areT3-dependent transcription factors. We previously showed thatintestinal epithelial cell proliferation is controlled by thyroidhormones and the TR ${alpha}$ gene. To analyze the mechanisms responsible,we studied the expression of genes belonging to and/or activatedby the Wnt/ß-catenin pathway, a major actor in thecontrol of physiological and pathological epithelial proliferationin the intestine. We show that T3-TR ${alpha}$ 1 controls the transcriptionof the ß-catenin gene in an epithelial cell-autonomousway. This is parallel to positive regulation of proliferation-controllinggenes such as type D cyclins and c-myc, known targets of theWnt/ß-catenin. In addition, we show that the regulationof the ß-catenin gene is direct, as TR binds in vitroand in chromatin in vivo to a specific thyroid hormone-responsiveelement present in intron 1 of this gene. This is the firstreport concerning in vivo transcriptional control of the ß-cateningene. As Wnt/ß-catenin plays a crucial role in intestinaltumorigenesis, our observations open a new perspective on thestudy of TRs as potential tumor inducers.

INTRODUCTION

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Introduction

The mammalian gut develops from the association of the endodermand the splanchnic mesoderm. The adult features are acquiredduring well-defined fetal and postnatal developmental steps,which are completed at the weaning (16, 18). The pluristratifiedgut endoderm is remodeled to form a monolayer of rapidly renewingcolumnar epithelial cells, organized in the small intestinein the typical finger-shaped structure (49). From a functionalpoint of view, this defines specific spatial and functionalunits: the villi, which constitute the differentiated compartment,and the crypts of Lieberkühn, where proliferation takesplace. The main characteristic of the intestinal epitheliumis its continuous cell renewal, which is fuelled by multipotentstem cells within the crypts (49). The rate of crypt cell division,migration, and villus cell differentiation must be tightly controlledto ensure epithelial homeostasis (49).

Several pathways have been described to play an important rolein the control of epithelial proliferation in the intestine,during early development as well as during the continuous renewingin adulthood (3, 49). Among these pathways, the Wnt/ß-cateninpathway has received major attention, as it is involved in thenormal and in the pathological proliferation of the gut (4,50). The signal transducing components of the Wnt pathway arereceptors belonging to the frizzled and LRP protein families(35, 42). One target of the Wnt pathway is the ß-cateninprotein. In the absence of Wnt signal, ß-catenin isdestabilized by phosphorylation from a cytoplasmic complex comprisingaxin, adenomatous polyposis coli, and glycogen synthase kinase3ß. The phosphorylated ß-catenin is finallydegraded by the proteasome. The action of this complex can beantagonized by disheveled, a cytoplasmic protein that is activatedby the frizzled receptors. In addition to Wnt, some other factorshave been shown to act in the stabilization of ß-catenin,such as insulin-like growth factor, epidermal growth factor,and human growth factor, through their respective thyrosin kinase-associatedreceptors (35). The main characteristic of the stabilized ß-cateninis its accumulation in the cytoplasm and migration into thenucleus, where it acts as a transcriptional coactivator by associatingwith transcription factors of the Tcf/Lef family (35). Intestinaltumors characterized by actively proliferating cells displayhigh levels of nuclear ß-catenin (42). Recently, vande Wetering et al. (52) described the presence of ß-cateninin the nuclei of crypt cells, strongly supporting its role inthe control of cell proliferation. The complex ß-catenin/Tcfhas been shown to control the expression of several genes involvedin cell cycle/proliferation control like cyclin D1, c-myc, andcdx1 (54) as well as, indirectly, cyclin D2 (20). It is worthnoting that mutations of the different partners of this pathwayare responsible for tumorigenesis in humans, as they constitutivelyactivate uncontrolled cell proliferation (42, 50). In fact,activation of this pathway as well as that of the ras oncogeneare sufficient to induce colon tumorigenesis (3). Surprisingly,to date, very little is known on the transcriptional regulationof the ß-catenin gene.

Several observations suggested that thyroid hormones (TH), T3and T4, are involved in the control of intestinal development(15). First of all, it has been shown that the gastrointestinaltract remodeling during amphibian metamorphosis is completelydependent on TH (48). In rodents, they have been shown to participatein the developmental processes responsible for the increasein the mucosa growth as well as in the onset of the adult-typedigestive enzymes of the absorptive enterocytes at weaning (15).The action of TH is mediated by T3 binding to thyroid hormonenuclear receptors, the TRs, which belong to the nuclear hormonereceptor family of transcription factors (21). The TRs are encodedby two genes, TR ${alpha}$ and TRß (22), each of which producesseveral protein isoforms (10). More recently, using congenitalhypothyroid and TR knockout mice, we pointed out that TH acton the intestinal postnatal development mainly through the controlof the proliferation of the intestine epithelial progenitorcells. This function depends on the TR ${alpha}$ 1 receptor (9, 13, 41).

To analyze the molecular targets of T3-activated TR ${alpha}$ 1 in theintestine, we focused here on the regulation of the Wnt/ß-cateninpathway. We showed that T3-TR ${alpha}$ 1 controls the expression of themurine ß-catenin gene, catnb, by binding on the chromatinof epithelial cells to a functional TRE sequence in intron 1.This effect is parallel to the activation of the Wnt/ß-catenintarget genes, including type D cyclins and c-myc. This workthen provides the first demonstration of the direct controlof transcription of the ß-catenin gene.

MATERIALS AND METHODS

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Materials and Methods

Animal treatment and tissue preparation. TR ${alpha}$ ^0/0 (13), TRß^–/– (12), Pax8^–/–(28), and the respective wild-type animals have been used inthis study. Mice were housed and maintained with approval fromthe animal experimental committee of the Ecole Normale Supérieurede Lyon (Lyon, France). TH deficiency in pups was induced byfeeding the mothers a low-iodine diet supplemented with 0.15%propylthiouracil (PTU; Harlan/Teklad) for 2 weeks. To induceTH deficiency in adult animals, they were fed the low-iodinediet supplemented with 0.15% PTU and 0.05% methimazole (Sigma)in the drinking water for 3 weeks. Hyperthyroidism was inducedby intraperitoneal injections of a mixture of T4 and T3 (2.5mg/kg T4 and 0.25 mg/kg T3 in 100 µl of phosphate-bufferedsaline) for the indicated length of time. Control animals werefed standard mouse chow. Animals were sacrificed at the indicatedages, and the intestine was quickly removed. The proximal anddistal parts of the small intestine were fixed in 10% bufferedformalin for immunohistochemistry or frozen in liquid nitrogenand used for RNA and/or protein extraction. Blood was also recoveredfor serum preparation, and the levels of free T3 and T4 wereanalyzed by a VIDAS enzyme-linked assay kit (Biomerieux). Thedata on T3 and T4 measurements are reported in Tables S1 andS2 in the supplemental material.

Primary cell culture of intestine epithelial cells. Primary intestinal cell cultures were derived from 10-day postnatalmice. After sacrifice, the whole small intestine was removed.The epithelium was isolated as intact organoids by enzymaticdissociation using collagenase type XI (Sigma) and dispase (BoehringerMannheim) followed by physical disaggregation (8). Organoidswere plated in Dulbecco's modified Eagle medium (DMEM; Invitrogen)supplemented with 2.5% heat-inactivated fetal calf serum (Gibco)depleted of T3 and T4 (45), 20 ng/ml epidermal growth factor(Sigma), and insulin-transferrin-selenium diluted 1/100 (Sigma).Cultures were maintained on 24-multiwell tissue culture dishes.For immunolabeling experiments, coverslips were inserted inthe wells. Culture surfaces were coated with type 1 collagen.The purity of the epithelial colonies was analyzed by immunolabelingfor specific markers: epithelial cells by anticytokeratins (ICN);fibroblasts by antivimentin (Sigma); smooth muscle cells byanti-smooth muscle actin (Sigma). Cells were analyzed after5 days of culture. For the analysis of T3 action in vitro, 10^–7M T3 or vehicle alone was added to the culture medium for theindicated length of time. Cycloheximide (15 µg/ml) wasadded as indicated. For proliferation studies, 10 µM bromodeoxyuridine(BrdU) was added to the culture medium during an overnight incubation.

RNA extraction and analysis. RNA was extracted from tissues using the QIAGEN RNeasy kit (QIAGEN)and from cell cultures using the Stratagene Absolutely RNA nanoprepkit (Stratagene). To avoid the presence of contaminating DNAin RNA samples, DNase digestion (according to the specific instructionsof the manufacturer; QIAGEN or Stratagene) was performed inall preparations. Northern blot analysis was carried using aHybond N⁺ membrane (Amersham), and DNA probes for ß-catenin,cyclin D1, cyclin D2, c-myc, and hypoxanthine phosphoribosyltransferase(HPRT) were labeled by random priming using [³²P]ATP. Real-timequantitative PCR analysis used random hexanucleotide primingand amplification in a Lightcycler apparatus (Bio-Rad Laboratories)using SYBR green PCR master mix according to the manufacturer'sinstructions (QIAGEN). The primers used are listed in TableS3 in the supplemental material. The data from the PCR werenormalized to that of 36B4 levels in each sample. Semiquantitativereverse transcription (RT)-PCR was used to analyze TR ${alpha}$ 1 expression;HPRT has been used as internal control. The primers were asfollows: TR ${alpha}$ 1, 5'-GGAGATGATTCGCTCACTGCAG and 5'-CGACTTTCATGTGGAGGAAG(product size, 720 bp); HPRT, 5'-GCTGGTGAAAAGGACCTCT and 5'-CACAGGACTAGAACACCTGC(product size, 240 bp).

Cell line culture and transfection. Cos1 cells were cultured in DMEM supplemented with 5% heat-inactivatedfetal calf serum. The different constructs were cloned in thepGL2 basic expression vector (Promega). The wild-type or mutated36 bp of the catnb gene comprising TRE-int1 (from bp 2140 to2175 of intron 1) were cloned upstream of a simian virus 40minimal promoter (29). The cells were transfected using theExgen transfection reagent (Euromedex). After transfection,cells were maintained in thyroid hormone-depleted serum (45).T3 (10^–7 M) or vehicle alone was added to the culturemedium 24 h before the end of the culture. Luciferase activitywas measured 48 h after transfection using the luciferase dualsystem (Promega).

Electrophoretic mobility shift assay (EMSA). (i) In vitro protein synthesis. Full-length cDNA cloned in pSG5 (Stratagene) coding for mouseTR ${alpha}$ 1, rat TRß1, and mouse RXR ${gamma}$ were transcribed/translatedin vitro using the TNT kit (Promega). Each cDNA was under thecontrol of the T7 promoter.

(ii) EMSA. The single-strand oligonucleotides were labeled with T4 polynucleotidekinase (Fermentas) in the presence of [ ${gamma}$ -³²P]ATP. After annealingwith the complementary cold oligonucleotides, the probes werepurified on a 10% acrylamide gel and the specific bands wereeluted overnight at 4°C in Tris-EDTA. Binding reactionswere performed for 20 min using the radiolabeled DNA probes(20,000 cpm) and the in vitro-transcribed TR and/or RXR in 10%glycerol, 10 mM HEPES, 30 mM KCl, 4 mM spermidine, 0.1 mM EDTA,0.25 mM dithiothreitol, 1 mM Na₂PO₄, and poly(dI-dC) (1.5 µg).Unlabeled specific and nonspecific competitor oligonucleotideswere included at the indicated molar excess in the binding reactions.Where indicated, anti-TR ${alpha}$ 1 antibodies (antibodies raised againsta C-terminal peptide and affinity purified with the same peptide)have been included in the reaction mix for 30 min on ice. Afterbinding, reaction samples were loaded on a 5% nondenaturingpolyacrylamide gel and electrophoresed for 2 h at 180 V, followedby gel fixation, drying, and exposure to X-ray film.

Western blot and immunolabeling. Whole-protein extracts were obtained by homogenizing mouse intestinein 5 mM HEPES (pH 7.9), 26% glycerol, 1.5 mM MgCl₂, 0.2 mM EDTA,0.5 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride.NaCl was added to a final concentration of 300 mM before centrifugation.Supernatant proteins were separated on a 10% acrylamide-bisacrylamide (29:1) gel and transferred to a nitrocellulose membrane(Hybond ECL) before incubation with the first antibody. Thiswas followed by an incubation with secondary anti-rabbit oranti-mouse immunoglobulin G-horseradish peroxidase (Promega).The signal was analyzed by using the enzymatic chemiluminescencedetection kit (Amersham).

Antibodies. We used the following primary antibodies: mouse monoclonal anti-cyclinD2 (Sigma), rabbit anti-ß-catenin (Santa Cruz), mouseanti-cyclin D1 (Santa Cruz), rabbit anti-c-myc (a gift fromCalame Kathryn, Columbia University College of Physicians andSurgeons, New York), and mouse anti-ß-actin (Sigma).Immunohistochemical analysis of ß-catenin (Santa Cruz),TR ${alpha}$ 1 (antibodies raised against a C-terminal peptide and affinitypurified with the same peptide), and Ki67 (Novocastra) was performedon 10% buffered formalin-fixed paraffin-embedded tissue sections.Immunolabeling for ß-catenin and BrdU (Roche) hasbeen done on 2% paraformaldehyde fixed cell cultures. Secondaryfluorescent antibodies were from Jackson Laboratories. Confocalanalysis has been performed on a Zeiss Axiovert microscope,with fluorescence microscopy on a Zeiss Axioplan microscope.

ChIP and DNA analysis. The chromatin immunoprecipitation (ChIP) study was performedon collagenase-dispase-separated epithelial fragments from 3-to 5-day-old mouse intestines. The monolayer of cells was incubatedwith 1% formaldehyde in DMEM for 5 min at 37°C. The reactionwas blocked by the addition of glycine to a final concentrationof 125 mM, followed by two washes of cold phosphate-bufferedsaline (Invitrogen) containing protease inhibitors (Roche).ChIP was conducted using the kit from Upstate according to themanufacturer's instructions. Briefly, 500 ng of sonicated DNA(fragments of 200 to 1,000 bp, by using a Vibra Cell by Sonicsand Materials, Inc.) were incubated, respectively, with 10 µlof anti-TR ${alpha}$ 1, anti-TRß1 (Affinity Bioreagents), andpreimmune sera overnight at 4°C. At the end of the reactionand washing steps, DNA was extracted by phenol-chloroform andsuspended in 30 µl of H₂O. Four microliters of sampleswas used for conventional PCR (Taq from Eurogentec). For quantitativePCR we used the Lightcycler apparatus (Bio-Rad Laboratories)and the SYBR green PCR master mix from QIAGEN. The primers arelisted in Table S4 in the supplemental material. All of theamplified fragments have been controlled by cloning and sequencing.

RESULTS

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Results

TR ${alpha}$ gene controls gut epithelial proliferation starting early after birth. Our previous study indicated the major involvement of the TR ${alpha}$ gene in the control of epithelial proliferation in the intestineduring weaning time (40, 41). We extended here this observationand analyzed the rate of cell proliferation in the TR ${alpha}$ ^0/0 knockoutmice (13) compared to the wild type (WT) throughout fetal andpostnatal intestinal development until adulthood (Fig. 1A).In TR ${alpha}$ ^0/0 mutants, we observed a 25% decrease in the number ofproliferating cells at postnatal day 1 and in 8-week-old adultanimals and a decrease of nearly 50% at 7, 14, and 21 days afterbirth compared with the respective WT animals. This was observedin both the proximal (Fig. 1A) and distal small intestine (notshown). Looking for the expression of the TR ${alpha}$ 1 receptor, we couldobserve that TR ${alpha}$ 1 mRNA was expressed during this same period(Fig. 1B). Moreover, TR ${alpha}$ 1 protein, analyzed by an immunohistochemicalapproach, was specifically present in the crypt cells, as shownfor the 14-day-old intestine (Fig. 1C to H; see Fig. S1C toH in the supplemental material).

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FIG. 1. Analysis of crypt cell proliferation and TR ${alpha}$ 1 expression in the small intestine. (A) Study of cell proliferation during development in the proximal small intestine. Ki67 immunolabeling was performed as previously described (41). The number of Ki67-positive nuclei has been evaluated on well-oriented sections from 4 to 5 different animals per age. Histograms represent means ± standard deviations. Statistical analysis was conducted by using two-tailed Student t test. *, P < 0.05; **, P < 0.001. P, postnatal day; W, weeks. (B) Analysis of TR ${alpha}$ 1 expression by semiquantitative RT-PCR during intestine development. HPRT was used as an internal control. E, embryo; PN, postnatal. (C to H) TR ${alpha}$ 1 protein immunolabeling (E and H) on a 2-week-old mouse intestine. In panel C, the phase-contrast picture is shown. All nuclei were stained with Hoechst (D). In panels F and G, the merging of each simple staining is shown. Controls for TR ${alpha}$ 1 antibody specificity are illustrated in Fig. S4 in the supplemental material. Bars, 15 µm (C to F); 7 µm (G and H). Abbreviations: ve, villus epithelium; ce, crypt epithelium. The white dotted bar defines the limit between the crypt and the villus compartments. Some villi (red dots) and crypts (yellow dots) are highlighted in panels C to E.

Thyroid hormone controls the expression of the Wnt/ß-catenin pathway. Focusing on 2-week-old animals, we studied the expression ofseveral components of the Wnt pathway, including ß-catenin;ß-catenin destabilizers adenomatous polyposis coli,glycogen synthase kinase 3ß, and axin; ß-cateninstabilizer disheveled; ß-catenin transcription cofactorTCF4 and its repressors Groucho and Chybby (1, 51). Using RNAsextracted from euthyroid, hypothyroid, and hyperthyroid intestines,we observed that the expression of these genes was unaffectedby the TH status (not shown), except for ß-catenin.In fact, chemical-induced or congenital hypothyroidism resultedin a decrease of ß-catenin mRNA and protein (Fig.2A and B). TH injections for 2 days resulted in an increaseof both mRNA and protein levels. We also analyzed the expressionof the downstream targets of this pathway, cyclin D1 and c-myc(1), as well as the indirect target cyclin D2 (20), in animalsof different TH status (Fig. 2A and B). All of these genes,expressed mainly in crypt cells where they control the cellproliferation (5, 25, 52), showed a decreased expression atthe mRNA and protein levels in hypothyroid conditions and astrong increase after TH injection.

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FIG. 2. Study of TH action in vivo. (A) Representative Northern blot analysis of the indicated mRNAs in 2-week-old intestine of euthyroid (Pax8^+/+) (Ctr), congenital hypothyroid (Pax8^–/–) (Hypo), and hyperthyroid (Pax8^–/– TH-injected) (Hyper) animals. HPRT mRNA was used as an internal control. (B) Representative Western blot analysis of indicated proteins in animals as described for panel A. (C) Kinetic analysis of gene induction by TH evaluated by Northern blotting. PTU-treated animals were injected with TH for the indicated time. 18S RNA was the internal control. Histograms (means ± standard deviations) summarize densitometry analyses from three independent experiments. (D) Quantitative real-time RT-PCR analysis of ß-catenin mRNA in intestines of TR ${alpha}$ ^0/0, TRß^–/–, and WT animals displaying different TH status. The picture is representative of two independent experiments, with 3 animals/condition. Histograms represent means ± standard deviations. (E) Representative Western blot analysis of indicated proteins in animals as described for panel D. Statistical analysis was conducted by using two-tailed Student t test. **, P < 0.001. C, control; P, PTU treated; T, TH injected (inj).

We then analyzed the kinetic profile of mRNA induction to clarifythe hierarchy of the gene induction. We gave a single pulseof TH to hypothyroid animals and recovered the intestine atdifferent time points: 1 h, 3 h, 5 h, and 7 h after injection.The results are illustrated in Fig. 2C. Cyclins D1 and D2 andc-myc (not shown) mRNA expression were not changed over a periodof 7 h after injection (Fig. 2C). On the contrary, ß-cateninmRNA started to be increased as early as 1 h after TH injections,being statistically significant after 3 h and reaching a maximumlevel 5 h after the injection (Fig. 2C). These data indicatethat ß-catenin induction by TH is an earlier eventthan with the other studied genes.

We also quantified ß-catenin mRNA and protein expressionin TR ${alpha}$ and TRß mutant mice having different TH statusto evaluate whether TH responsiveness depended on a specificTR pathway (Fig. 2D and E). Wild-type and TRß^–/–animals showed a decreased ß-catenin mRNA and proteinexpression under hypothyroid conditions and an induction afterTH treatment for 6 h (not shown) or 2 days. Untreated TR ${alpha}$ ^0/0mice, which showed a decreased level of ß-cateninmRNA compared to wild-type mice under the same conditions, displayedunchanged levels of this mRNA and protein under hypothyroidor hyperthyroid conditions. Similar analysis was performed onwild-type and TR ${alpha}$ ^0/0 adult animals. The results showed that ß-cateningene and protein expression was regulated in a similar mannerin the adult and the developing intestine (see Fig. S2A andB in the supplemental material). The parallel analysis of thedownstream controlled gene cyclin D1 at the mRNA (not shown)and protein levels (Fig. 2E; see Fig. S2B in the supplementalmaterial) showed a trend of regulation similar to that of ß-catenin.It is worth pointing out that both ß-catenin and cyclinD1 expression are much more affected by hypothyroidism thanby lack of the TR ${alpha}$ gene (compare wild-type PTU with TR ${alpha}$ ^0/0). Thisis probably due to the aporeceptor effect of the nonligandedTR ${alpha}$ 1 in the absence of T3 (9). Altogether, these data demonstratedthat the regulation of ß-catenin expression by THdepended specifically on the TR ${alpha}$ 1 receptor in the developingand the adult intestine.

In vitro analysis of the ß-catenin induction by thyroid hormone and TR ${alpha}$ gene. To analyze a homogeneous cell population and to understand whetherthe responsiveness to TH through the TR ${alpha}$ gene is an epithelialcell autonomous process, we used a model of epithelial primarycultures from WT and TR knockout mice. The enzymatic separationbetween the epithelium and the connective tissue (8) provideda highly pure epithelial cell population (see Fig. S3 in thesupplemental material). BrdU incorporation analysis showed thatthis culture system preserved a population of proliferatingcells, presumably epithelial progenitors. However, due to thesmall size of the epithelial organoids, this model could onlybe used for immunolabeling and quantitative real-time RT-PCRexperiments.

First, we tested whether the cell proliferation in vitro wasaffected by the addition of T3 in the culture medium (Fig. 3A).Primary cultures established from WT as well as TRß^–/–intestine showed a strong increase in the rate of BrdU-positivecells when maintained for 2 days in the presence of T3 comparedwith untreated cells. In contrast, TR ${alpha}$ ^0/0-derived cell culturesshowed a lower level of proliferating cells than the WT anddid not respond to T3 treatment.

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FIG. 3. In vitro study of TH action. (A) Rate of cell proliferation in epithelial primary cultures from WT and TR knockout intestines, after BrdU incorporation and immunolabeling. Total nuclei stained with propidium iodide and BrdU-positive nuclei were counted under the microscope. Histograms (means ± standard deviations) represent results from three independent experiments conducted (each) on 12 1-cm wells. (B) ß-Catenin mRNA quantification by real-time RT-PCR, evaluated at different times of T3 treatment. The results (means ± standard deviations) are the summary of those for four independent experiments, each conducted in duplicate. (C) ß-Catenin and cyclin D1 mRNA expression in primary cultures maintained during 24 h in the presence (+) or absence (–) of T3 and/or cycloheximide (Cyclohex). Data (means ± standard deviations) summarize the results of two independent experiments conducted in triplicate. Two-tailed Student t test was used. *, P < 0.05; **, P < 0.001; ***, P < 0.0001.

The in vitro model was then used to analyze specific gene expressionby quantitative real-time RT-PCR. We studied ß-cateninexpression in vitro in control or T3-treated cells for differentlengths of time (Fig. 3B). The mRNA level increased after T3treatment, reaching statistical significance after 6 h. In vivostudies showed a hierarchy in the induction of gene expressionby TH. If the induction of the Wnt/ß-catenin targetgenes by T3-TR ${alpha}$ 1 depends on the previous induction of the ß-catenin,the use of in vitro model could help to answer to this question.For this reason, we treated the cells with cycloheximide and/orT3 for 24 h and analyzed the expression of ß-cateninand cyclin D1. In non-cycloheximide-treated cells, we observedan induction of both mRNAs compared to the control (Fig. 3C).The addition of cycloheximide specifically suppressed the inductionby T3 of cyclin D1 expression. On the contrary, cycloheximidehad no effect on ß-catenin up regulation by T3 (Fig.3C). The small decrease of gene expression in the cycloheximide-treatedcells is probably due to a generalized action of the drug. Thesedata demonstrate that the positive regulation of the ß-cateningene expression does not need protein synthesis, strongly suggestinga direct effect on ß-catenin but not on the cyclinD1 gene.

Characterization of a functional TRE in intron 1 of the ß-catenin gene: in vitro and in vivo studies. The data on the regulation of ß-catenin gene by THand TR ${alpha}$ 1, in vivo and in vitro, suggested that this gene maybe a direct target of the TR ${alpha}$ 1 receptor. Looking in silico forprediction of TRE sequences in the mouse ß-cateningene catnb, we found a sequence of 36 bp (referred as TRE-int1),spanning from nucleotide 2140 of the first intron and containingfour putative TRE sites (Fig. 4A). While the TRE-A is a perfectDR4 (6), the sites TRE-B, TRE-C, and TRE-D show degenerate DR4structures. To evaluate whether the TR ${alpha}$ 1 receptor binds in vitroto these TREs, we performed EMSAs. Compared to a synthetic positivecontrol DR4 (29), the TRE-int1 has the same ability to bindto TR ${alpha}$ 1 as a monomer, homodimer, and TR ${alpha}$ 1/RXR heterodimer (Fig.4C and D). These complexes were supershifted after incubationwith an antibody against TR ${alpha}$ 1 (Fig. 4D). We also tested by EMSAdifferent mutant TRE-int1 sequences (Fig. 4B). Mutant 2,5, mutant6, and to a lesser extent, mutant 3 retained their ability tobind to TR ${alpha}$ 1 as a monomer, homodimer, and heterodimer (Fig. 4E).Mutant 1,4 had a lower ability, and mutant 2,3,4 was unableto bind to TR ${alpha}$ 1.

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FIG. 4. Molecular analysis of the thyroid hormone responsive element present in the catnb gene. (A) Structure of the four putative DR4 found in intron 1. Divergences from canonical DR4 are indicated in boldface type. (B) WT TREint-1, showing the GT sequences (1 to 6) substituted in the different TRE mutants. (C to E) EMSA analysis using the labeled probes indicated and in vitro-transcribed/translated proteins indicated. The specificity of the binding was assessed by adding specific or nonrelated cold sequences at the indicated molar excess (Comp. 10x or Comp. 100x). +, present; –, absent.

Next, we analyzed the enhancer properties of the ß-cateninTREs in transient-transfection experiments. We cloned the 36-bpTRE-int1 sequence upstream of a minimal simian virus 40 promoter(29) in a luciferase reporter vector and then transfected itin Cos1 cells (Fig. 5). The expression of the luciferase wasanalyzed in the presence or absence of either TR ${alpha}$ 1 expressionvector and/or T3. As Cos1 cells contain low levels of endogenousTR ${alpha}$ 1, induction of the DR4 positive control by T3 was observedonly in cultures cotransfected with the TR ${alpha}$ 1 expression vector.A similar response was observed with the TRE-int1 reporter,showing that it is functionally responsive to T3. When we comparedthe different TRE-int1 mutant constructs, it was clear thatonly those preserving the TRE-A, alone or in combination withTRE-B and C, displayed a responsiveness to T3 similar to thatof the TRE-int1. Altogether, these data strongly suggested thatTRE-A is the functional binding site for TR ${alpha}$ 1.

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FIG. 5. Transient-transfection analysis in Cos1 cells. Two hundred nanograms of the luciferase expression vector was transfected alone or in combination with 200 ng of pSG5-hTR ${alpha}$ 1 expression vector. Cells were treated or not with 10^–7 M T3 for 16 h. Bars represent means ± standard deviations (n = 6).

To check whether the TRE-int1 is a binding site for TR ${alpha}$ 1 in vivo,we investigated the binding by in vivo ChIP assay. We performedthese experiments on collagenase-dispase-isolated fragmentsof the intestinal epithelium, freshly prepared from newbornanimals. Figure 6 summarizes the set up and the results of theseexperiments. The sonicated chromatin has been incubated withanti-TR ${alpha}$ 1, anti-TRß1, or preimmune sera. The precipitatedDNA from these different experimental conditions and in thestarting inputs was then analyzed by conventional and real-timePCR to reveal the presence of specific DNA templates. All ofthe amplified products have been controlled by cloning and sequencing(not shown). Interestingly, using the Catnb1 and Catnb2 primers,which amplify DNA fragments of the catnb gene, including theTRE-int1 (Fig. 6A), we showed a specific band only in the samplesincubated with the anti-TR ${alpha}$ 1 antibody (Fig. 6B, lane b). A bandof the same size was obviously present in the starting inputsamples (Fig. 6B, lane d). To verify the specificity of thisresult, we performed a PCR analysis using the Catnb3 and Catnb4primers, which amplify fragments of the intron 1 outside theTRE (Fig. 6A). Moreover, we used primers specific to promotersof the phosphoriboprotein gene 36B4 and of the villin gene,for which no regulation by TH has been described. Using theseoligonucleotides, we only amplified DNA fragments in the startinginputs (Fig. 6B, lanes d), indicating that the anti-TR ${alpha}$ 1 antibodydoes not bind the chromatin aspecifically. To quantify the amountof the DNA precipitated by the TR ${alpha}$ 1 antibodies, we also performedreal-time PCR. Using the Catnb1 (Fig. 6C) and Catnb2 (not shown)primers, we amplified specific DNA fragments. The amounts areexpressed as percentages of the starting inputs. No specificamplification could be observed in the samples incubated withthe anti-TRß1 or preimmune sera or in the differentsamples when we used the Catnb3 (Fig. 6C), Catnb4, and p36B4(not shown) primers. These data clearly demonstrated that, inintestinal epithelial cells in vivo, TR ${alpha}$ 1 binds to the ß-cateningene at the level of the TRE-int1.

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FIG. 6. In vivo ChIP. (A) Scheme of intron 1 of the catnb gene displaying the different primers used for this study. S (sense) and AS (antisense) numbers refer to oligonucleotide primers of the respective Catnb-amplified fragment numbers. E1 and E2 refer to exons 1 and 2, respectively. The arrow upstream of E1 indicates the promoter of the gene. Note that Catnb1 and Catnb2 primers amplify DNA fragments comprising the TRE. Catnb3 and Catnb4 were used as negative controls, as the amplified fragments are, respectively, 500 bp and 3,000 bp away from the TRE. (B) Study by conventional PCR of the DNA purified from the different samples before and after ChIP. The picture is representative of the results from two independent experiments. On the right part of each panel, the fragment amplified on the catnb gene or on the promoters of 36B4 and villin genes is indicated: a, preimmune serum; b, anti-TR ${alpha}$ 1; c, anti-TRß1; d, starting input; e, negative control for PCR mix. (C) Quantitative real-time PCR study of the purified DNA before and after ChIP. The amount of the indicated products is illustrated as a percentage of that measured in the starting inputs. The picture is representative of the results of two independent experiments.

DISCUSSION

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Discussion

In this paper and in previous reports, we pointed out that T3-activated-TR ${alpha}$ 1receptor controls the proliferation of the intestinal epithelialcells in vivo and in vitro. In an attempt to identify the molecularbasis of this control, we focused here on the analysis of theWnt/ß-catenin pathway, since it plays a central rolein the control of normal and pathological cell proliferationin the gut (42). Interestingly, we observed that the TR ${alpha}$ 1 receptordirectly controls the transcription of the ß-cateningene, the central actor of this pathway. In fact, in vivo andin vitro models treated with TH displayed an increase in ß-cateninmRNA and protein expression. This was parallel to the up-regulationof cyclins D1 and D2 and c-myc, well-known proliferation controllinggenes (5, 25, 52) and targets of the Wnt/ß-cateninpathway (1, 20). Several findings support the assertion thatthe ß-catenin gene, but not cyclin D or c-myc, isdirectly regulated by TH. First, induction of the ß-cateninmRNA in vivo and in vitro is achieved after a short time oftreatment by TH. Second, the cycloheximide addition to the culturemedium does not prevent the up-regulation of the ß-cateninmRNA in vitro by T3, as it is the case for cyclin D1 mRNA. Third,TH injections to TR ${alpha}$ ^0/0 animals, which are unable to stimulateß-catenin expression, also fail to increase the cyclinD1 mRNA compared to the WT animals (data not shown). Finally,we also provided the experimental proof that in epithelial cellsof the intestine in vivo, TR ${alpha}$ 1 is fixed on the TRE-int1 presentin the catnb gene. This is the first report of in vivo ChIPanalysis which shows the occupancy of a TRE by a TR receptorin mammals. A binding of TRE by TRs has already been reportedfor Xenopus laevis target genes (44).

The regulation of the transcription of the ß-cateningene by T3 is likely to go through the TRE-int1. In fact, itis the unique responsive element found within the promoter environment.Moreover, its intronic location is not surprising, since severalreports described TREs located downstream of the promoters (7,38, 46). We also showed that the TRE-int1 is composed of severalTREs organized in tandem, reminiscent of the TREs present inthe rat growth hormone gene (37).

Despite the well-described posttranslational control of theß-catenin, very little is known at the gene transcriptionlevel. A recent paper described the cloning and analysis ofthe rat and human ß-catenin regulatory regions ofthis gene (23). Interestingly, several AP1 binding sites andone TCF binding site have been described, suggesting that severalputative factors may influence the transcription of this gene.However, no physiological functions for these regulatory regionshave been reported until now. We showed here that the TRE inintron 1 of the catnb gene is functional in vivo and in vitro.

ß-Catenin is a central actor in gut epithelial cellhomeostasis, and its subcellular localization defines specificfunctions in cell adhesion (membrane bound) or in target geneactivation (nuclear presence) (36, 52). We reported in our studythat TH deprivation or supplementation in animals and in cellsaffects ß-catenin gene and protein expression. Morespecifically, TH treatment allows an increase of its levels.The availability of free ß-catenin, which migratesinto the nuclei, is controlled by several mechanisms involvingstabilizing and destabilizing phosphorylations as well as destabilizingdephosphorylations (36). In TH-stimulated models, we observedan increase of ß-catenin-positive nuclei (not shown),strongly suggesting that this treatment positively affects itsstabilization. However, the exact mechanisms responsible forthis remain to be clarified and will be the subject of futureinvestigations.

In the nucleus, ß-catenin activates the transcriptionof target genes, as it binds to the Tcf/Lef family of transcriptionfactors (35). Here we show that some of its targets, activatorsof cell proliferation, are positively regulated by TH in vivoand in vitro. This strongly suggests that the increase of cellproliferation induced by TH may be dependent on the activationof ß-catenin expression. Is the increase of the ß-cateninexpression per se sufficient to induce cell proliferation? Thisis a crucial question. Several reports indicated that increasingthe level of ß-catenin leads to increased cell proliferationin various systems, such as the intestine (47, 55), liver (32,34), and vestibular cells (19). This strongly supports our owndata showing that the increase of ß-catenin expressionis one key molecular event related to the activation of cellproliferation. However, it is worth pointing out that a negativecontrol by TH of the Wnt pathway was described in an in vitromodel of pituitary cells (33). In this model, the positive actionof the TH on cell proliferation was parallel to a decrease ofß-catenin expression. However, there was no indicationof a direct negative control of the ß-catenin geneby TRs in these cells. The different model systems analyzedand differences in the cellular and molecular microenvironmentmay account for the discrepancy with our results. It is worthpointing out that our results are consistent with the well-establishedfact that the Wnt/ß-catenin pathway is a positiveregulator of the intestine cell proliferation.

Our observations suggested that TH signaling in the intestineis essential throughout intestine development and in adulthood.However, a major role is played during postnatal development,when an extensive growth takes place (39). Indeed, this periodalso corresponds in the mouse to a surge of TH, which declinesstarting from the third week to reach a steady-state adult level(14). According to this, TR ${alpha}$ knockout mice show more intestinalimpairment during the first 3 weeks after birth than in adulthood.Moreover, the absence of TH production during early postnataldevelopment, as in the congenital hypothyroid Pax8^–/–mice (28), has dramatic effects on intestine development, muchmore so than in TR ${alpha}$ mutants (9). This can be explained by theaporeceptor function of TR ${alpha}$ 1 in absence of TH, acting as a transcriptionrepressor of target genes (9, 26). In this light, the repressionof ß-catenin and of its downstream targets observedin hypothyroid animals (both congenital and chemical induced),may help explain some traits of the intestine phenotype. Indeed,removal of the TR ${alpha}$ 1 receptor in Pax8^–/– animals waspreviously reported to induce a recovery of intestine development(9).

Together, these data indicated that T3-activated TR ${alpha}$ 1 plays anessential role in postnatal intestine development as well asin adult homeostasis. Our present data show that the actionon the ß-catenin gene control is one of the molecularprocesses involved.

Until now few studies pointed to a function of TH and TR ingastrointestinal physiopathology (11, 15, 17, 27, 31). Somedata showed a correlation between altered levels of TH and theincidence of breast and colon cancer in humans (43). On theother hand, it has been reported that hyperthyroxinemia caninfluence the rate of colon cancer in an experimental modelof carcinogenesis in rats (24). Finally, in some cases, theabsence (30) or the mutation (53) of the TRs have been associatedwith gastrointestinal tumors. In our study, we show that THand TR ${alpha}$ 1 receptor control epithelial cell proliferation and theexpression of components of the Wnt pathway. As ß-cateninand some of its target genes are involved in intestinal tumorigenesis,our data open new perspectives in the function of the TR ${alpha}$ 1 receptoras a potential tumor inducer, as is the case for other nuclearreceptors (2).

ACKNOWLEDGMENTS

We thank the animal (PBES) and microscopy (PLATIM) facilitiesof the IFR 128 Biosciences. We also thank the ANIGENE platformof Rhône-Alpes Génopole. We gratefully acknowledgeNadine Aguilera for animal handling. We thank N. Davidson, K.Gauthier, and B. Pain for critical reading of the manuscriptand O. Bakker for help in immunolabeling for TR ${alpha}$ 1.We are gratefulto A. Rezza for participation in experiments with adult animals.

This work was supported by a grant from the Ligue Nationalecontre le Cancer to J.S. (Equipe Labellisée). E.K. wasa fellow of the Ligue Nationale contre le Cancer.

FOOTNOTES

* Corresponding author. Mailing address: IFR 128, Laboratoire de Biologie Moléculaire de la Cellule, CNRS UMR 5161, INRA UMR 1237, Ecole Normale Supérieure de Lyon, 46, Allee d'Italie, Lyon 69364, France. Phone: 33 4 72728616. Fax: 33 4 72 72 80 80. E-mail: Michela.Plateroti{at}ens-lyon.fr.

Supplemental material for this article may be found at http://mcb.asm.org.

REFERENCES

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