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First published online 27 June 2007
doi: 10.1242/dev.02875
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Research Report |
1 Department of Genetic Medicine and Development, University of Geneva Faculty
of Medicine, 1 Rue Michel-Servet, CH-1211 Geneva 4, Switzerland.
2 Department of Human Molecular Genetics and Biochemistry, Sackler School of
Medicine, Tel-Aviv University, Israel.
3 Department of Cell Biology, Cancer Institute, Tokyo 135-8550, Japan.
4 Unitat de Recerca en Biologia Cel·lular i Molecular (URBCM), Institut
Municipal d'Investigació Mèdica (IMIM), Universitat Pompeu
Fabra, Dr Aiguader, 88 08003 Barcelona, Spain.
5 Genetics and Stem Cell Laboratory, Swiss Institute for Experimental Cancer
Research (ISREC), Ch. des Boveresses 155, CH-1066 Epalinges,
Switzerland.
6 Division of Diabetes, Digestive and Kidney Disease, Division of Diabetes
Metabolism and Endocrinology, Department of Clinical Molecular Medicine,
Department of Internal Medicine, Kobe University Graduate School of Medicine,
7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan.
7 Ecole Polytechnique Fédérale de Lausanne (EPFL), School of Life
Sciences, CH-1015 Lausanne, Switzerland.
* Author for correspondence (e-mail: Pedro.Herrera{at}medecine.unige.ch)
Accepted 18 May 2007
SUMMARY
ß-catenin signaling is heavily involved in organogenesis. Here, we investigated how pancreas differentiation, growth and homeostasis are affected following inactivation of an endogenous inhibitor of ß-catenin, adenomatous polyposis coli (Apc). In adult mice, Apc-deficient pancreata were enlarged, solely as a result of hyperplasia of acinar cells, which accumulated ß-catenin, with the sparing of islets. Expression of a target of ß-catenin, the proto-oncogene c-myc (Myc), was increased in acinar cells lacking Apc, suggesting that c-myc expression is essential for hyperplasia. In support of this hypothesis, we found that conditional inactivation of c-myc in pancreata lacking Apc completely reversed the acinar hyperplasia. Apc loss in organs such as the liver, colon and kidney, as well as experimental misexpression of c-myc in pancreatic acinar cells, led to tumor formation with high penetrance. Surprisingly, pancreas tumors failed to develop following conditional pancreas Apc inactivation. In Apc-deficient acini of aged mice, our studies revealed a cessation of their exaggerated proliferation and a reduced expression of c-myc, in spite of the persistent accumulation of ß-catenin. In conclusion, our work shows that ß-catenin modulation of c-myc is an essential regulator of acinar growth control, and unveils an unprecedented example of Apc requirement in the pancreas that is both temporally restricted and cell-specific. This provides new insights into the mechanisms of tumor pathogenesis and tumor suppression in the pancreas.
Key words: Pancreas, Growth, Apc, ß-catenin, c-myc, ICAT, Mouse
INTRODUCTION
The size of the pancreas, a compound acinar exocrine gland also containing
endocrine islets, is determined by intrinsic factors, such as the number of
early progenitor cells (Stanger et al.,
2007
), and by extrinsic signals. The Wnt/ß-catenin signaling
pathway is one genetic mechanism controlling body and organ size and shape:
pancreas size and the proportions of its different cell types are altered when
the stability of ß-catenin is modified
(Heiser et al., 2006).
However, the mechanism of these effects conveyed by ß-catenin has not
been identified.
ß-catenin binds 
-catenin in adherens junctions and, by
regulating the transcriptional activity of T cell factors (TCFs), is a key
effector of Wnt signaling (reviewed in
Harris and Peifer, 2005). Wnt
signaling induces a conformational change in ß-catenin to favor TCF
binding over that of -catenin
(Gottardi and Gumbiner,
2004a). In the absence of Wnt signaling, free cytoplasmic
ß-catenin is sequentially phosphorylated by a complex containing Apc and
degraded. Upon Wnt binding to its receptor, on the contrary, the
unphosphorylated form accumulates in the cytoplasm before being translocated
into the nucleus, where it binds TCFs, thus activating the transcription of
genes involved in cell proliferation
(Harris and Peifer, 2005;
Polakis, 1999).
Because Apc is involved in ß-catenin degradation
(Polakis, 1999;
van Es et al., 2001), Apc
inactivation creates a permissive condition whereby free unphosphorylated
ß-catenin may accumulate, thus mimicking active Wnt signaling
(Staal et al., 2002). Here, we
show that inactivating Apc in all pancreatic epithelial cells of
early primordia induces a pancreatomegaly resulting from the selective,
c-myc-dependent, increased proliferation of acinar cells between
birth and 6 months of age. Interestingly, this very mutation in liver, colon
and kidney is always tumorigenic (Andreu et
al., 2005; Colnot et al.,
2004; Sansom et al.,
2005; Shibata et al.,
1997), but not in the pancreas.
MATERIALS AND METHODS
Mice
Animals bearing exon 15 (anciently 14) of Apc flanked by two loxP
sites (`floxed') (Shibata et al.,
1997) were crossed with mice expressing Cre under the control of a
Pdx1 promoter (Herrera,
2000; Herrera et al.,
2002). Tail DNAs were analyzed as described, using P3, P4 and P5
primers (Shibata et al.,
1997). c-myc-floxed, Pax6-Cre and
Smad4-floxed mice are described elsewhere
(Trumpp et al., 2001;
Ashery-Padan et al., 2004;
Herrera et al., 2002;
Yang et al., 2002). All
experiments were approved by the `Office Vétérinaire' of the
State of Geneva.
Gene expression analyses
Adult pancreas RNAs were extracted as described
(Glisin et al., 1974), whereas
embryonic and isolated islets RNAs were extracted with the RNeasy micro kit
(Qiagen). Total RNA was DNase-I-treated according to the manufacturer
(Ambion). First-strand cDNA synthesis was performed using SuperScript II
reverse transcriptase (Invitrogen Life Technologies). Real-time RT-PCR primers
were designed with Primer express 2.0 (Applied Biosystems); three housekeeping
genes were used as controls (Eef1a1, Rps9 and ß-tubulin). PCRs were done
in triplicate, with five specimens per condition, and were labeled with SYBR
green master mix (Applied Biosystems). Fluorescence was quantified with the
Prism 7900 HT sequence detection system (Applied Biosystems). Raw Ct
(threshold cycle) values obtained with SDS 2.0 (Applied Biosystems) were used
to calculate the normalization factor and the fold change with the geNorm
script, as published (Vandesompele et al.,
2002). No change was scored when P
0.05. All
experiments were performed at the Genomics Platform of our Medical School.
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Specificity of the different immunostainings was confirmed with sections in which primary antibodies were omitted. Sections were examined with a Nikon epifluorescence microscope (Eclipse TE200) equipped with a Nikon DS-L1 camera, or with a Zeiss LSM 510 confocal microscope.
Western blotting
Pancreata or islets from ApcloxP/loxP,
ApcloxP/loxP;Pdx1-Cre+/-,
ApcloxP/loxP;Pax6-Cre+/- and
c-mycloxP/loxP;ApcloxP/loxP;Pdx1-Cre+/-
mice were homogenized using a polytron in lysis buffer (50 mM Tris-HCl, pH
7.5, 250 mM NaCl, 1% Triton X-100, 1 mM EDTA, 1 mM DTT) containing complete
protease inhibitors (Roche) and incubated for 30 minutes on ice. Lysates were
clarified by centrifugation and protein concentration was determined. Samples
were fractionated by SDS-PAGE and transferred to Immobilon P membranes
(Millipore) for immunoblotting with mouse monoclonal
anti-active-ß-catenin 8E7 antibody (Upstate, 1/400) or with rabbit
anti-ß-catenin (phospho Y142; Abcam, 1/500) and with rabbit polyclonal
anti-ß-tubulin antibody (Abcam ab6046, 1/2000). Detection was performed
using peroxidase-conjugated anti-mouse or anti-rabbit IgGs (Promega, 1/5000).
Bands were visualized by chemiluminescence (ECL, Amersham) according to the
manufacturer's instructions.
BrdU treatment
Animals were given BrdU (Sigma) intraperitoneally (50 µg/g of body
weight) 2 hours prior to sacrifice.
Glucose tolerance
Animals were fasted overnight (16 hours) and injected intraperitoneally
with 2 g glucose (Fluka) per kg of body weight. Glycemia was measured using
Glucometer DEX (Bayer) strips. Insulinemia was determined by ELISA (Kit
Mercodia Ultrasensitive Rat Insulin ELISA).
Pancreatic glucagon and insulin content
Pancreatic protein extracts were prepared by adding acid-ethanol solution
(74% ethanol, 1.4% HCl) and then performing homogenization. Samples were
sonicated and centrifuged, and the supernatant was used for radioimmunoassay
experiments performed following the manufacturer's instructions (Glucagon RIA
Kit, Linco, for glucagon; and ELISA Kit Mercodia Ultrasensitive Rat Insulin
ELISA for insulin).
Islet isolation
Islets from 1-month-old ApcloxP/loxP,
ApcloxP/loxP;Pdx1-Cre+/- and
ApcloxP/loxP;Pax6-Cre+/- mice were isolated as
described above (with collagenase type V Sigma #C-9263) and purified on a
Ficoll gradient (Sigma Histopaque #1077)
(Wollheim et al., 1990).
Amylase activity
Adult mouse pancreata were homogenized in 3 ml of PBS and centrifuged for 1
minute at 70.86 g. Blood samples from the retro orbital sinus
were collected into lithium-heparin-treated vials and centrifuged for 5
minutes at 784 g. Amylase activity was assessed by enzymatic
photometry using -amylase CC FS (y; DiaSys) relative to the protein
content determined by the Bradford test.
Morphometry
Three 8-week-old animals per group were analyzed. Paraffin sections
obtained at 200-µm intervals were immunostained with anti-insulin antibody.
Photographs were obtained with an EOS D30 digital camera (Canon) and analyzed
with the National Institutes of Health (NIH) ImageJ 1.60 software. ß-cell
area was expressed as a percentage of total pancreatic area.
Statistics
All results were reported as mean±s.e.m. (standard error of the
mean). Groups were compared with independent t-tests (unpaired and
two-tailed), reported as P values. All tests were performed using the
GraphPad Prism software.
RESULTS AND DISCUSSION
Apc loss leads to postnatal pancreatomegaly due to acinar hyperplasia
In order to investigate the role of ß-catenin signaling in pancreas
organogenesis and homeostasis, we took advantage of an existing mouse line in
which exon 15 of the two Apc alleles was flanked by loxP sites
[designated ApcloxP/loxP or `control' mice
(Shibata et al., 1997)]. These
mice were bred to Pdx1-Cre+/- transgenics
(Herrera, 2000;
Herrera et al., 2002), which
express Cre recombinase in all pancreatic epithelial cell types from embryonic
day (E)10.5 (Fig. 1A). In
ApcloxP/loxP;Pdx1-Cre+/- mice
(ApcP-/- hereafter), Apc was selectively
invalidated in acinar, ductal and islet cells
(Fig. 1B).
Control and heterozygous
(ApcloxP/+;Pdx1-Cre+/-) mice showed
undistinguishable phenotypes. ApcP-/- animals appeared
normal throughout embryogenesis and at birth but, from 3 weeks of age, a
marked pancreatomegaly ensued, with a pancreas-to-body weight ratio three- to
five-fold higher than in control mice (Fig.
1C,D). The increased size of the pancreas persisted during the
whole period of study, and is consistent with previous observations involving
the forced expression of ß-catenin in pancreata
(Heiser et al., 2006). Acinar,
ductal and islet cells appeared histologically normal in
ApcP-/- fetuses and young adults (1-4 months old; see Fig.
S1 in the supplementary material), yet the islets of Langerhans appeared
diluted (i.e. more scarce within a hyperplastic exocrine compartment). Cell
density was comparable in control and ApcP-/- young adults
(data not shown), indicating that pancreatomegaly was due to acinar cell
hyperplasia, rather than to acinar cell enlargement (hypertrophy). Cell
proliferation and differentiation were normal in fetal pancreata but, from
birth until 6 months of age, acinar cells had an increased proliferation rate
(Fig. 1E); the rates of cell
proliferation were normal in ducts and islets (data not shown).
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Although adult islet density was reduced, the absolute ß-cell mass in ApcP-/- animals was unaffected, as were total pancreatic insulin and glucagon contents, and glucose homeostasis (see supplementary Fig. S1B and Fig. S3 in the supplementary material).
Altogether, these observations indicate that only acinar cells are
sensitive to Apc loss and that the endocrine-to-exocrine tissue ratio [1-99%
(Orci, 1982)] is not crucial
in long-term pancreas homeostasis.
Sensitivity of acinar cells to ß-catenin signaling is restricted to a postnatal competence period
Apc inactivation is expected to induce the accumulation of unphosphorylated
ß-catenin (Staal et al.,
2002). Surprisingly, despite the efficient inactivation of Apc in
ApcP-/- fetuses (E15.5), the expression and distribution
of ß-catenin was not changed compared to controls
(Fig. 2A,B); accordingly,
expression of ß-catenin target genes was not, or only slightly,
upregulated (Table 1). By
contrast, in the postnatal pancreas, Apc loss elicited the nuclear
accumulation of ß-catenin in acinar cells from birth
(Fig. 2A). This unexpected
difference confirms the compartmentalization of the effects of Apc loss to
postnatal acinar lineages.
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).
These transcripts were markedly increased in 2-month-old
ApcP-/- total pancreatic extracts; by contrast, this was
not the case in islets isolated from 1-month-old ApcP-/-
animals (data not shown).
In young adult ApcP-/- mice, and more markedly in aged
animals, ß-catenin accumulation was apparent in a fraction of ductal and
islet cells, and was diffuse, both cytoplasmic and nuclear
(Fig. 2A,B). The refractoriness
of islet cells to ß-catenin signaling was further established with mice
bearing the Apc inactivation exclusively in islets through the use of
a Pax6-Cre transgene (Ashery-Padan
et al., 2004; Herrera et al.,
2002) (ApcloxP/loxP;Pax6-Cre+/-).
In these mutants, no islet dysplasia or hyperplasia was observed (see Fig. S4A
in the supplementary material). Despite the efficient downregulation of
Apc transcripts (80%) in
ApcloxP/loxP;Pax6-Cre+/- islets, the expression
of ß-catenin target genes, such as in ApcP-/- islets,
was not augmented (see Fig. S4B in the supplementary material).
In conclusion, these results reveal a precise spatiotemporal pattern in the pancreas for ß-catenin signaling: it remains low or inactive during pancreas development but, after birth, is activated in acinar cells only.
Interestingly, ß-catenin signaling (i.e. `activation' of
ß-catenin, or its nuclear translocation) can occur in the absence of Wnt
signaling via other mechanisms (Harris and
Peifer, 2005; Willert and
Jones, 2006). ß-catenin-(phospho Y142) is an indicator of
Wnt-independent ß-catenin signals
(Brembeck et al., 2004).
Phosphorylation of ß-catenin at tyrosine 142, mediated by hepatocyte
growth factor (Hgf) receptors (Met), has been shown to occur in murine
hepatomegaly (Apte et al.,
2006), human hepatoblastomas
(Ranganathan et al., 2005) and
human colorectal carcinoma cells (Rasola
et al., 2006). In the pancreas, we found that
ß-catenin-(phospho Y142) levels were undetectable in pancreatic primordia
and in isolated islets, but high in acinar cells, whether control or
ApcP-/- (Fig.
2B). This expression pattern correlates with that of
unphosphorylated nuclear ß-catenin in ApcP-/-
pancreata.
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Deletion of c-myc is sufficient to abolish the ApcP-/- phenotype
To test whether the increased c-myc expression observed in
ApcP-/- mutants is required for the acinar overexpansion,
we performed the double inactivation of Apc and c-myc using
mice bearing, in addition to the two loxP-flanked Apc alleles, two
loxP-flanked c-myc alleles
(Trumpp et al., 2001).
Remarkably, mice simultaneously lacking Apc and c-myc
(ApcP-/-;c-mycP-/-) in pancreas displayed a
complete reversal of the ApcP-/- phenotype, with no
pancreatomegaly in spite of the accumulation of nuclear ß-catenin in
acinar cells (Fig. 3A-C). These
mice showed normal relative ß-cell area
(Fig. 3D) and pancreatic
insulin content at 2 months of age.
Together, these results indicate that the proliferative effect of
ß-catenin on acinar cells after Apc loss requires increased
c-myc activity. This is the first evidence, together with two reports
on Apc inactivation in the intestine
(Ignatenko et al., 2006;
Sansom et al., 2007), which
appeared while this work was under evaluation for publication, for an in vivo
molecular mechanism (mediated by c-myc inactivation) involved in the
reversal of a `Wnt gain-of-function' (i.e. Apc deficiency) phenotype.
ApcP-/- pancreata become `resistant' to ß-catenin signaling and escape tumorigenesis
Mice bearing the same Apc mutation in liver, colon or kidney
(Andreu et al., 2005;
Colnot et al., 2004;
Sansom et al., 2005;
Shibata et al., 1997) always
develop tumors. Similarly, continued overexpression of c-myc in
acinar cells under the control of an elastase promoter is tumorigenic
(Sandgren et al., 1991), and
human pancreatic cancer cells have high levels of c-myc expression
(Buchholz et al., 2006).
However, contrary to these observations, tumor formation was prevented in
ApcP-/- pancreata, despite the high levels of
ß-catenin.
In ApcP-/- animals, pancreatomegaly remained stable and, up to 1 year of age, mice were in good health, had unchanged pancreatic mass and normal endocrine function (see Fig. S3D in the supplementary material). In 1-year-old mice, hypertrophic acinar cells, with dysplastic nuclei, were observed focally (Fig. 2A; and see below and Fig. S5 in the supplementary material). However, E-cadherin expression was always normal (data not shown) and no tumors developed.
The absence of pancreatic tumors was further explored in mice lacking,
simultaneously, Apc and Smad4 in the pancreas. Smad4, a central transducer of
signals conveyed by Tgfß ligands, is often mutated or deleted in
colorectal cancer and pancreatic carcinoma
(Bardeesy et al., 2006;
Hahn et al., 1996;
Hua et al., 2003;
Shattuck-Brandt and Dubois,
1999; Tang et al.,
2002). However, mice lacking Smad4 in the pancreas have
no pancreatic tumors (Simeone et al.,
2006). In the present study, we analyzed the cumulative effect of
the concurrent loss of both Apc and Smad4, and no
spontaneous pancreatic tumor developed during the first year of life.
Whereas nuclear ß-catenin in acinar cells persisted in 1-year-old mice (Fig. 2A), the expression of c-myc and other genes that are upregulated in young pancreata returned to normal levels in mature animals (Table 1), indicating the acquisition of `resistance' to signaling via ß-catenin. This correlates with the normalization of acinar cell proliferation after 5 months of age in ApcP-/- animals, and the absence of tumorigenesis (Fig. 1E and see Fig. S6 in the supplementary material).
The first months of life thus represent a competence window, a sensitive
period during which acinar cells may undergo ß-catenin-induced
proliferation. The spontaneous downregulation of ß-catenin signaling in
the exocrine pancreas in aged mice, despite the persistent presence of
abundant nuclear ß-catenin, defines the beginning of a
ß-catenin-unresponsive phase (summarized in Fig. S6 in the supplementary
material). Our observations indeed suggest a mechanism of tumor suppression,
or rather of signal adaptation, after Apc loss: a resetting of the threshold
upon continuous signaling by ß-catenin. A similar negative-feedback
mechanism after inactivation of another tumor suppressor gene, NF1
(whose malfunction underlies the familial cancer syndrome neuro - fibromatosis
type I), was recently reported
(Courtois-Cox et al.,
2006).
The refractoriness of islet cells to convey growth signals through
ß-catenin might also help understand why insulinomas, which very often
display a loss of APC protein expression
(Arnold et al., 2007), are
largely benign tumors (Gonzalez-Gonzalez
and Recio-Cordova, 2006), and why ß-cells do not
transdifferentiate into ductal cells in pancreatic metaplastic lesions
(Strobel et al., 2007).
The blockade of ß-catenin/TCF activity, revealed by the persistence of
nuclear ß-catenin in acini without increased c-myc expression or
cell proliferation, possibly results from the activity of ß-catenin
competitors [i.e. ICAT (also known as Ctnnbip1 - Mouse Genome Informatics) and
Duplin (also known as Chd8 - Mouse Genome Informatics)], TCF repressors
[Groucho (Tle1), Hbp1, CtBP1], or from TCF post-translational modifications
(Kikuchi et al., 2006). Among
the negative modulators of ß-catenin that we analyzed, we found that ICAT
(inhibitor of ß-catenin and Tcf4), which prevents the binding of
ß-catenin to TCF (Gottardi and
Gumbiner, 2004b; Tago et al.,
2000), has a pancreatic expression pattern consistent with the
observed spatial and temporal oscillations of c-myc expression: islet
cells maintain high levels of ICAT throughout life; in acinar cells, ICAT is
downregulated in mature mice, but not in ApcP-/- animals
(see Fig. S5 in the supplementary material). Whether ICAT or other repressors
are involved in this ß-catenin blockade will be addressed in further
studies.
In conclusion, using an in vivo system in which endogenous ß-catenin
signaling is allowed or favored (i.e. the ablation of Apc), we report that
early pancreatic progenitor cells, and islet cells, exhibit strong inhibition
of excessive ß-catenin signaling (see Fig. S6B in the supplementary
material). Others have shown that forced excessive or defective ß-catenin
signaling disrupts normal acinar organogenesis
(Dessimoz et al., 2005;
Heiser et al., 2006;
Murtaugh et al., 2005;
Papadopoulou and Edlund, 2005;
Wells et al., 2007). Later in
life, upregulation of ß-catenin inhibitors/competitors, leading to
impaired ß-catenin/TCF activity, would block signaling in acini (see
supplementary material Fig. S6B).
Whether ß-catenin signaling does play a role during pancreas
development, growth and maturation remains to be clearly determined. Here, we
have demonstrated that, at least in a favorable situation for ß-catenin
signaling (such as after Apc loss), the definitive size of the pancreas at
complete maturation appears to be intrinsically determined by the progressive
unresponsiveness to ß-catenin signaling in acinar cells; in addition,
this endogenous constraint probably explains why tumor formation is prevented
in pancreas but not in liver, colon or kidney bearing the same Apc
mutation (Andreu et al., 2005;
Colnot et al., 2004;
Sansom et al., 2005;
Shibata et al., 1997), and
might be of therapeutic relevance.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/15/2719/DC1
ACKNOWLEDGMENTS
We are most grateful to Pierre Vassalli, Seung K. Kim and Ariel Ruiz i Altaba for insightful comments and fruitful discussions and support, as well as to Paolo Meda, Pierre Maechler, Jean-Dominique Vassalli, Annie Rodolosse and Matthias Hebrok. We thank Cara J. Gottardi for the kind gift of the affinity-purified anti-ICAT antibody and Chris Wright for anti-Pdx1 antibody. We thank Géraldine Dussex for devoted and skillful technical help and, with Olivier Fazio, for the help in managing the colony of mice. P.L.H. is recipient of grants from the JDRF (#5-2005-12; #1-2005-31), NIH/NIDDK [(DK072522-01) Beta Cell Biology Consortium], Swiss National Science Foundation (#3100A0-103867/1; and the NCCR Frontiers in Genetics) and the 6FP EU Integrated Project Beta Cell Therapy for Diabetes; F.X.R. is recipient of grant SAF2004-01137 from Ministerio de Educación y Ciencia, Spain. R.A.-P. is recipient of grants from the Israel Science Foundation and the German Israeli Foundation.
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