1 Institut Cochin, INSERM U567, CNRS UMR8104, Université Paris V, 24 rue
du Fb St-Jacques, 75014 Paris, France
2 UMR-S INSERM U490, 45 rue des St-Pères, 75006 Paris, France
3 INSERM U434, 27 rue Juliette Dodu, 75010 Paris, France
4 Institut Curie, UMR 144, 75005 Paris, France
* Author for correspondence (e-mail: perret{at}cochin.inserm.fr)
Accepted 14 January 2005
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SUMMARY |
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Key words: APC, ß-Catenin, Intestine, Paneth cells, Differentiation
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Introduction |
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Numerous factors that regulate the stem cell compartment and stem cell
differentiation have been identified
(Sancho et al., 2003;
van de Wetering et al., 2002
).
Wnt/ß-catenin signaling appears to control intestinal homeostasis.
Activation of the ß-catenin signaling pathway results in the formation of
the ß-catenin-Tcf complex, which controls the transcription of several
target genes (Bienz and Clevers,
2000
; Giles et al.,
2003
). This signaling pathway is tightly regulated by several
inhibitors, including the well-known tumor suppressor gene APC
(Polakis, 1997
). Inactivation
of Wnt signaling, either by ablation of the Tcf4 gene or by ectopic
expression of Dkk1, a secreted Wnt inhibitor, results in depletion of stem
cell compartments in the small intestine, indicating that ß-catenin
signaling plays a role in stem cell maintenance
(Korinek et al., 1998
;
Kuhnert et al., 2004
;
Pinto et al., 2003
). Familial
adenomatous polyposis (FAP), an autosomal dominant condition characterized by
the development of hundreds or thousands of polyps in the colon and rectum, is
caused by constitutive activation of Wnt signaling by mutations in the
APC gene. Mutations in APC are also responsible for most
sporadic colorectal cancers (Fodde et al.,
2001
). APC has been defined as the gatekeeper of the intestine
(Kinzler and Vogelstein,
1996
). The distribution of APC is in agreement with the role of
ß-catenin signaling in maintaining stem cell properties and controlling
differentiation in the intestine. A gradient of APC is observed along the
crypt-villus axis, where it counteracts ß-catenin signaling and allows
differentiation.
Enhanced ß-catenin signaling is common to normal intestinal stem cell
compartments and colorectal cancer (Batlle
et al., 2002; Sancho et al.,
2003
). This suggests that colorectal cancers associated with APC
mutations result from excessive proliferation and/or lack of differentiation.
It is still unclear whether tumor formation occurs in the crypt or in
well-differentiated cells of the villus
(Preston et al., 2003
;
Shih et al., 2001
). Sequential
analyses of the cellular and molecular consequences of Apc
inactivation may elucidate the nature of the pre-malignant disease. Therefore,
we analyzed the immediate effects of Apc loss in the intestinal
epithelium all along the crypt-villus axis. We used a conditional-knockout
approach based on the Cre/loxP system and an inducible Cre recombinase
(CreERT2) driven by the villin promoter to disrupt the Apc
gene in adult Apc-floxed mice (Colnot et
al., 2004a
). The villin promoter directs transgene expression
specifically in the intestinal epithelium
(el Marjou et al., 2004
).
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Materials and methods |
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Collection of patient samples
Ten primary colorectal cancers and the adjacent tissues were collected at
the Laennec Hospital (Paris, France).
Immunohistochemistry
Immunostaining was performed on a paraffin wax-embedded Swiss roll, as
previously described (Colnot et al.,
2004b). We used polyclonal primary antibodies directed against APC
C20 (Santa Cruz Biotechnologies, 1/250), caspase3 (Cell Signalling, 1/200),
Ki67 (Novocastra, 1/300), c-Myc N-262 (Santa Cruz, 1/50), chromogranin A+B
(Progen, 1/50) and Villin (S. Robine, Institut Cochin, Paris), and monoclonal
primary antibodies directed against ß-catenin (BD Biosciences, 1/100),
E-cadherin (Zymed, 1/400) and cyclin D1 (Dako, 1/50). For APC immunostaining,
a catalyzed signal amplification system based on the Dako CSA kit was used.
Frozen sections were stained with the Cre antibody (Covance, 1/400) and
UEA1-FITC (Sigma, 1/100).
Western blotting
Western blots were performed as previously described, and probed with
anti-ß-catenin (1/500), polyclonal anti-c-Myc N-262 (1/200) and
monoclonal anti-cylin D1 (1/250) antibodies
(Ovejero et al., 2004).
In situ hybridization
Partial cryptdin 5 and lysozyme cDNAs were obtained by RT-PCR and then
subcloned. In situ hybridization was performed using a digoxigenin-labeled
probe (Roche) and detected using an anti-digoxigenin antibody (Roche,
1/4000).
RNA extraction and analyses
Extraction of total RNA and reverse transcription were performed as
previously described (Ovejero et al.,
2004). Cre recombinase activity was detected by allele-specific
RT-PCR (Colnot et al., 2004b
).
Real-time quantitative RT-PCR was carried out with a LightCycler instrument
using the LightCycler-fastStart DNA Master SYBR Green I Kit (Roche
Diagnostics). Quantification was performed in duplicate and expressed relative
to 18s rRNA. For mouse samples, gene expression levels are expressed relative
to in wild-type small intestines. For human samples, data are expressed as the
ratio of gene expression levels in the tumor to gene expression levels in the
non-tumor counterpart.
Cell lines and transfection
Human embryonic kidney cells 293T were grown in DMEM with 10%
penicillin-streptomycin (Invitrogen). After 24 hours in culture (80%
confluence), 293T cells were cotransfected with 0.2 µg of the indicated
reporter plasmid plus 0.5 µg of 89ß-catenin expression vector,
and/or 0.25 µg of Tcf4 expression vector and 20 ng of TK-Renilla reporter
vector, using Lipofectamine 2000 (Invitrogen). Total amounts of plasmids were
kept constant by adding the empty DNA vector when necessary. Reporter activity
was determined by using the dual luciferase reporter assay system (Promega).
Experiments were performed in duplicate and repeated at least three times.
Plasmid constructs
Cryptdin/defensin promoter constructs were synthesized from mouse and human
genomic DNA by PCR and subcloned into the pGL3-basic luciferase reporter
vector (Promega). The proximal mutated Tcf-binding sites at nucleotides -159
(C-binding site), -141 (B-binding site) and -130 (A-binding site) from AT to
GC (HD6C and HD6ABC) were constructed from the HD6-200 plasmid using the
QuikChange Site-directed mutagenesis kit (Stratagene).
Oligonucleotides
The sequences of the primers used in this study are available upon
request.
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Results |
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Loss of Apc along the crypt-villus axis of the small intestine disturbs only the crypt architecture
Four days after one injection of TAM, the mutant mice appeared unwell.
Given the moribund phenotype of the mutant mice, we killed them. The proximal
distal extension of the small intestine of mutant mice appeared normal in
length but was consistently wider than that of control mice. No apparent
macroscopic phenotype was observed in the colon.
Histological studies revealed signs of dysplasia in the crypt compartment
all along the small intestine of mutant mice
(Fig. 2A). These signs included
nuclear pseudostratification, enlargement of the nuclei, prominent nucleoli,
and basophil accumulation. We observed numerous apoptotic bodies
(Fig. 3A), and an impairment of
differentiation associated with loss of mucosecretion
(Fig. 2A). The crypt
compartment of mutant mice was greatly expanded. Histological analysis
revealed no effect of TAM injection in the control mice
(Fig. 2A). The phenotype
appeared less drastic in the colon (Fig.
2B). We observed some rare lesions in the proximal colon, which
presented similar histological characteristics to the small intestinal
dysplasia (Fig. 2B). The reason
of the distinct severity of phenotype in mutant mice between the small
intestine and the colon is unclear. It could be due to a longer cell cycle in
the large intestine than in the small intestine
(Potten and Hendry, 1995).
However it is important to note that all mouse models with activated
ß-catenin signaling present less adenomatous polyps in the colon than in
the small intestine (Harada et al.,
1999
; Yamada et al.,
2002
). As the colonic lesions appeared infrequent, we choose to
focus our studies on the effect of Apc loss on the small intestine.
|
|
We then analyzed the small intestine of control and mutant mice at various time points after a single TAM injection (from day 2 to day 4) (Fig. 2C). No histological changes were observed in the control intestinal epithelium at any stage. Although the mutant epithelium appeared grossly normal on day 2, an enlarged crypt compartment was visible on day 3 and was more pronounced on day 4. We examined the proliferative status of the small intestine epithelium by immunohistochemistry for the S-phase marker Ki-67 (Fig. 2C). In the control epithelium, proliferative cells were restricted to the crypt compartment. By contrast, in the mutant mucosa, the Ki-67-positive zone was greatly expanded between days 2 and 4, with Ki-67 staining present in all the cells of the abnormal crypts (at day 4, 28.6±1.7 Ki-67-positive cells per crypt section in the control; 94.2±7.6 Ki-67-positive cells per crypt section in the mutant; P=0.04 Mann Whitney). Ki-67 staining was restricted to the expanded proliferative compartment and was negative in the villi of mutant mice (Fig. 2C).
Only occasional apoptotic cells were found in control crypts. Hematoxylin and Eosin staining revealed a much higher number of apoptotic cells in the elongated crypts of the mutant mice. Caspase 3 immunostaining confirmed that apoptosis was enhanced in the mutant (3.3±0.8 caspase 3-positive cells per crypt section in the control, 11.2±0.8 caspase 3-positive cells per crypt section in the mutant; P=0.03 Mann Whitney) (Fig. 3A).
BrdU experiments were carried out to study the kinetics of epithelial cell migration within the gastrointestinal tracts of mutant and control mice. Mice were given a single BrdU injection 2 days after TAM injection and were killed 2 or 24 hours later. We evaluated the rate of cell migration by comparing the labeling of BrdU at 2 and 24 hours. At 24 hours, the epithelial cells from the mutant mice had clearly not migrated as far as those from the control mice (Fig. 3B). Thus, BrdU labeling showed that Apc loss slowed down the migration of epithelial cells along the crypt-villus axis in the mutant epithelium (Fig. 3B).
These findings show that inactivation of Apc is sufficient to alter the intestinal epithelium architecture dramatically within three days, especially in the crypt compartment, resulting in a large dysplasia-like zone. These morphological changes were associated with an increase in cell proliferation and cell death, and with impaired cell migration along the crypt-villus axis. By contrast, no proliferative phenotype was induced in the villi compartment, which did not reveal any morphological changes.
Loss of Apc induces distinct patterns of ß-catenin signaling along the crypt-villus axis
All the cellular defects described above were restricted to the
proliferative compartment; they did not appear to affect the differentiated
villi. We thus analyzed the subcellular distribution of ß-catenin as an
indicator of activation of the ß-catenin signaling pathway. As expected,
cytoplasmic and nuclear ß-catenin staining was observed in the aberrant
highly proliferative area (Fig.
4A). However, ß-catenin cell membrane staining was stronger
in the upper part of the mutant villi than in the control, and precipitates
accumulated in the cytosol at the basolateral membrane
(Fig. 4A). To analyze whether
aberrant activation of ß-catenin signaling was readily induced all along
the crypt-villus axis, we analyzed the expression of two well-established
target genes of ß-catenin/Tcf4 signaling: Myc (previously known as c-myc)
and cyclin D1 (He et al.,
1998; Tetsu and McCormick,
1999
). As expected, the accumulation of ß-catenin in the
small intestine of the mutant mice was associated with the induction of Myc
and cyclin D1, both at the RNA and protein levels
(Fig. 4B). Interestingly, four
days after TAM injection, Myc and cyclin D1 were not found in the same
compartments along the crypt-villus axis in the small intestine of the mutant
mice. Myc was localized mainly in the aberrant crypt compartment and in the
bottom part of the villi all along the small intestine
(Fig. 4C, parts a-c), whereas
cyclin D1 was mainly present in the upper part of the villi, where
ß-catenin accumulated at the membrane
(Fig. 4C, parts b-d;
Fig. 4A). It is possible that
ß-catenin nuclear staining was not observed because this gene is
transiently expressed or expressed at a level too low to be readily
detected.
|
Effects of Apc loss on intestinal epithelial differentiation: commitment to Paneth cell lineage
We then investigated the consequences of Apc loss on the differentiation of
all four epithelial lineages found in the small intestine: enterocytes, goblet
cells, enteroendocrine cells and Paneth cells. We used the villin marker for
the enterocyte lineage. In controls, villin was uniformly localized at the
brush border of enterocytes all along the villus
(Fig. 5A). In TAM-treated
mutants, only the epithelial cells of the upper part of villi, corresponding
to the non-proliferative area, expressed villin. Staining with UEA-I, a marker
for goblet cells, showed that goblet cells were dramatically less numerous in
the intestinal epithelium of the mutant mice than in that of the control mice.
Similarly, chromogranin labeling, indicative of differentiation along the
enteroendocrine lineage, was slightly weaker in the mutant mice
(Fig. 5A). These results
indicate that loss of Apc rapidly induces excessive proliferation, associated
with a lack of differentiation to the three epithelial intestinal lineages,
enterocytes, goblet and enteroendocrine. This result is in agreement with the
role of the Wnt pathway in maintaining stem cell properties in the intestine
(van de Wetering et al.,
2002).
|
Cryptdin/defensin family genes are direct targets of ß-catenin
To analyze further this commitment to the Paneth cell lineage, we
investigated whether the strong and rapid induction of lysozyme and crytpdins
could be controlled by ß-catenin signaling. In contrast with lysozyme, a
GenBank search for regulatory elements revealed multiple consensus
ß-catenin/Tcf-binding sites in the promoters of several cryptdin genes,
all of which are located close to the TATA box
(Fig. 6A). The probability of
uncovering a Tcf-binding consensus sequence (T/A T/A CAA T/A G) by chance is
one per 2048 nucleotides. We found at least one Tcf motif in the first 300 bp
of the promoter regions of several cryptdin genes. In addition, both human
cryptdin genes, defensin 5 and 6 (HD5, HD6), contain at least three
Tcf-binding sites in the first 300 nucleotides that includes the promoter.
This situation is reminiscent of the transcriptional control of a group of
hair-specific keratin genes. Indeed, all these genes have a Lef/Tcf1 consensus
motif positioned close to the promoter
(Zhou et al., 1995
). This was
the first clue suggesting the involvement of the ß-catenin signaling in
hair follicle lineage differentiation.
|
Our data indicate that Apc is a crucial determinant of intestinal epithelium cell fate. Activation of the ß-catenin signaling pathway following Apc loss alters the terminal differentiation of the enterocyte, goblet and enteroendocrine lineages, and promotes differentiation along the Paneth cell lineage. This de novo differentiation is partly mediated by the transcriptional control of specific markers of Paneth cells: the cryptdin/defensin genes by ß-catenin/Tcf signaling.
HD6 is induced in human colon cancers
The demonstration that the cryptdin/defensin genes are the direct targets
of ß-catenin and are overexpressed in colon tumors of Apc+/-
mice led us to investigate whether these genes are also induced in human colon
cancers. We thus analyzed the levels of defensins HD5 and HD6
(Fig. 7). First, we compared
Myc and cyclin D1 levels in 10 tumor samples and in patient-matched normal
tissues (Fig. 7). Real-time
RT-PCR revealed that Myc and cyclin D1 mRNA levels were elevated in nine out
of ten tumors. This is probably due to enhanced ß-catenin-mediated
transcription. We found elevated HD6 mRNA levels in all nine of the tumors in
which Myc and cyclin D1 had accumulated. However, virtually no HD5 mRNA was
detectable in any of the samples. Given that the regulatory sequences of these
two genes do not share a high degree of homology (except Tcf-binding sites),
we hypothesise that transcriptional regulations of HD5 and HD6 are different.
HD5 expression may be activated by multiple pathways. These results suggest
that HD6 expression and ß-catenin activation are related events in human
colon cancer, and that HD6 could be used as a new molecular marker of human
colon tumors.
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Discussion |
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Similar results were recently obtained by Sansom et al., using a
conditional deletion of Apc by expression of an inducible Cre recombinase
dependent on ß-naphthoflavone treatments
(Sansom et al., 2004). However
in Sansom's study, the Apc deletion was restricted to the proliferative
compartment of the small intestine, which meant it was not possible to analyze
the consequence of Wnt deregulation in differentiated villus cells. Our
experiments show that the crypt and the villus react differently to
ß-catenin signaling. In the crypt compartment, which contains the
progenitor stem cells and the cellular amplification pools, the immediate
consequences of Apc loss were severe. Within three days, we detected an
expanded compartment with dysplastic-like cells. Myc expression was strongly
induced in this compartment, confirming the crucial in vivo role of Myc in the
maintenance of the crypt progenitor phenotype
(van de Wetering et al.,
2002
). By contrast, the villus cells appeared to be resistant to
morphological alterations. At least in the first four days after Apc loss, the
villus cells presented no proliferation or morphological changes, although
activation of the ß-catenin signaling pathway was supported by the
induction of cyclin D1. However, this was not sufficient to allow the
differentiated epithelial cells of the villus to re-enter the cell cycle, as
Ki67 stained very few cells in this compartment. Accordingly, in human
colorectal cancer, cyclin D1 is predominantly expressed in non-proliferative
cells at the invasion front (Jung et al.,
2001
). Thus, our results support the bottom-up proposal based on
adenomatous crypts from FAP patients: tumorigenesis takes place among the stem
cell population in the crypt base and the transformed stem cells subsequently
expand (Preston et al., 2003
).
The local microenvironment or niche may be critical for inducing proliferation
in response to activation of ß-catenin signaling.
The distinct biological responses of the proliferative and the
differentiated compartments imply that ß-catenin signaling induces
different target genes. Indeed, we observed different expression patterns for
two canonical target genes of ß-catenin. Several hypotheses, which
possibly overlap, can be proposed to explain these molecular differences: the
level of ß-catenin signaling and the cell context (i.e. the cell type and
the cell environment). The heterogeneity of target gene transcription may be
the result of distinct critical thresholds of ß-catenin signaling.
Several studies have indicated that a minimum amount of ß-catenin is
necessary for the transcriptional activation of target genes
(Kielman et al., 2002;
Muller et al., 2002
). Using
distinct ES-cell lines harboring different APC mutations, Kielman et al.
showed that the differentiation capacity of ES cells depends on the amount of
active nuclear ß-catenin (Kielman et
al., 2002
). Alternatively, complex regulation could be due to
crosstalk with other signaling pathways resulting from the dialogue between
epithelial cells and their environment
(Israsena et al., 2004
;
Muller et al., 2002
). Our
results demonstrate that the effects of ß-catenin signaling on gene
expression have context-specific outcomes in terms of cell behavior.
Another important point highlighted by our results concerns the role of Apc
in cell fate in the intestinal epithelium. We were not surprised that enhanced
ß-catenin/Tcf4 activity, known to impose a crypt progenitor phenotype,
prevented the terminal differentiation for three epithelial intestinal
lineages: enterocytes, goblet cells and enteroendocrine cells. However, our
results provide strong evidence that Apc promotes, rather than prevents, the
differentiation of the Paneth cell lineage. This was shown by the strong
induction of Paneth cell markers both in the aberrant crypt areas and in the
colonic tumors of Apc+/- mice. In addition, we demonstrated that
the cryptdin/defensin genes are targets of ß-catenin signaling. Previous
studies demonstrated that activation of ß-catenin-Tcf signaling is
implicated in the proper allocation of Paneth cells via the regulation of
expression of Eph ligand and receptors
(Batlle et al., 2002). Our
studies show that it might also play a role in commitment to the Paneth cell
lineage. Thus, ß-catenin signaling might control this lineage at two
levels: cell fate determination and the spatial localization in the intestine.
To date, three specific Paneth cell markers have been described as target
genes of ß-catenin signaling: MMP7, Eph and cryptdin genes.
Interestingly, Wnt signaling has been shown to regulate positively the
defensin/cryptdin genes in two other systems: ES cell lines with mutations in
the Apc gene express different Paneth cell markers, such as cryptdin
(Kielman et al., 2002
), and
activation of the ß-catenin signaling pathway in lung progenitors can
induce a switch in lineage commitment and, in particular, is responsible for
the expression of Paneth cell markers, MMP7 and cryptdins
(Okubo and Hogan, 2004
). Our
results and these studies suggest that Wnt signaling can induce
differentiation towards the Paneth lineage.
Although ß-catenin signaling induces different Paneth cell markers, it
does not appear to be sufficient for the complete differentiation of Paneth
cells, as shown by the presence of typical secretory granules
(Batlle et al., 2002;
Crawford et al., 1999
). We thus
propose that ß-catenin activation leads to commitment to the Paneth cell
lineage. This is consistent with the results of a previous study showing that,
during mouse development, the differentiation of the Paneth cell lineage
involves the sequential expression of specific markers
(Bry et al., 1994
).
Cryptdin-positive cells first appear in the intervillus epithelium of the
small intestine at E15. Cells with the morphological appearance of Paneth
cells only appear on P7. Our model makes it possible to mimic the first step
of Paneth cell lineage differentiation.
It is possible that immature Paneth cells are a molecular target that may
allow the early detection of colon cancer. Accordingly, our experiments with
human cancer samples showed that HD6 might be such a molecular marker. Despite
extensive examinations, the biological role of Paneth cells has not been
clearly defined. Many studies have suggested that Paneth cells play a role in
intestinal host defense through their production of antimicrobial factors such
as cryptdin/defensin (Porter et al.,
2002). There is also weaker evidence suggesting that Paneth cells
participate in stem cell protection and crypt formation. The expression
patterns of Paneth cell products support the notion that the emergence of this
cell lineage in the small intestine coincides with crypt morphogenesis
(Bry et al., 1994
). In our
study, we observed an association between increased proliferation in the
crypts and the accumulation of `Paneth-like cells' following Apc gene
ablation, which reinforces this hypothesis.
Our study confirms that Apc is a crucial gatekeeper in intestinal homeostasis in vivo. Loss of function of the gene encoding this tumor suppressor rapidly disrupts the balance between cell proliferation, death and differentiation, and impairs cell migration. These events are believed to be the hallmarks of the initiation of intestinal tumorigenesis. Activation of the ß-catenin signaling pathway also promotes commitment to the Paneth cell lineage.
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ACKNOWLEDGMENTS |
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