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Institut National de la Santé et de la Recherche Médicale U129, ICGM, 75014 [B. R., S. S-K., A. P., A-l. P., A. K., C. P.]; Service d’Anatomopathologie, Hôpital Robert Debré, 75019 [D. B., M. P.]; Institut National de la Santé et de la Recherche Médicale U246, Faculté de Médecine Xavier Bichat, Institut Fédératif de Recherche 02, 75810 [A. V.], Paris, France
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ABSTRACT |
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N131ß-catenin) in the intestine. These mice had multifocal dysplastic lesions in the small intestine, reminiscent of the early lesions observed in the mouse models of familial adenomatous polyposis. The number of apoptotic cells in the villi of these transgenic mice was 3–4-fold higher than in nontransgenic mice. Expression of the truncated ß-catenin mutant in the kidney led to the development of severe polycystic kidney disease. Our findings support the concept that deregulation of the ß-catenin signaling pathway is the major oncogenic consequence of adenomatous polyposis coli mutations in intestinal neoplasia.
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Introduction |
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-catenin and seems to be essential for cadherin-mediated cell adhesion (1)
. The similarity between the ß-catenin gene and the Drosophila segment polarity gene Armadillo (Arm) suggests that ß-catenin may also act as a signal transducer of the evolutionary conserved Wnt/Wingless signaling system (1
, 2)
. Free ß-catenin accumulates in response to Wnt signaling and binds to LEF/TCF transcription factors to modulate the activity of target genes involved in the control of cell proliferation. It has been demonstrated recently that this signaling pathway can be activated inappropriately and, thus, contribute to the formation of various epithelial tumors (3, 4, 5)
. In particular, most colorectal tumors are initiated by inactivation of both alleles of the APC3
tumor suppressor gene, resulting in the synthesis of a truncated protein that lacks part of the region involved in targeting ß-catenin for destruction by the ubiquitin-proteasome pathway (2)
. Several studies suggest that APC affects intestinal tumorigenesis mainly as a result of its regulation of ß-catenin signaling (3
, 4)
. Additional evidence for the potential oncogenic role of ß-catenin in intestinal neoplasia is provided by the finding that mutations in the ß-catenin gene itself occur in colon cancer cell lines with no detectable mutations in the APC gene, which lead to stabilization and accumulation of the protein (4
, 6
, 7) . We have, therefore, analyzed the oncogenic potential of ß-catenin in the intestine by creating transgenic mice expressing an activated form of ß-catenin in the epithelial cells of the intestine. The ß-catenin mutant was a NH2-terminal truncation mutant (N131ß-catenin; see Fig. 1A
; Ref. 8
) that had lost both the GSK-3ß phosphorylation site involved in the control of protein stability and the binding site for -catenin necessary for the adhesive properties of ß-catenin (1
, 8)
.
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Materials and Methods |
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N131-ß-catenin construct was produced by inserting the blunted XbaI-SalI fragment encoding the myc-tagged-N131ß-catenin (Ref. 8; kindly provided by W. Birchmeier, Berlin, Germany) into the plasmid containing the calbindin promoter sequence (4.9 kbp of DNA from -4580 to +365 nucleotides) at a single EcoRV restriction site (9
, 10)
. The level of transgene expression was increased by inserting two blunted BamHI-EcoRI copies of the aldolase B enhancer fragment at the 5' end of the CaBP9K promoter sequence (11)
. This chimeric construct was named EAB/9K/N131-ß-catenin construct. Transgenic mice were generated by microinjection of the DNA construct in fertilized mouse eggs of (C57/B6XDBA) F1 hybrid. They were identified by Southern blotting.
Preparation of Tissue Samples.
Mice were killed by cervical dislocation and about one-third of the small intestine was analyzed by removing 3–8-cm fragments from the proximal (duodenum), middle (jejunum), and distal (ileum) parts of the small intestine. Segments 1–15, 3–5 mm long, of each small intestinal fragment were fixed in 4% (v/v) formaldehyde for histological analysis; the remainder were quick-frozen in liquid nitrogen for RNA analysis. The entire large intestine was removed: the colon was divided into proximal and distal segments. As for the small intestine, segments 1 and 2, 3–5 mm long, were fixed in 4% (v/v) formaldehyde for histology, and the remainder were quick-frozen in liquid nitrogen for RNA analysis. Kidneys were removed, cut longitudinally, and processed for histological or RNA analyses.
Northern Blots.
Total RNA was extracted from the mouse tissues (duodenum, jejunum, ileum, cecum, proximal colon, distal colon, and kidney) by the guanidium thiocyanate single-step procedure and analyzed by Northern blotting.
Histological Analysis.
H&E-stained sections were prepared from the duodenum, jejunum, ileum, proximal colon, distal colon, and kidney and examined for histopathological abnormalities and to score the apoptotic and M phase cells in sections with normal morphology. Cell proliferation was analyzed by immunohistochemistry with formalin-fixed sections stained with polyclonal anti-Ki67 antibody (dilution, 1:400; Novocastra, New Castle, United Kingdom).
Quantitation of Apoptotic and M Phase Cells.
M phase and apoptotic cells were scored in 70–120-well oriented crypt-villus units for each mouse. The crypt-villus units are defined as in (Ref. 12). Data were expressed as the average number of M phase or apoptotic cells/villus or crypt. All sections were analyzed in a single-blinded fashion. The five N131-ß-catenin transgenic mice (E29, E187, E222, E311, and E313), together with five nontransgenic mice, were analyzed by scoring the M phase and apoptotic cells in the crypts and villi in various parts of the small intestine (duodenum, jejunum, and ileum). In the colon, 40 colonic crypts and 600 epithelial differentiated cells of the surface epithelial cuff were scored for apoptotic and M phase cells. Only the sections of the transgenic mice with normal morphology were scored. The values were then averaged and analyzed using Student’s unpaired t test.
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Results |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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N131-ß-Catenin Mutant in Transgenic Mice.N131ß-catenin mutant was driven by the calbindin-D9K (CaBP9K) promoter and its regulatory sequences (active in the differentiated epithelial cells of the villus and also in the kidney; Refs. 9
and 10
), linked to the enhancer of the aldolase B gene (11)
, into fertilized eggs. Thirteen founders harboring the EAB/9K-N131ß-catenin construct were created (Fig. 1A)
. At weaning, when we checked the first two founders, one was dead and the other (E29) was moribund. We, therefore, sacrificed all the new founders at 3–4 weeks of age. Eight of the 11 new founders seemed healthy, two were moribund (E313 and E187), and one was dead. Transgene expression was analyzed by Northern blotting. A high level of transgene expression was observed in the intestine and kidney of five founders (E29, E187, E222, E311, and E313; Fig. 1B
). Transgene expression was barely detectable in the six remaining founders (data not shown). Only the mice expressing the N131-ß-catenin mutant at a high level were analyzed further. The pattern of expression of the N131-ß-catenin mutant along the intestine was slightly different in the various highly-expressing founders tested, but the expression of the ß-catenin mutant was higher than (or at least equal to) that of the endogenous ß-catenin gene (Fig. 1B
and data not shown). Although the transgene was less active in the colon than in the small intestine, it remained similar to that of the endogenous ß-catenin gene (Fig. 1B)
. The observed variations in the profile of transgene expression most likely reflect position insertion effects.
Intestinal Lesions in N131ß-Catenin Transgenic Mice.
We first analyzed H&E stained sections of the intestine of the EAB/9K-N131ß-catenin founders. We found dysplastic lesions in three founders (E29, E222, and E187). They were of different grades but always multifocal and restricted to the small intestine. We found no lesions in the colon (Table 1
and Fig. 2
). The duodenum of the E222 founders showed adenomatous proliferation of high-grade dysplasia reminiscent of microadenomas developing early in the APC+/- mice (Fig. 2, D–F
; Refs. 13
and 14
). There were dysplasia of low grade in the duodenum of the E187 founder (Table 1)
. The ileum of the E29 founder showed a high-grade dysplasia different from the pattern of the E222 founder, with histological lesions confined to the villi that were highly basophilic (Table 1
and Fig. 2, G–I
). The villus architecture was undisturbed (Fig. 2G)
. Cells had piled up on top of one another all along the villus, suggesting that abnormal migration and proliferation had occurred (Fig. 2, H and I)
. Some villi were composed of highly vacuolated cells (Fig. 2, G–I)
. Lesions developed in regions of the intestine where the transgene expression was severalfold higher than that of the endogenous ß-catenin gene, but some other parts of the intestine with high transgene activity had villi with normal morphology. Therefore, as in the mouse model where a truncated ß-catenin is targeted to the skin, expression of the truncated ß-catenin in the intestine is not sufficient in itself to cause dysplastic and malignant lesions (15)
. No similar intestinal lesions were observed in nontransgenic littermates of the same age (data not shown).
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. Hence, the different types of lesions occurring in our transgenic mice may be due to differences in their sites of development, at the top of the crypt for the adenomatous polyps (E222, E187; Figs. 2, D–F
; and Fig. 3, B and C
) and in the villi for the highly basophilic lesions (E29; Fig. 2, H and I
). The precise site of lesion occurrence along the villus axis could be stochastic or could depend on the variability of the pattern of transgene expression in the different founders.
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N131ß-Catenin Expression on Cell Proliferation and Apoptosis.N131ß-catenin was analyzed by immunohistochemistry using the nuclear proliferation marker Ki-67 (Fig. 3)
. Cell proliferation was not limited to the crypt, but extended to the villus in all intestinal lesions analyzed (Fig. 3, B–D)
. In contrast, Ki-67 staining was restricted to the crypt and was not found in the N131ß-catenin villi with normal morphology (Fig. 3A)
.
The effect of N131ß-catenin expression on cell proliferation and apoptosis along the crypt-villus axis was determined in all other parts of the intestine devoid of detectable lesions by scoring the number of M phase and apoptotic cells in 70–150 crypt-villus units of the five highly expressing N131ß-catenin founders. There was a statistically significant 3–4-fold increase in apoptotic cells in the villi of N131ß-catenin mice than in normal nontransgenic mice (Student’s t test, P < 0.001); the increase in apoptosis was observed in the five founders mice expressing a high level of the transgene (Fig. 3E)
. In contrast, no significative change in apoptosis or cell proliferation was observed in the crypts of the N131ß-catenin transgenic mice (Fig. 3, F and G)
. This is consistent with the specificity of the regulatory regions directing expression of the transgene in the epithelial cells of the villi (10)
. The colon showed no significant changes in cell proliferation or apoptosis either in the colonic crypts or in the surface epithelial cuff of the colon (data not shown).
N131ß-Catenin Expression in the Kidney Is Associated with the Development of Polycystic Kidney Disease.
All five N131ß-catenin founders analyzed showed elevated transgene expression in the kidney that was at least similar to the level shown in Fig. 1
for the E29 founder. The kidneys of all of these founders had dramatic alterations with numerous cysts (Fig. 2, J and K)
.
The medulla was almost lost (Fig. 2J)
in two founders, E187 and E222. The great disruption of the kidney structure was probably responsible for the premature death of the transgenic mice.
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Discussion |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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N131ß-catenin in the small intestine of transgenic mice is associated with the development of dysplastic lesions. All the intestinal lesions showed abnormal morphology with increased cell proliferation and apoptosis confirming the oncogenic activity of the ß-catenin mutant. The lesions developed in two founders were similar to the early lesions seen in the mouse models containing an APC gene defect obtained by chemical mutagenesis or by homologous recombination in embryonic stem cells (APC+/- mice; Refs. 13
, 14
, and 16
). In these mice, the wild-type APC allele is lost before polyps develop (13
, 16)
. The lesions in the small intestine of the N131ß-catenin transgenic mice are reminiscent of the lesions developed in the nascent intestinal polyps of APC+/- mice (14)
, indicating that control of ß-catenin activity is a major target in the tumor suppressor activity of APC in intestinal tumorigenesis (2)
. However, the intestinal lesions were focal and rather infrequent. The reasons are presently unknown, but our transgenic animals were killed when they were 3–4 weeks of age. Thus, there may not have been enough time for cell transformation in response to the oncogenic activation by ß-catenin. A new transgenic mouse model without transgene expression in the kidney should reduce the early mortality and allow us to analyze the oncogenic properties of ß-catenin in the intestine over a longer period.
Overexpression of the N131ß-catenin in the small intestine villi resulted in a significant increase in apoptosis. This is consistent with recent data showing that Armadillo (the Drosophila ß-catenin homologue) also induces apoptosis in the retina (17)
, indicating that ß-catenin is involved in controlling programmed cell death. The link between apoptosis and the oncogenic action of ß-catenin is still unknown. Apoptosis could result from targeting a deregulated proliferative signal to postmitotic cells, as has been previously reported (18)
. Or a anoïkis-like mechanism could help increase apoptosis because the mutant we used in our experiments lacked the -catenin binding site and, thus, could affect cell adhesion.
There seems to be no change in phenotype (dysplasia or changes in apoptosis) in the colon of the N131ß-catenin transgenic mice. Although we examined only a few transgenic mice, this is consistent with the fact that very few polyps develop in the colon of APC+/- mice compared with the small intestine (19)
.
A recent study by Wong et al. (12)
found no oncogenic effect as a result of the overexpression of a NH2-terminally deleted ß-catenin mutant in the epithelial cells of the small intestine villi. This could be due to a difference in the genetic background of the mice used in their study. Indeed, this pathology has been described to be very sensitive to gene modifiers (20)
. There are also many differences in the experimental settings of the two studies. The regulatory regions directing the transgenes and even the ß-catenin mutant used are different. Hence, the precise pattern of gene expression (e.g., both the activity and timing) could affect the initiation of tumorigenesis. Lastly, the ß-catenin mutant used by Wong et al. (12)
retained the -catenin binding site, whereas our transgene did not. Thus, a disturbance in cadherin-mediated cell adhesion could be involved in the phenotype of our N131ß-catenin mutant mice. Intestinal adenomas have been found in transgenic mice expressing a dominant negative N-cadherin mutant in the crypt-villous unit of the small intestine (21)
.
Expression of the N131ß-catenin in the kidney leads to the development of polycystic kidney disease. The cause of this disease is presently unknown, but it has been suggested that increased epithelial cell proliferation is involved, which would indicate that the kidney is another target of ß-catenin misregulation. The Wnt pathway has already been implicated in kidney morphogenesis (22)
.
In conclusion, we have found strong evidence that ß-catenin is a key factor in controlling the homeostasis of intestinal epithelium, indicating that ß-catenin may be the main target of the tumor suppressor function of APC in intestinal tumorigenesis.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by the Institut National de la Santé et de la Recherche Médicale (INSERM), la Ligue Nationale contre le Cancer, l’Association pour la Recherche contre le Cancer (ARC), la Fondation pour la Recherche Médicale, and a grant from the European Community (BIO4-CT-96005052). 
2 To whom requests for reprints should be addressed, at INSERM U129, 24 rue du Faubourg St. Jacques, 75014 Paris, France. Phone: 33-1-44-41-24-12; Fax: 33-1-44-41-24-21; E-mail: perret{at}icgm.cochin.inserm.fr
3 The abbreviations used are: APC, adenomatous polyposis coli.
Received 4/23/99. Accepted 7/ 2/99.
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REFERENCES |
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716) knockout mice. Cancer Res., 57: 1644-1649, 1997.This article has been cited by other articles:
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) and in the five highly expressing 
). E, average apoptotic cells/villus section (*, P < 0.001). F, apoptotic cells/crypt section. G, average of the M phase/crypt section. No M phase cells were seen in normal morphology villi of