The frizzled-related sFRP2 gene is a target of thyroid hormone receptor α1 and activates β-catenin signaling in mouse intestine

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The frizzled-related sFRP2 gene is a target of thyroid

hormone receptor α1 and activates β-catenin signaling in

mouse intestine

Elsa Kress, Amélie Rezza, Julien Nadjar, Jacques Samarut, Michelina

Plateroti

To cite this version:

Elsa Kress, Amélie Rezza, Julien Nadjar, Jacques Samarut, Michelina Plateroti. The frizzled-related

sFRP2 gene is a target of thyroid hormone receptor α1 and activates β-catenin signaling in mouse

intestine. Journal of Biological Chemistry, American Society for Biochemistry and Molecular Biology,

2009, 284 (2), pp.1234-1241. �10.1074/jbc.M806548200�. �hal-02665755�

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The Frizzled-related sFRP2 Gene Is a Target of Thyroid

Hormone Receptor

␣1 and Activates ␤-Catenin Signaling in

Mouse Intestine

*

□S

Received for publication, August 22, 2008, and in revised form, November 6, 2008 Published, JBC Papers in Press, November 10, 2008, DOI 10.1074/jbc.M806548200

Elsa Kress

1

, Amelie Rezza

1

, Julien Nadjar, Jacques Samarut, and Michelina Plateroti

2

From the Universite´ de Lyon, Universite´ Claude Bernard Lyon 1, Ecole Normale Supe´rieure de Lyon, INRA, CNRS, Institut de

Ge´nomique Fonctionnelle de Lyon, 69364 Lyon, France

The thyroid hormone receptor TR

␣

1 regulates intestinal development and homeostasis by controlling epithelial prolifer-ation in the crypts. This involves positive control of the Wnt/

␤

-catenin pathway. To further investigate the effect of thyroid hor-mone-TR

␣

1 signaling on the intestinal epithelium proliferating compartment, we performed a comparative transcription pro-file 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

regu-lated genes and to group them into organized functional net-works. 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 hor-mones. In vitro and in vivo analyses showed that the expression of sFRP2 is directly regulated by TR

␣

1 and that it activates

␤

-catenin signaling via Frizzled receptors. Indeed, sFRP2 stabi-lizes

␤

-catenin, activates its target genes, and enhances cell pro-liferation. In conclusion, these new data, in conjunction with our previous results, indicate a complex interplay between TR

␣

1 and components of the Wnt/

␤

-catenin pathway. More-over, we describe in this study a novel mechanism of action of sFRP2, responsible for the activation of

␤

-catenin signaling.

The thyroid hormones (TH),

3

_T

3

and T

4

, control cell

prolif-eration, cell differentiation, and apoptosis depending on tissue

targets (1). This is well illustrated during TH-dependent

amphibian metamorphosis (2). The action of TH is mediated by

T

3

binding to nuclear receptors (TRs), encoded by the TR␣ and

TR

␤ genes (3). The TRs activate or repress transcription of

target genes by binding to specific DNA sequences named

thy-roid hormone-responsive elements (TRE) (1). In some organs

of the gastrointestinal tract, TH-TRs stimulate cell

prolifera-tion (4 – 8).

The intestinal epithelium is characterized by continuous and

rapid cell renewal, fuelled by adult stem cells located in the

crypts of Lieberku¨hn (9). 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 (9, 10). This continuous cell

renewal is regulated by several intrinsic (i.e. transcription

fac-tors) and extrinsic (i.e. growth factors and hormones)

compo-nents. A complex interplay between different signaling

path-ways maintains epithelial homeostasis (11, 12). We recently

showed that the TR␣1 receptor controls the proliferation of

intestinal epithelium progenitors (8). The mechanism behind

this effect involves direct transcriptional regulation of the

Ctnnb1

gene, which encodes

␤-catenin, the intracellular

medi-ator of the canonical Wnt pathway (13).

Canonical Wnt is activated when Wnt proteins bind to the

Frizzled receptors, allowing stabilization and nuclear

translo-cation of

␤-catenin. ␤-Catenin then binds to the TCF/LEF

fam-ily of transcription factors to regulate the expression of Wnt

target genes (13). Because some of these factors control

intes-tinal progenitor cell proliferation (12, 13), this pathway is

thought to be a key regulator of intestinal homeostasis.

To analyze the TH-responsive genes in the intestinal

epithe-lium, 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

␤-catenin expression by TH. In

fact, we identified a new target of TR␣1, the secreted

Frizzled-related protein sFRP2, which in these cells behaves as a positive

regulator of

␤-catenin stabilization and signaling.

EXPERIMENTAL PROCEDURES

Animal Treatment and Tissue Preparation—For

microdis-section, we used TR

␣

0/0

_{(14), TR}

_␤

⫺/⫺

_{(15), and wild type}

ani-mals, housed and maintained with approval from the animal

experimental committee of the Ecole Normale Superieure de

Lyon (Lyon, France), and in accordance with European

legisla-*

This work was supported in part by the Reseau National des Genopoles Project 186, Agence Nationale pour la Recherche Grant ANR-06-BLAN-0232-01, Program Equipe Labelise´e of the Ligue Nationale Contre le Can-cer, and the European Network of excellence CRESCENDO. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

□S _{The on-line version of this article (available at http://www.jbc.org) contains}

supplemental Materials and Methods, Figs. S1–S8, and Table S1.

1_{Studentship supported by the Ligue Nationale Contre le Cancer.}

2_{To whom correspondence should be addressed: Institut de Ge´nomique}

Fonctionnelle de Lyon, Ecole Normale Supe´rieure de Lyon, 46 Alle´e d’Italie, 69364 Lyon Cedex 07, France. Tel.: 33-472728536; Fax: 33-472728080; E-mail: Michela.Plateroti@ens-lyon.fr.

3_{The abbreviations used are: TH, thyroid hormone; WT, wild type; TRE,}

thy-roid hormone-responsive element; RT-QPCR, reverse transcription-quanti-tative PCR; TR, thyroid hormone receptor; BrdUrd, bromodeoxyuridine; GFP, green fluorescent protein; PTU, propylthiouracil; IP, immunoprecipi-tation; T3, triiodothyronine; T4, thyroxine.

at INRA Institut National de la Recherche Agronomique on June 14, 2018

http://www.jbc.org/

(3)

tion on animal care and experimentation. TH deficiency in

pups was induced by 0.15% propylthiouracil (PTU) in the diet

(Harlan/Teklad) as described previously (8). A group of

PTU-treated animals received a single IP injection of TH (2.5 mg/kg

T

₄

and 0.25 mg/kg T

₃

in 100

␮l of phosphate-buffered saline)

24 h before sacrifice. Control animals were fed with standard

mouse chow. Animals were euthanized at 14 days, and

seg-ments of proximal, median, and distal small intestine were

embedded together in Tissue-Tek mounting medium and

fro-zen 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%

paraformalde-hyde for immunohistochemistry or frozen in liquid nitrogen for

RNA and/or protein extraction.

Isolation of Villus-Crypt Epithelial Fractions—The

sequen-tial isolation of mouse small intestinal epithelial cells along the

villus-crypt axis has been described previously and validated

(16). 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

T

3

and T

4

(Biomerieux).

Laser Capture Microdissection and GeneChip

Analysis—Tis-sue-Tek-embedded intestine fragments were prepared for

LCM by using the protocol described previously (17). Details

for RNA extraction, labeling, and hybridization can be found in

the supplemental Materials and Methods

.

Statistics

—For pairwise comparisons (WT-PTU-treated

ver-sus

WT-Control; WT-TH-injected versus WT-Control;

WT-TH versus WT-PTU; TR␣

0/0

_versus

_{WT-Control; TR␤}

⫺/⫺

versus

WT-Control), we used Affymetrix Microarray Suite

soft-ware 5.0 and two-tailed Student’s T-Test (Zoe softsoft-ware). 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

supplemen-tal Materials and Methods.

Primary Culture of Intestine Epithelial Cells

—Intestinal

epi-thelial primary cultures were derived from 4- to 6-day-old

neo-natal mice, using the protocol and culture conditions described

previously (8). Either 2

⫻ 10

⫺7M

T

₃

or the vehicle alone was

added to the culture medium for the indicated length of time.

For proliferation studies, 10

␮

M

BrdUrd was added to the

cul-ture 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

␮g/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%

paraform-aldehyde for immunofluorescence.

RNA Preparation and Analysis

—RNA was extracted from

primary cultures with Absolutely RNA nanoprep kit

(Strat-agene), and from epithelial fractions with total RNA and

pro-tein isolation kit (Macherey-Nagel). Reverse transcription was

performed using 1

␮g 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

Meth-ods for details of quantitative PCR.

Immunostaining and Western Blot

—Immunolabeling for

␤-catenin (Santa Cruz Biotechnology) and BrdUrd (Roche

Applied Science) was performed on 2%

paraformaldehyde-fixed cell cultures. Staining for

␤-catenin was also performed on

5-␮m 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

epi-thelial 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

cen-trifugal filter devices (Centricon, Millipore). Proteins were

sep-arated and analyzed as described previously (8). We used the

following primary antibodies: anti-sFRP2 (Santa Cruz

Biotech-nology), anti-␤-catenin (Santa Cruz BiotechBiotech-nology),

anti-acti-vated

␤-catenin (Upstate), anti-actin (Sigma), anti-Dishevelled

1 (Santa Cruz Biotechnology), and anti-phospho-GSK3

␤

(Ser-9; Cell Signaling).

Chromatin Immunoprecipitation

—The chromatin

immuno-precipitation study was performed on collagenase-dispase

sep-arated epithelial fragments from 3- to 6-day-old mouse

intes-tine as described previously (8). For conventional PCR, we used

the Euroblue Taq (Eurobio). Oligonucleotides are listed in

sup-plemental Fig. S5B. All amplicons were sequenced.

RESULTS

Crypt Cell Isolation and Microarray Data

—Crypt cells were

from wild type (WT) animals with normal or perturbed TH

status, as well as from TR␣ and TR␤ knock-out mice (14, 15).

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

homo-log-like 1) genes, which encode regulators of cell cycle

progres-sion and mitotic checkpoint, respectively (18, 19). 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

canon-ical pathways represented in the set of input genes

(supplemen-tal 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

prolifera-tion,” 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

Thyroid Hormone and Wnt

http://www.jbc.org/

(4)

nodes. One is Fos, which is up-regulated by TH treatment,

coherent with its positive control of cell proliferation (20). The

second is Ctnnb1, encoding

␤-catenin, already described as a

TH-TR␣1 target (8). An interesting result was the positive

rela-tionship between the Frizzled-related protein sFRP2 and

␤-catenin, in agreement with work described previously (21).

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/␤-catenin pathway. Moreover, the action of sFRP2 on

canonical Wnt has been described in contradictory terms,

either repression or activation (21–25). 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.

More-over, they decreased in TR␣

0/0

_and

increased in TR␤

⫺/⫺

_compared

with WT control crypts. It is worth

noting that TR

␤

⫺/⫺

mice are

con-genitally hyperthyroid (15). We also

quantified sFRP2 mRNA and

pro-tein in the intestines of TR

␣ and

TR␤ mutant mice with altered TH

status to evaluate whether TH

re-sponsiveness depended on a specific

TR subtype (supplemental Fig. S4, A

and B

). WT and TR

␤

⫺/⫺

animals

both

showed

decreased

sFRP2

mRNA and protein expression

under hypothyroid conditions and

an induction of sFRP2 after TH

treatment. In TR␣

0/0

_{mice, sFRP2}

expression was unchanged by

alter-ation 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.

Finally, we quantified by Western

blot sFRP2 protein levels in

frac-tionated villus-enriched or

crypt-enriched cells from the villus-crypt

axis (16) (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

injec-tion increased the protein

specifi-cally 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 TR

␣1 is the only

TR receptor expressed and exclusively present in crypt cells.

This observation was made both in this study and previously

(8). Altogether, these data demonstrated that the regulation of

sFRP2 expression by TH depended specifically on the TR␣1

receptor.

To analyze whether the regulation of sFRP2 by TH was

epi-thelial cell-autonomous, we used intestinal epithelium primary

cultures maintained in the absence or presence of T

₃

for 24 h

(Fig. 2C). In WT cells, sFRP2 mRNA levels were clearly and

significantly increased by T

3

treatment. We obtained a similar

result by short term T

₃

treatment (6 h, Fig. 2D), strongly

sug-gesting that this gene might be a direct target of TR␣1. The

mRNA levels of sFRP2 in TR

␣

0/0

_{cells maintained in control}

condition were significantly decreased compared with WT

control cells. As expected, the up-regulation of sFRP2 mRNA

by T

₃

was abolished in cells from TR

␣

0/0

_{mutant mice (Fig. 2C).}

FIGURE 1. Validation of microarray data by RT-QPCR on RNA from intestinal crypts. A and B, differentially

regulated genes. We used a multiplex method to simultaneously quantify genes of interest and internal control (36B4) mRNAs. Values (mean_{⫾ S.D., n ⫽ 3) represent fold change, after normalization to the WT control} condition. WT-C, wild type control; WT-PTU, wild type PTU-treated; WT-TH, wild type TH-injected. **, p⬍ 0.01, compared with WT-C condition; $$, p⬍ 0.01, compared with WT-PTU and TR␣0/0_{conditions, by Student’s t test.}

C, molecular relationships between transcripts in the identified network ”cell cycle/cell proliferation“ by

Inge-nuity Pathway Analysis. This network had the highest significant score. Genes or gene products are repre-sented as nodes, and connections between genes are supported by information in the Ingenuity Pathways Knowledge Base. The color indicates up-regulation (red) or down-regulation (green). Different shapes of nodes represent different functional classes of gene products (rhomboid, enzymes; ovals, transcription factors;

trian-gles, kinases; trapeziums, transport and carrier proteins; and circles, others). *, validated. $, nonvalidated.

http://www.jbc.org/

(5)

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 TR

␣1 in

vivo

, we carried out chromatin IP assays on freshly isolated

intestinal epithelium. Sonicated chromatin was incubated with

anti-TR

␣1, anti-TR␤1, or preimmune serum. The DNA

precip-itated 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-TR

␣1 antibody. A similar result was

obtained for the positive control Ctnnb1-TRE (8). We also

quantified by QPCR the DNA precipitated by the TR␣1

anti-body, 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 TR␣1 to sFRP2-TRE

was also demonstrated in vitro by gel shift analysis

(supplemen-tal Fig. S7). Altogether, these data clearly demonstrated that

TR␣1 binds sFRP2-TRE in intestinal epithelial cells both

in vitro

and in vivo.

TH-stimulated sFRP2 Stabilizes

␤-Catenin through Frizzled

and Promotes Cell Proliferation

—sFRP2 action on Wnt/

␤-cate-nin appears different depending on the cellular/tissue system.

Our results showing strong positive regulation by TH-TR␣1 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

␤-catenin as well as its function on

epithelial progenitor cell proliferation.

We treated WT or TR␣

0/0

_{cells with recombinant sFRP2 and}

monitored the mRNA levels of c-Myc (Fig. 4A) and cyclin D1

(not shown), which are established targets of

␤-catenin (26, 27).

A concentration of 50 ng/ml in the culture medium

signifi-cantly stimulated the expression of both targets. This

stimula-tion 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 TR␣

0/0

_intestine,

indi-cating that stimulation of

␤-catenin targets by exogenous

sFRP2 was independent of TR

␣1 signaling.

FIGURE 2. In vivo and in vitro regulation of the Sfrp2 gene. A, validation of microarray data by RT-QPCR on RNA from intestinal crypts. Histograms illus-trate 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 TR␣0/0

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 con-trol animals; 1_{⬘–4⬘, fractions from WT animals injected with TH. 50}␮g of pro-tein 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) summa-rize densitometry analyses (by ImageQuant) from four independent experi-ments. 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 T3in primary cultures by RT-QPCR analysis. Cells were treated

with T3during 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.

FIGURE 3. Molecular 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; TR␣1, anti-TR␣1; TR␤1,

anti-TR␤1; SI, starting input; Ctrl PCR, negative control for PCR mix.

Thyroid Hormone and Wnt

http://www.jbc.org/

(6)

Increased levels of

␤-catenin targets depend on increased

availability of

␤-catenin (13). Because the levels of ␤-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

␤-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

␤-catenin as detected with this antibody

(28, 29). Fig. 4C illustrates the results of Western blot analysis,

showing that T

₃

or sFRP2 treatment up-regulated the level of

both total and stabilized

␤-catenin. It is worth noting that T

3

treatment induced a clear-cut increase of sFRP2 in culture

medium (Fig. 4D), suggesting that activation of

␤-catenin

tar-gets might depend on T

3

-stimulated

expression of sFRP2. To verify this

hypothesis we co-treated the cells

with T

3

and with an anti-sFRP2

antibody. This blocking treatment

specifically blunted the stimulation

of c-Myc (Fig. 4E) and cyclin D1

(not shown) mRNA expression by

T

3

, whereas an anti-GFP antibody

was ineffective. Control cells treated

only with the anti-sFRP2 antibody

showed unchanged levels of

␤-catenin targets compared with

untreated cells (not shown). Finally,

T

₃

was still able to induce

␤-catenin

mRNA expression, even in the

pres-ence of the anti-sFRP2 antibody

(Fig. 4B). We also checked for

intra-cellular signaling molecules such as

Dishevelled 1 (Dvl1) and

Ser-9-phospho-Gsk3␤ expression by

Western blot. Interestingly, T

3

or

sFRP2 treatment in vitro causes an

increase in phosphorylated Dvl1 as

well

as

Ser-9-phospho-GSK3␤

compared with the control

(supple-mental Fig. S8A

). Moreover, the

increase

was

similar

to

that

obtained by treating the cells with

Wnt3a (supplemental Fig. S8A).

These different treatments did not

affect the mRNA levels of Dvl1 and

GSK3␤ (not shown).

To determine the mechanism by

which sFRP2 stimulated

␤-catenin

signaling, we focused on Frizzled

receptors (Fz). Our transcription

profile data showed that intestinal

crypts expressed Fz4 and Fz7,

which can both transduce Wnt

signal (30, 31). We then analyzed

the effect of soluble chimeric

Ch-Fz4 and Ch-Fz7, which act as Wnt

inhibitors (32). Fig. 5A illustrates

the expression levels of c-Myc mRNA in epithelial cells

maintained in different culture conditions. As expected,

treatment with either sFRP2 or Wnt3a significantly

increased the level of c-Myc mRNA compared with control

cells, whereas each chimeric Fz was ineffective. By

co-treat-ing the cells with either Wnt3a or sFRP2 and each chimeric

Fz, the up-regulation of c-Myc mRNA expression was

signif-icantly blunted. A similar result was obtained in cells

co-treated with T

₃

and each chimeric Fz (Fig. 5B). These data

clearly show the involvement of Fz receptors in transducing

the positive effect of recombinant or T

₃

-stimulated sFRP2 on

␤-catenin.

The consequence of

␤-catenin stabilization in intestinal

epithelial progenitors is nuclear translocation and activation

FIGURE 4. sFRP2 stabilizes␤-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␤-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 inde-pendent 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␤-catenin in primary cultures treated either with T3or 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␤-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 T3during

24 h. 500␮g 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 T3and 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.

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of cell proliferation (13). We analyzed the effects of sFRP2 on

both processes by treating the primary cultures with T

3

or by

adding sFRP2 exogenously to the culture medium (Fig. 6,

A–R). As summarized in Fig. 6, Q and R, in the absence of

treatment, nearly 5% of cells showed nuclear

␤-catenin; 15%

were proliferating BrdUrd-positive cells, and a small fraction

was double-stained (Fig. 6D). In contrast, upon increasing

the levels of sFRP2 either in T

3

- or sFRP2-treated cultures,

the frequency of cells displaying nuclear

␤-catenin and

incorporating BrdUrd greatly and significantly increased.

Finally, most of the cells displayed nuclear co-staining for

BrdUrd and

␤-catenin (Fig. 6, H and L versus D). When an

antibody against sFRP2 was added to T

3

-treated cells, the

number of cells displaying nuclear

␤-catenin and BrdUrd

were significantly reduced to control levels (Fig. 6, Q and R).

These data clearly show that the Sfrp2 gene, transcriptionally

induced by TR␣1, acts as a positive activator of ␤-catenin

signaling by stabilizing it and by increasing cell proliferation

of the intestinal epithelial progenitors in vitro.

To analyze whether TH treatment induced

␤-catenin

nuclear translocation in vivo, we perturbed the TH status of

WT animals. Immunohistochemical analysis of intestinal

sections showed an induction of

␤-catenin expression upon

TH treatment (Fig. 7, I–L versus A–D). This was

accompa-nied by a clear expression of

␤-catenin in some nuclei of

crypt cells. These data were further confirmed by Western

blot performed on protein extracts from the villus-crypt

fractions. As reported in Fig. 7M and in agreement with

oth-ers (13), the activated

␤-catenin was mainly expressed by the

crypt fraction (Fig. 7, M lane and histogram 4). When the

animals were injected with TH, the levels of activated

␤-catenin were greatly and significantly increased in the

crypt fraction (Fig. 7M, compare lane and histograms 4

⬘

ver-sus 4). It is worth noting that sFRP2 was also significantly

up-regulated in this same cellular fraction (Fig. 2B), as well as

Dvl1 and Ser-9-phospho-Gsk3␤ (supplemental Fig. S8B

).

These data strongly suggest that by increasing the expression

of sFRP2, TH can activate the

␤-catenin signaling in vivo as

well as in vitro.

DISCUSSION

The thyroid hormones regulate intestinal development. This

has previously been described in detail for amphibian

meta-morphosis (2). In mouse models lacking the expression of TRs,

we showed that the TR␣ gene controls intestinal development

during maturation at weaning as well as intestinal homeostasis

in adulthood. More precisely, TH-TR␣1 activates the

prolifer-ation of intestinal progenitors (7, 8, 33). One mechanism

involves positive control of the

␤-catenin signaling. The current

work extended this finding.

To globally characterize genes and signaling pathways

regu-lated by TH-TR␣1 in intestinal epithelial progenitors, we used a

comparative transcription profile approach on laser

microdis-sected crypt cells. This allowed us to compile a comprehensive

list of differentially regulated genes. Some of these were already

known to be TH targets, including Idh3a and Apobec1 in the

liver (34) and Bub1b and Ccna1 in intestine, during

TH-de-pendent amphibian metamorphosis (35). In agreement with

other studies, we showed a similar number of genes that were

up- or down-regulated and different patterns of TH-mediated

regulation (1, 34, 36).

In this study we focused on the Sfrp2 gene, which we

identi-fied as a new target gene of TH-TR␣1. It likely undergoes direct

regulation by T

₃

through the TRE characterized in its promoter

region. In fact, this is the unique responsive element found

within the promoter environment. It is composed of two direct

repeats separated by eight nucleotides, which is quite

uncom-FIGURE 5. Frizzled receptors are involved in the transduction of sFRP2

signal. A and B, RT-QPCR analysis of c-Myc from primary cultures treated with

soluble molecules in the medium as indicated. C, control; S, sFRP2 50 ng/ml;

W, Wnt3a 10 ng/ml; F4, Ch-F4 300 ng/ml; F7, Ch-Fz7 300 ng/ml; T, T3.

Histo-grams illustrate mean⫾ S.D. (n ⫽ 4) from two independent experiments each

conducted in duplicate. Statistical analysis was conducted by using the Stu-dent’s t test. *, p⬍ 0.05, and **, p ⬍ 0.01 compared with the control condition; $, p⬍ 0.05 compared with the single sFRP2 treatment; ##, p ⬍ 0.01 compared with the single Wnt3a treatment;€€, p ⬍ 0.01 compared with the single T3

treatment.

FIGURE 6. Immunofluorescence analysis of epithelial primary cultures.

A–P, double staining for␤-catenin and BrdUrd in different culture conditions:

control (A–D), T3(E–H), sFRP2 (I–L), and T3⫹ anti-sFRP2 antibody (M–P). Cells

were labeled by Hoechst (A, E, I, and M),␤-catenin (B, F, J, and N), and BrdUrd (C, G, K, and O). The merging of␤-catenin and BrdUrd staining is shown in D, H, and L, and P. Q and R, summary (mean⫾ S.D.) of the scoring of specific immu-nolabeling in three independent experiments, obtained by counting the pos-itive cells under the microscope (60 cells per experimental condition). For statistical analysis, the Student’s t test was used. *, p⬍ 0.05; **, p ⬍ 0.01.

Arrows point to some double-stained nuclei. Magnification, bar⫽ 12␮m.

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mon. However, it is constituted by two perfect canonical

half-sites (TGGTCA-5

⬘). Whether TRs can bind to half-sites is still

under debate, and all possible conformations of TRE have been

described in the literature as follows: direct repeats, inverted

repeats, and everted repeats (37). In this respect, more studies

are necessary to clearly understand the kinetics and the

chem-istry of TR binding on target sequences in vivo.

The functional link between TH-TRs and canonical Wnt

has been the focus of previous studies (8, 38, 39), which show

that depending on cell type TH can either activate or repress

components of the Wnt pathway. Indeed, in some organs,

TH induced cell differentiation (40), although in others, it

induces cell proliferation (5). We showed that in highly

pro-liferating progenitors of crypts, TH-TR␣1 induced the

expression of the Ctnnb1 gene, which encodes

␤-catenin.

This in turn activated its targets cyclins D1 and D2 as well as

c-Myc (8), all positive regulators of cell proliferation (41–

43). The nuclear action of

␤-catenin depends on its

stabili-zation (13). Our previous data showed the up-regulation of

␤-catenin and its molecular targets, but we did not define

how

␤-catenin was stabilized. We describe here another

component of the Wnt pathway as a direct target of the

TH-TR␣1. It is the Frizzled-related sFRP2 that, in the crypt

cells, behaves as an activator of the

␤-catenin. Altogether, in

the intestinal epithelial progenitors, TH act simultaneously

on Ctnnb1 and Sfrp2 genes to increase and stabilize the levels

of the

␤-catenin and finally to stimulate the cell

proliferation.

Since their discovery, sFRP proteins have been considered

competitors of canonical Wnt, because of their sequence

homology with membrane Frizzled receptors (22, 23).

How-ever, several papers have suggested a potential role for sFRP2 in

stabilizing

␤-catenin (21, 24, 25). A study on metanephric

development also showed that sFRP2 blocked

sFRP1-depend-ent inhibition of Wnt (44). However, in this system, sFRP2 did

not act directly on Wnt. As it is increasingly recognized in

sig-naling systems, the molecular and cellular contexts for signal

transmission may be crucial in determining the final outcome

(45, 46). Our results in vitro excluded a functional interference

between sFRP2 and canonical Wnt3a ligand and clearly

dem-onstrated a positive action of sFRP2 on

␤-catenin. Moreover, in

accord with recent reports (47– 49), our results showed that its

action implicated Fz receptors. This is the first study describing

a functional interaction between sFRP2 and Fz receptors

result-ing in positive regulation of

␤-catenin signaling. It is tempting

to speculate that sFRP2, by binding to Fz, activates

␤-catenin

and intestinal progenitors proliferation through the classical

Wnt pathway (13). However, we cannot exclude an action

involving other signaling pathways (50) as well as noncanonical

Wnt (13). It is worth noting the current lack of methodology for

directly modifying gene expression in intestinal epithelial

pri-mary cultures. Therefore, new strategies and tools will need to

be developed to test our hypothesis and to define the

mecha-nisms of this functional interaction.

Several reports have indicated that mutant TRs are

involved in various cancers (51–53). Other data showed

cor-relations between altered levels of TH and human breast and

colon cancers (54, 55). Finally, it has been shown that the

mutation (56) or aberrant expression (57) of TRs is

associ-ated with gastrointestinal tumors. However, there is no

con-clusive evidence that their proliferative action is linked to

tumorigenesis. Here we described the complex interplay

between TH-TR␣1 and other signaling pathways, key

regu-lators of intestinal epithelial progenitor homeostasis. By

focusing on Wnt, we demonstrated a new modality of

␤-catenin stabilization by sFRP2. Given the key role of

canonical Wnt in intestinal homeostasis, our work opens a

new perspective in the study of TR

␣1 as a potential inducer

of cell transformation leading to tumorigenesis.

FIGURE 7.␤-Catenin study in wild type animals. A–L, confocal microscopy analysis after immunolabeling. Sections from euthyroid (A–D), hypothyroid (D–H), and hyperthyroid (I–L) animals were stained with␤-catenin antibodies (A, E, and I). Nuclei in B, F, and J were counterstained with propidium iodide.

C, D, G, H, K, and L, merging of the two single stainings is shown. To minimize

differences, all the slides have been simultaneously processed and the con-focal images taken with same laser settings. Magnification: A–C, E–G, and I–K,

bar⫽ 7␮m; D, H, and L, bar ⫽ 3 ␮m. White dotted lines delineate individual

crypts. White solid lines locate the muscle layer under the crypts. White arrows in I–L indicate nuclear␤-catenin. C, crypts; ml, muscle layers. J, representative Western blot analysis of activated nonphosphorylated␤-catenin in epithelial cells fractionated from the villus-crypt axis. The picture is representative of three independent experiments. M, representative Western blot analysis of activated nonphosphorylated␤-catenin in epithelial cells fractionated from the villus-crypt axis. 1⬘–4⬘, fractions from WT animals injected with TH.

Histo-grams in the lower panel (mean⫾ S.D., n ⫽ 3) summarize densitometry

anal-yses (by ImageQuant) from three independent experiments. Data are normal-ized 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 corre-sponding untreated fraction.

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Acknowledgments—We acknowledge Nadine Aguilera for animal handling. We thank Drs. C. Thibault and P. Kastner of the Affymetrix platform, Illkirch, France. We especially thank C. Rey and G. Cavillon of the Neurobiotech, Lyon, France, and C. Savouret of the Stratagene technical services. We are especially grateful to Drs N. Davidson, T. Osborne, and B. Pain for helpful discussion and the critical reading of the manuscript.

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Thyroid Hormone and Wnt

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Elsa Kress, Amelie Rezza, Julien Nadjar, Jacques Samarut and Michelina Plateroti

-Catenin Signaling in Mouse Intestine

β

and Activates

doi: 10.1074/jbc.M806548200 originally published online November 10, 2008

2009, 284:1234-1241.

J. Biol. Chem.

10.1074/jbc.M806548200

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