- Email: [email protected]
Min mice
FEBS Letters 582 (2008) 2911–2915
Betacellulin stimulates growth of the mouse intestinal epithelium and increases adenoma multiplicity in Apc+/Min mice Maik Dahlhoffa, David Horstb, Markus Gerhardc, Frank T. Kolligsd, Eckhard Wolfa,e, Marlon R. Schneidera,* a
Institute of Molecular Animal Breeding and Biotechnology, Gene Center, LMU Munich, Feodor-Lynen-St. 25, 81377 Munich, Germany b Pathologisches Institut, LMU Munich, Germany c Second Department of Internal Medicine and Gastroenterology, Technical University of Munich, Germany d Department of Medicine II, LMU Munich, Munich, Germany e Laboratory for Functional Genome Analysis (LAFUGA), Gene Center, LMU Munich, Germany Received 29 May 2008; accepted 14 July 2008 Available online 24 July 2008 Edited by Jesus Avila
Abstract We employed transgenic mice overexpressing betacellulin (BTC) to study its effects in the gut. BTC stimulated crypt cell proliferation and markedly increased intestinal size, while the crypt-villus architecture was preserved. Introduction of a dominant negative epidermal growth factor receptor (EGFR) completely abolished the intestinal hyperplasia. BTC increased polyp multiplicity but did not change the mean size or the histological quality of intestinal polyps in Apc+/Min mice. Analysis of intact and cleaved caspase-3 levels indicated that BTC has anti-apoptotic effects in the intestinal epithelium. We conclude that increased BTC levels support the survival of nascent adenomas in Apc+/Min mice, resulting in a larger total polyp number at later stages. Ó 2008 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: Epidermal growth factor receptor; Betacellulin; Transgenic mice; Intestine; Apc+/Min
1. Introduction The epidermal growth factor receptor (EGFR, ERBB1) is of essential importance for the homeostasis of epithelial tissues. Canonical EGFR activation involves ligand-induced homodimerization or heterodimerization with the related ERBB2 (neu), ERBB3 or ERBB4 receptors, with the formed dimers inducing potent intracellular signaling [1]. The EGFR ligands include amphiregulin (AREG), betacellulin (BTC), epidermal growth factor (EGF), epigen (EPGN), epiregulin (EREG), heparin-binding EGF-like growth factor (HBEGF) and transforming growth factor alpha (TGFA) [2]. The EGFR and its ligands are widely expressed in the intestinal epithelium and play important roles in the regulation of
* Corresponding author. Fax: +49 89 218076849. E-mail address: [email protected] (M.R. Schneider).
Abbreviations: AREG, amphiregulin; BTC, betacellulin; DSS, dextran sulfate sodium; EGF, epidermal growth factor; EGFR, EGF receptor; EPGN, epigen; EREG, epiregulin; HBEGF, heparin-binding EGF-like growth factor; TGFA, transforming growth factor alpha; BTC-tg, betacellulin transgenic; PFA, Paraformaldehyde; WT, wild-type
gastrointestinal physiology and pathology [3]. While targeted inactivation of the Egfr gene in mice results in strain-dependent lethality, favorable genetic backgrounds allow development to the early postnatal period. In these mice, although the intestinal crypt architecture and cell lineages are preserved, villi are reduced in size and number, and cell proliferation is decreased [4,5]. In contrast, mutations in the genes encoding the EGFR ligands have resulted in much milder phenotypes. Mice lacking TGFA [6] or EREG [7] show solely an increased susceptibility to intestinal damage caused by oral administration of dextran sulfate sodium (DSS), and even triple knockout mice lacking EGF, TGFA and AREG show only minor defects in gastrointestinal development [8]. The effects elicited by overexpressing EGFR ligands in the intestinal epithelium are also relatively mild: EGF transgenic mice show enhanced intestinal adaptation after small bowel resection [9], while TGFA overproduction resulted in increased crypt cell proliferation [10] and protected transgenic mice from DSS-induced colitis [11]. The levels of EGFR or its ligands are often increased in tumors as compared to the unaffected surrounding tissue [12], and deregulation of EGFR signaling is frequently associated with tumorigenesis [13,14]. For instance, the hypomorphic allele Egfrwa2 [15] and the antimorphic allele Egfrwa5 reduce the multiplicity of adenomas in the Apc+/Min mouse model of familial adenomatous polyposis. However, treatment of Apc+/Min mice with recombinant EGF over 4 weeks failed to increase polyp number [16]. Betacellulin is widely expressed in the gastrointestinal tract [8,17]. Infusion of BTC into rats resulted in a moderate increase in intestinal growth, but, surprisingly, no increase in cell proliferation was detected [18]. We recently generated transgenic mouse lines producing higher-than-normal levels of this EGFR ligand under control of the b-actin promoter [19]. In the initial analysis of these lines we observed transgene expression in the intestine, but overt effects of BTC in this tissue were not evident at the ages examined. Here, we report that BTC transgenic (BTC-tg) mice develop a remarkably enlarged intestine and increased crypt cell proliferation. Moreover, we employed the Egfrwa5 [20] and Apc+/Min [21] mouse models to evaluate whether the phenotypical consequences of BTC overabundance are EGFR-dependent and whether increased levels of BTC would alter the growth of adenomas in double mutant mice.
0014-5793/$34.00 Ó 2008 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2008.07.026
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2. Materials and methods 2.1. Animals Transgenic mice overexpressing BTC have been described previously in detail [19]. Hemizygous BTC-tg mice from line 2 were mated to wildtype (WT) FVB/N animals, the resulting litters were weaned at an age of 3 weeks, and tail tips were used for genotyping by PCR. Egfrwa5 mice expressing an antimorphic Egfr allele [20] were donated by the Medical Research Council (Oxfordshire, UK) via Dr. David Threadgill (University of North Carolina, NC, USA). C57BL/6J-Apc+/Min mice were purchased from the Jackson Laboratory. The animals were maintained under specific pathogen-free conditions in a closed barrier facility, and had free access to a standard rodent diet (V1534, Ssniff, Soest, Germany) and water. All experiments were approved by the local veterinary authority and were carried out according to the German Law of Animal Protection. 2.2. Tissue collection Mice were killed by cervical dislocation and the gastrointestinal tract was removed. After flushing with ice-cold PBS, the small intestine was cut into halves and the caecum and colon were separated.
M. Dahlhoff et al. / FEBS Letters 582 (2008) 2911–2915 For routine histological evaluation, swiss-rolls [22] were prepared, fixed in 4% paraformaldehyde (PFA), dehydrated and embedded in paraffin. Sections perpendicular to the plane of the tract were further processed. Mice in litters from BTC-tg vs. Apc+/Min matings were monitored daily. Individual animals showed signs of illness at the age of 5 months and all animals were killed at this time point. Intestinal segments were cut longitudinally, laid open on Whatmann 3MM paper, and fixed overnight in 4% paraformaldehyde (PFA). After replacing PFA with 70% ethanol, the number of polyps and their maximum diameter were recorded under a dissection microscope at 10x magnification. 2.3. Immunohistochemistry and in situ hybridization A goat anti-betacellulin antibody was employed for spatial localization of the growth factor (R&D Systems, Wiesbaden, Germany). For visualization of proliferating cells, mice were injected i.p. with BrdU (30 mg/kg body weight) 2 h before killing. Proliferating cells were detected with a rat anti-BrdU antibody (Oxford Biotechnology, Kidlington, UK). In situ hybridization was carried out as described [23].
Fig. 1. Betacellulin overexpression causes phenotypical changes in the intestine of transgenic mice. (A) Macroscopic view of the intestine of a 6month-old control animal and a transgenic littermate. (B) Hematoxylin–eosin staining demonstrating the increase in villus length in the small intestine. (C) Periodic acid-Schiff staining of small intestinal sections showing normal number and distribution of Goblet cells. (D) In situ hybridization showing increased BTC expression in transgenic tissue. (E) Immunohistochemical localization of BTC. (F) Immunohistochemical detection of BrdU incorporation in intestinal crypt cells. (G) Quantitative analysis indicated a significant increase in the number of BrdU-positive cells in BTC-tg mice as compared to gender-matched control littermates. A total of 40 mid-jejunal crypts per animal were counted (n = 2 mice/group). *P < 0.05.
M. Dahlhoff et al. / FEBS Letters 582 (2008) 2911–2915 2.4. Western blot analysis Western blots showing levels of caspase-3, cleaved caspase-3, or GAPDH were carried out with 25 lg protein from the small intestinal mucosa of BTC-tg or control mice. All antibodies and reagents were obtained from Cell Signaling (Frankfurt, Germany). 2.5. Statistical analysis Data are presented as means ± S.D. Groups were compared using two-tailed Student t-tests and P < 0.05 was considered significant.
3. Results and discussion 3.1. Anatomical and histological changes elicited by BTC in the intestine Differences in intestinal size between WT and BTC-tg mice became macroscopically visible at the age of 16 weeks and increased further with age (Fig. 1A). Quantitative analysis revealed that the small intestine of transgenic mice was significantly reduced in length and an increase in weight was detected both in the small and large intestine (Table 1). Analysis of well-oriented histological sections from corresponding regions (i.e. at identical distances from pyloric sphincter) of intestinal rolls revealed a significant increase in the length of villi in BTC-tg mice as compared to WT animals (Table 1 and Fig. 1B). Evaluation of PAS-stained histological sections showed a normal distribution of Goblet cells (Fig. 1C). In addition, no changes in the distribution or localization of Paneth cells were observed (data not shown), indicating normal cell type distribution along the crypt-villus axis. Histological evaluation of the intestinal epithelium of control and transgenic animals at different ages (up to 12 months) did not reveal pathological alterations such as fibrosis, metaplastic or neoplastic lesions. Although transgene-derived BTC expression in the intestine was reported previously [19], no information was available about its spatial localization. In situ hybridization revealed considerably higher amounts of Btc message in the intestinal epithelium of transgenic mice as compared to control animals (Fig. 1D), and this was confirmed for BTC protein by immunohistochemistry (Fig. 1E).
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firmed a significantly higher number of BrdU-positive cells per crypt in the transgenic group (Fig. 1G). This finding contrasts to the results obtained after the administration of recombinant BTC [18]. These disparate findings can have multiple explanations, including the different actions exerted by the full-length, membrane-bound peptide (our studies) and the soluble, mature peptide [18]; furthermore, our transgenic mice have been exposed to higher-than-normal levels of BTC since the embryonic development, while Howarth et al. [18] administered the peptide to adult animals and only for a limited number of days. Finally, our finding of increased crypt cell proliferation under a condition of increased EGFR ligand availability is in accordance with the observation of reduced proliferation in mice lacking the EGFR [4,5]. 3.3. Effects of BTC in the intestine are EGFR-dependent Since BTC is also able to activate ERBB4 [24], we used a genetic approach to evaluate to which extent the observed effects of BTC are mediated by the EGFR. Specifically, we crossed BTC-tg mice to a mouse strain carrying the antimorphic allele Egfrwa5, whose product acts as a dominant negative EGFR [20]. At both macroscopic and histological levels, the intestinal phenotype observed in BTC-tg animals was completely abrogated in double mutant mice (data not shown). Further, the weight of small and large intestine of double mutant mice returned to the levels observed in WT or Egfrwa5 animals (Fig. 2) indicating that the phenotypical alterations observed in the intestine of BTC-tg mice are to a great extent, if not completely, a consequence of increased EGFR activation. 3.4. BTC overproduction increases the multiplicity of adenomas in Apc+/Min mice To test the potential involvement of BTC in tumor formation, we introduced the BTC transgene into mice carrying an Apc+/Min allele, a widely used model of intestinal tumorigenesis [21]. First signs of illness were apparent in some animals at the age of 5 months. All mice were killed at this age and the total number of polyps and their size were recorded. No polyps were
3.2. BTC stimulates cell proliferation in the intestinal crypt Immunohistochemical detection of BrdU incorporation revealed a greatly expanded zone of labeled cells in the crypts of transgenic mice (Fig. 1F) and quantitative analysis conTable 1 Intestinal phenotype of BTC-tg mice and control littermates Control
Transgenic
Small intestine Length (cm) Wet weight (g) Villus lengtha (lm)
40.6 ± 2.4 1.26 ± 0.11 204.5 ± 9.3
33.7 ± 1.8** 2.00 ± 0.16*** 463.7 ± 2.6**
Large intestine Length (cm) Wet weight (g)
7.7 ± 0.7 0.290 ± 0.03
7.3 ± 0.6 0.352 ± 0.06*
Data shown are means ± S.D., n = 4 mice/group. a Crypts in 10 mid-jejunal segments (0.5–0.7 mm each) were analyzed; n = 2 animals per group. * P < 0.05. ** P < 0.01. *** P < 0.001.
Fig. 2. The phenotypical alterations observed in the intestine of BTCtg mice are EGFR-dependent. The weight of small (A) and large (B) intestine of double mutant mice (BTC + Wa5) returned to the levels observed in control or Egfrwa5 (Wa5) animals. All mice were littermates and were analyzed at the age of 9 weeks (n = 3–5/genotype). Values shown represent means ± S.D. *P < 0.05; **P < 0.01.
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found in mice with intact Apc alleles, independently of the BTC status (data not shown). While tumors were observed in only approximately 60% of the Apc+/Min mice at 5 months, 100% of double mutant mice showed at least one polyp (Fig. 3A). More importantly, BTC overproduction had a profound effect on the number of polyps arising in Apc+/Min mice (Fig. 3B). In contrast to the increased polyp multiplicity, we did not detect a significant effect of BTC on tumor size (Fig. 3C). Representative polyps were excised including adjacent normal tissue and examined by a pathologist blinded to the experimental group. No differences in the morphology and the degree of dysplasia were observed (data not shown). Increased BTC levels had no influence on the frequency of polyps of the large intestine, which were observed only sporadically independently of the BTC status. Since the number of polyps observed was rather low, we genotyped the employed lines for Modifier of Min 1 (Mom1) allele status, an important modulator of polyp multiplicity [25]. Parental BTC-tg mice were found to be homozygous for the resistant Mom1r allele, while Apc+/Min mice were homozygous for the susceptible Mom1s allele (data not shown). Thus, F1 animals obtained from these crossings were 100% heterozygous Mom1r/s (data not shown), providing an explanation for the overall low polyp number. Our findings are in contrast to the unchanged number of intestinal polyps after EGF administration to Apc+/Min mice [16]. However, these authors administered EGF over a rather short period (4 weeks). In addition, the signaling pathways initiated by EGF or BTC are considerably distinct, with BTC having a much more pronounced anti-apoptotic activity [26].
M. Dahlhoff et al. / FEBS Letters 582 (2008) 2911–2915
More importantly, our results are in line with the significant reduction in polyp multiplicity, but not in tumor size and quality due to decreased EGFR activity, as described in Apc+/Min mice homozygous for the hypomorphic allele Egfrwa2 [15] or carrying the antimorphic allele Egfrwa5 [20]. In the former report [15], the authors proposed the concept that nascent tumors – which are highly susceptible to being lost – require a threshold level of EGFR activity to become established. Once a certain growth stage has been reached, tumor survival can be achieved by the already initiated downstream activity or by parallel, EGFR-independent tumorigenic events. Our results confirm for the first time an important prediction of this model, namely that increasing EGFR activity due to greater ligand availability during this initial stage would increase final polyp number. 3.5. BTC has anti-apoptotic effects on intestinal epithelial cells Western blots carried out employing protein extracts from the small intestine epithelium revealed markedly lower levels of activated caspase-3 in samples from BTC-tg mice, as compared to control mice (Fig. 3D). This result confirms previous observations indicating that BTC has stronger anti-apoptotic actions as compared to EGF [26]. Furthermore, this finding suggests that BTC overexpression creates an anti-apoptotic environment in the gut epithelium, supporting the survival of newly formed adenomas. In summary, the present study uncovers a remarkable, EGFR-dependent mitogenic effect of BTC in the intestinal epithelium. An additional important finding is the absence of overt pathological (and specially neoplastic) alterations in
Fig. 3. BTC overexpression in Apc+/Min mice increases adenoma multiplicity. (A) Percentage and absolute numbers of mice in both groups showing at least one adenoma. (B) Polyp number in the small intestine of Apc+/Min mice with or without the BTC transgene. Each dot represents the total polyp number of an individual animal. The horizontal line represents the mean value of each genotype. (C) The mean polyp size in each single mouse is represented by a dot, while the horizontal line shows the mean for each genotype. (D) Western blots showing levels of caspase-3, cleaved caspase-3, or GAPDH and quantitative analysis indicating a significant decrease in the cleaved caspase-3/caspase-3 ratio in BTC-tg mice as compared to control littermates.
M. Dahlhoff et al. / FEBS Letters 582 (2008) 2911–2915
the intestinal epithelium after long-term BTC excess in this tissue, which suggest a potential therapeutical application of these growth factor (or derivatives) for supporting cell proliferation after gut resection or reparative processes after tissue injury. The observed increase in polyp number after introduction of the BTC transgene into the Apc+/Min background is most probably a general effect of the EGFR as a ‘‘survival factor’’ for nascent adenomas. Acknowledgements: The authors would like to thank Dr. Ingrid Renner-Mu¨ller, Petra Renner and Tanja Hndawy (animal facility) as well as Joseph Millauer and Stephanie Riesemann (genotyping and expression studies) for their expert assistance. We are indebted to the MRC and to Dr. David Threadgill for providing us with the Egfrwa5 mutant mice. Note added in proof: While this manuscript was being considered, a report describing an increase in the number of polyps (but unaltered polyp size or progression) in the small intestine of transgenic Apc+/Min mice overexpressing the EGFR ligand TGFA was published (Bilger et al., Carcinogenesis, in press).
References [1] Amit, I., Wides, R. and Yarden, Y. (2007) Evolvable signaling networks of receptor tyrosine kinases: relevance of robustness to malignancy and to cancer therapy. Mol. Syst. Biol. 3, 151. [2] Harris, R.C., Chung, E. and Coffey, R.J. (2003) EGF receptor ligands. Exp. Cell Res. 284, 2–13. [3] Barnard, J.A., Beauchamp, R.D., Russell, W.E., Dubois, R.N. and Coffey, R.J. (1995) Epidermal growth factor-related peptides and their relevance to gastrointestinal pathophysiology. Gastroenterology 108, 564–580. [4] Threadgill, D.W., Dlugosz, A.A., Hansen, L.A., Tennenbaum, T., Lichti, U., Yee, D., LaMantia, C., Mourton, T., Herrup, K. and Harris, R.C. (1995) Targeted disruption of mouse EGF receptor: effect of genetic background on mutant phenotype. Science 269, 230–234. [5] Miettinen, P.J., Berger, J.E., Meneses, J., Phung, Y., Pedersen, R.A., Werb, Z. and Derynck, R. (1995) Epithelial immaturity and multiorgan failure in mice lacking epidermal growth factor receptor. Nature 376, 337–341. [6] Egger, B., Procaccino, F., Lakshmanan, J., Reinshagen, M., Hoffmann, P., Patel, A., Reuben, W., Gnanakkan, S., Liu, L., Barajas, L. and Eysselein, V.E. (1997) Mice lacking transforming growth factor alpha have an increased susceptibility to dextran sulfate-induced colitis. Gastroenterology 113, 825–832. [7] Lee, D., Pearsall, R.S., Das, S., Dey, S.K., Godfrey, V.L. and Threadgill, D.W. (2004) Epiregulin is not essential for development of intestinal tumors but is required for protection from intestinal damage. Mol. Cell Biol. 24, 8907–8916. [8] Troyer, K.L., Luetteke, N.C., Saxon, M.L., Qiu, T.H., Xian, C.J. and Lee, D.C. (2001) Growth retardation, duodenal lesions, and aberrant ileum architecture in triple null mice lacking EGF, amphiregulin, and TGF-alpha. Gastroenterology 121, 68–78. [9] Erwin, C.R., Helmrath, M.A., Shin, C.E., Falcone Jr., R.A., Stern, L.E. and Warner, B.W. (1999) Intestinal overexpression of EGF in transgenic mice enhances adaptation after small bowel resection. Am. J. Physiol. 277, G533–G540. [10] Acra, S.A., Bulus, N., Bogatcheva, G., Coffey, R.J. and Barnard, J.A. (1998) Increased intestinal epithelial proliferation in metallothioneine-transforming growth factor alpha transgenic mice. Regul. Pept. 74, 105–112.
2915 [11] Egger, B., Carey, H.V., Procaccino, F., Chai, N.N., Sandgren, E.P., Lakshmanan, J., Buslon, V.S., French, S.W., Buchler, M.W. and Eysselein, V.E. (1998) Reduced susceptibility of mice overexpressing transforming growth factor alpha to dextran sodium sulphate induced colitis. Gut 43, 64–70. [12] Messa, C., Russo, F., Caruso, M.G. and Di Leo, A. (1998) EGF, TGF-alpha, and EGF-R in human colorectal adenocarcinoma. Acta Oncol. 37, 285–289. [13] Normanno, N., De Luca, A., Bianco, C., Strizzi, L., Mancino, M., Maiello, M.R., Carotenuto, A., De Feo, G., Caponigro, F. and Salomon, D.S. (2006) Epidermal growth factor receptor (EGFR) signaling in cancer. Gene 366, 2–16. [14] Zandi, R., Larsen, A.B., Andersen, P., Stockhausen, M.T. and Poulsen, H.S. (2007) Mechanisms for oncogenic activation of the epidermal growth factor receptor. Cell Signal. 19, 2013–2023. [15] Roberts, R.B., Min, L., Washington, M.K., Olsen, S.J., Settle, S.H., Coffey, R.J. and Threadgill, D.W. (2002) Importance of epidermal growth factor receptor signaling in establishment of adenomas and maintenance of carcinomas during intestinal tumorigenesis. Proc. Natl. Acad. Sci. USA 99, 1521–1526. [16] Bashir, O., FitzGerald, A.J., Berlanga-Acosta, J., Playford, R.J. and Goodlad, R.A. (2003) Effect of epidermal growth factor administration on intestinal cell proliferation, crypt fission and polyp formation in multiple intestinal neoplasia (Min) mice. Clin. Sci. (London) 105, 323–330. [17] Kallincos, N.C., Xian, C.J., Dunbar, A.J., Couper, R.T. and Read, L.C. (2000) Cloning of rat betacellulin and characterization of its expression in the gastrointestinal tract. Growth Factors 18, 203–213. [18] Howarth, G.S., Bastian, S.E., Dunbar, A.J. and Goddard, C. (2003) Betacellulin promotes growth of the gastrointestinal organs and effects a diuresis in normal rats. Growth Factors 21, 79–86. [19] Schneider, M.R., Dahlhoff, M., Herbach, N., Renner-Mueller, I., Dalke, C., Puk, O., Graw, J., Wanke, R. and Wolf, E. (2005) Betacellulin overexpression in transgenic mice causes disproportionate growth, pulmonary hemorrhage syndrome, and complex eye pathology. Endocrinology 146, 5237–5246. [20] Lee, D., Cross, S.H., Strunk, K.E., Morgan, J.E., Bailey, C.L., Jackson, I.J. and Threadgill, D.W. (2004) Wa5 is a novel ENUinduced antimorphic allele of the epidermal growth factor receptor. Mamm. Genome 15, 525–536. [21] Moser, A.R., Pitot, H.C. and Dove, W.F. (1990) A dominant mutation that predisposes to multiple intestinal neoplasia in the mouse. Science 247, 322–324. [22] Moolenbeek, C. and Ruitenberg, E.J. (1981) The ‘‘Swiss roll’’: a simple technique for histological studies of the rodent intestine. Lab. Anim. 15, 57–59. [23] Gregorieff, A., Grosschedl, R. and Clevers, H. (2004) Hindgut defects and transformation of the gastro-intestinal tract in Tcf4( / )/Tcf1( / ) embryos. EMBO J. 23, 1825–1833. [24] Riese, D.J., Bermingham, Y., van Raaij, T.M., Buckley, S., Plowman, G.D. and Stern, D.F. (1996) Betacellulin activates the epidermal growth factor receptor and erbB-4, and induces cellular response patterns distinct from those stimulated by epidermal growth factor or neuregulin-beta. Oncogene 12, 345–353. [25] Gould, K.A., Dietrich, W.F., Borenstein, N., Lander, E.S. and Dove, W.F. (1996) Mom1 is a semi-dominant modifier of intestinal adenoma size and multiplicity in Min/+ mice. Genetics 144, 1769–1776. [26] Saito, T., Okada, S., Ohshima, K., Yamada, E., Sato, M., Uehara, Y., Shimizu, H., Pessin, J.E. and Mori, M. (2004) Differential activation of epidermal growth factor (EGF) receptor downstream signaling pathways by betacellulin and EGF. Endocrinology 145, 4232–4243.
Betacellulin stimulates growth of the mouse intestinal epithelium and increases adenoma multiplicity in Apc+/Min mice Maik Dahlhoffa, David Horstb, Markus Gerhardc, Frank T. Kolligsd, Eckhard Wolfa,e, Marlon R. Schneidera,* a
Institute of Molecular Animal Breeding and Biotechnology, Gene Center, LMU Munich, Feodor-Lynen-St. 25, 81377 Munich, Germany b Pathologisches Institut, LMU Munich, Germany c Second Department of Internal Medicine and Gastroenterology, Technical University of Munich, Germany d Department of Medicine II, LMU Munich, Munich, Germany e Laboratory for Functional Genome Analysis (LAFUGA), Gene Center, LMU Munich, Germany Received 29 May 2008; accepted 14 July 2008 Available online 24 July 2008 Edited by Jesus Avila
Abstract We employed transgenic mice overexpressing betacellulin (BTC) to study its effects in the gut. BTC stimulated crypt cell proliferation and markedly increased intestinal size, while the crypt-villus architecture was preserved. Introduction of a dominant negative epidermal growth factor receptor (EGFR) completely abolished the intestinal hyperplasia. BTC increased polyp multiplicity but did not change the mean size or the histological quality of intestinal polyps in Apc+/Min mice. Analysis of intact and cleaved caspase-3 levels indicated that BTC has anti-apoptotic effects in the intestinal epithelium. We conclude that increased BTC levels support the survival of nascent adenomas in Apc+/Min mice, resulting in a larger total polyp number at later stages. Ó 2008 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: Epidermal growth factor receptor; Betacellulin; Transgenic mice; Intestine; Apc+/Min
1. Introduction The epidermal growth factor receptor (EGFR, ERBB1) is of essential importance for the homeostasis of epithelial tissues. Canonical EGFR activation involves ligand-induced homodimerization or heterodimerization with the related ERBB2 (neu), ERBB3 or ERBB4 receptors, with the formed dimers inducing potent intracellular signaling [1]. The EGFR ligands include amphiregulin (AREG), betacellulin (BTC), epidermal growth factor (EGF), epigen (EPGN), epiregulin (EREG), heparin-binding EGF-like growth factor (HBEGF) and transforming growth factor alpha (TGFA) [2]. The EGFR and its ligands are widely expressed in the intestinal epithelium and play important roles in the regulation of
* Corresponding author. Fax: +49 89 218076849. E-mail address: [email protected] (M.R. Schneider).
Abbreviations: AREG, amphiregulin; BTC, betacellulin; DSS, dextran sulfate sodium; EGF, epidermal growth factor; EGFR, EGF receptor; EPGN, epigen; EREG, epiregulin; HBEGF, heparin-binding EGF-like growth factor; TGFA, transforming growth factor alpha; BTC-tg, betacellulin transgenic; PFA, Paraformaldehyde; WT, wild-type
gastrointestinal physiology and pathology [3]. While targeted inactivation of the Egfr gene in mice results in strain-dependent lethality, favorable genetic backgrounds allow development to the early postnatal period. In these mice, although the intestinal crypt architecture and cell lineages are preserved, villi are reduced in size and number, and cell proliferation is decreased [4,5]. In contrast, mutations in the genes encoding the EGFR ligands have resulted in much milder phenotypes. Mice lacking TGFA [6] or EREG [7] show solely an increased susceptibility to intestinal damage caused by oral administration of dextran sulfate sodium (DSS), and even triple knockout mice lacking EGF, TGFA and AREG show only minor defects in gastrointestinal development [8]. The effects elicited by overexpressing EGFR ligands in the intestinal epithelium are also relatively mild: EGF transgenic mice show enhanced intestinal adaptation after small bowel resection [9], while TGFA overproduction resulted in increased crypt cell proliferation [10] and protected transgenic mice from DSS-induced colitis [11]. The levels of EGFR or its ligands are often increased in tumors as compared to the unaffected surrounding tissue [12], and deregulation of EGFR signaling is frequently associated with tumorigenesis [13,14]. For instance, the hypomorphic allele Egfrwa2 [15] and the antimorphic allele Egfrwa5 reduce the multiplicity of adenomas in the Apc+/Min mouse model of familial adenomatous polyposis. However, treatment of Apc+/Min mice with recombinant EGF over 4 weeks failed to increase polyp number [16]. Betacellulin is widely expressed in the gastrointestinal tract [8,17]. Infusion of BTC into rats resulted in a moderate increase in intestinal growth, but, surprisingly, no increase in cell proliferation was detected [18]. We recently generated transgenic mouse lines producing higher-than-normal levels of this EGFR ligand under control of the b-actin promoter [19]. In the initial analysis of these lines we observed transgene expression in the intestine, but overt effects of BTC in this tissue were not evident at the ages examined. Here, we report that BTC transgenic (BTC-tg) mice develop a remarkably enlarged intestine and increased crypt cell proliferation. Moreover, we employed the Egfrwa5 [20] and Apc+/Min [21] mouse models to evaluate whether the phenotypical consequences of BTC overabundance are EGFR-dependent and whether increased levels of BTC would alter the growth of adenomas in double mutant mice.
0014-5793/$34.00 Ó 2008 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2008.07.026
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2. Materials and methods 2.1. Animals Transgenic mice overexpressing BTC have been described previously in detail [19]. Hemizygous BTC-tg mice from line 2 were mated to wildtype (WT) FVB/N animals, the resulting litters were weaned at an age of 3 weeks, and tail tips were used for genotyping by PCR. Egfrwa5 mice expressing an antimorphic Egfr allele [20] were donated by the Medical Research Council (Oxfordshire, UK) via Dr. David Threadgill (University of North Carolina, NC, USA). C57BL/6J-Apc+/Min mice were purchased from the Jackson Laboratory. The animals were maintained under specific pathogen-free conditions in a closed barrier facility, and had free access to a standard rodent diet (V1534, Ssniff, Soest, Germany) and water. All experiments were approved by the local veterinary authority and were carried out according to the German Law of Animal Protection. 2.2. Tissue collection Mice were killed by cervical dislocation and the gastrointestinal tract was removed. After flushing with ice-cold PBS, the small intestine was cut into halves and the caecum and colon were separated.
M. Dahlhoff et al. / FEBS Letters 582 (2008) 2911–2915 For routine histological evaluation, swiss-rolls [22] were prepared, fixed in 4% paraformaldehyde (PFA), dehydrated and embedded in paraffin. Sections perpendicular to the plane of the tract were further processed. Mice in litters from BTC-tg vs. Apc+/Min matings were monitored daily. Individual animals showed signs of illness at the age of 5 months and all animals were killed at this time point. Intestinal segments were cut longitudinally, laid open on Whatmann 3MM paper, and fixed overnight in 4% paraformaldehyde (PFA). After replacing PFA with 70% ethanol, the number of polyps and their maximum diameter were recorded under a dissection microscope at 10x magnification. 2.3. Immunohistochemistry and in situ hybridization A goat anti-betacellulin antibody was employed for spatial localization of the growth factor (R&D Systems, Wiesbaden, Germany). For visualization of proliferating cells, mice were injected i.p. with BrdU (30 mg/kg body weight) 2 h before killing. Proliferating cells were detected with a rat anti-BrdU antibody (Oxford Biotechnology, Kidlington, UK). In situ hybridization was carried out as described [23].
Fig. 1. Betacellulin overexpression causes phenotypical changes in the intestine of transgenic mice. (A) Macroscopic view of the intestine of a 6month-old control animal and a transgenic littermate. (B) Hematoxylin–eosin staining demonstrating the increase in villus length in the small intestine. (C) Periodic acid-Schiff staining of small intestinal sections showing normal number and distribution of Goblet cells. (D) In situ hybridization showing increased BTC expression in transgenic tissue. (E) Immunohistochemical localization of BTC. (F) Immunohistochemical detection of BrdU incorporation in intestinal crypt cells. (G) Quantitative analysis indicated a significant increase in the number of BrdU-positive cells in BTC-tg mice as compared to gender-matched control littermates. A total of 40 mid-jejunal crypts per animal were counted (n = 2 mice/group). *P < 0.05.
M. Dahlhoff et al. / FEBS Letters 582 (2008) 2911–2915 2.4. Western blot analysis Western blots showing levels of caspase-3, cleaved caspase-3, or GAPDH were carried out with 25 lg protein from the small intestinal mucosa of BTC-tg or control mice. All antibodies and reagents were obtained from Cell Signaling (Frankfurt, Germany). 2.5. Statistical analysis Data are presented as means ± S.D. Groups were compared using two-tailed Student t-tests and P < 0.05 was considered significant.
3. Results and discussion 3.1. Anatomical and histological changes elicited by BTC in the intestine Differences in intestinal size between WT and BTC-tg mice became macroscopically visible at the age of 16 weeks and increased further with age (Fig. 1A). Quantitative analysis revealed that the small intestine of transgenic mice was significantly reduced in length and an increase in weight was detected both in the small and large intestine (Table 1). Analysis of well-oriented histological sections from corresponding regions (i.e. at identical distances from pyloric sphincter) of intestinal rolls revealed a significant increase in the length of villi in BTC-tg mice as compared to WT animals (Table 1 and Fig. 1B). Evaluation of PAS-stained histological sections showed a normal distribution of Goblet cells (Fig. 1C). In addition, no changes in the distribution or localization of Paneth cells were observed (data not shown), indicating normal cell type distribution along the crypt-villus axis. Histological evaluation of the intestinal epithelium of control and transgenic animals at different ages (up to 12 months) did not reveal pathological alterations such as fibrosis, metaplastic or neoplastic lesions. Although transgene-derived BTC expression in the intestine was reported previously [19], no information was available about its spatial localization. In situ hybridization revealed considerably higher amounts of Btc message in the intestinal epithelium of transgenic mice as compared to control animals (Fig. 1D), and this was confirmed for BTC protein by immunohistochemistry (Fig. 1E).
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firmed a significantly higher number of BrdU-positive cells per crypt in the transgenic group (Fig. 1G). This finding contrasts to the results obtained after the administration of recombinant BTC [18]. These disparate findings can have multiple explanations, including the different actions exerted by the full-length, membrane-bound peptide (our studies) and the soluble, mature peptide [18]; furthermore, our transgenic mice have been exposed to higher-than-normal levels of BTC since the embryonic development, while Howarth et al. [18] administered the peptide to adult animals and only for a limited number of days. Finally, our finding of increased crypt cell proliferation under a condition of increased EGFR ligand availability is in accordance with the observation of reduced proliferation in mice lacking the EGFR [4,5]. 3.3. Effects of BTC in the intestine are EGFR-dependent Since BTC is also able to activate ERBB4 [24], we used a genetic approach to evaluate to which extent the observed effects of BTC are mediated by the EGFR. Specifically, we crossed BTC-tg mice to a mouse strain carrying the antimorphic allele Egfrwa5, whose product acts as a dominant negative EGFR [20]. At both macroscopic and histological levels, the intestinal phenotype observed in BTC-tg animals was completely abrogated in double mutant mice (data not shown). Further, the weight of small and large intestine of double mutant mice returned to the levels observed in WT or Egfrwa5 animals (Fig. 2) indicating that the phenotypical alterations observed in the intestine of BTC-tg mice are to a great extent, if not completely, a consequence of increased EGFR activation. 3.4. BTC overproduction increases the multiplicity of adenomas in Apc+/Min mice To test the potential involvement of BTC in tumor formation, we introduced the BTC transgene into mice carrying an Apc+/Min allele, a widely used model of intestinal tumorigenesis [21]. First signs of illness were apparent in some animals at the age of 5 months. All mice were killed at this age and the total number of polyps and their size were recorded. No polyps were
3.2. BTC stimulates cell proliferation in the intestinal crypt Immunohistochemical detection of BrdU incorporation revealed a greatly expanded zone of labeled cells in the crypts of transgenic mice (Fig. 1F) and quantitative analysis conTable 1 Intestinal phenotype of BTC-tg mice and control littermates Control
Transgenic
Small intestine Length (cm) Wet weight (g) Villus lengtha (lm)
40.6 ± 2.4 1.26 ± 0.11 204.5 ± 9.3
33.7 ± 1.8** 2.00 ± 0.16*** 463.7 ± 2.6**
Large intestine Length (cm) Wet weight (g)
7.7 ± 0.7 0.290 ± 0.03
7.3 ± 0.6 0.352 ± 0.06*
Data shown are means ± S.D., n = 4 mice/group. a Crypts in 10 mid-jejunal segments (0.5–0.7 mm each) were analyzed; n = 2 animals per group. * P < 0.05. ** P < 0.01. *** P < 0.001.
Fig. 2. The phenotypical alterations observed in the intestine of BTCtg mice are EGFR-dependent. The weight of small (A) and large (B) intestine of double mutant mice (BTC + Wa5) returned to the levels observed in control or Egfrwa5 (Wa5) animals. All mice were littermates and were analyzed at the age of 9 weeks (n = 3–5/genotype). Values shown represent means ± S.D. *P < 0.05; **P < 0.01.
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found in mice with intact Apc alleles, independently of the BTC status (data not shown). While tumors were observed in only approximately 60% of the Apc+/Min mice at 5 months, 100% of double mutant mice showed at least one polyp (Fig. 3A). More importantly, BTC overproduction had a profound effect on the number of polyps arising in Apc+/Min mice (Fig. 3B). In contrast to the increased polyp multiplicity, we did not detect a significant effect of BTC on tumor size (Fig. 3C). Representative polyps were excised including adjacent normal tissue and examined by a pathologist blinded to the experimental group. No differences in the morphology and the degree of dysplasia were observed (data not shown). Increased BTC levels had no influence on the frequency of polyps of the large intestine, which were observed only sporadically independently of the BTC status. Since the number of polyps observed was rather low, we genotyped the employed lines for Modifier of Min 1 (Mom1) allele status, an important modulator of polyp multiplicity [25]. Parental BTC-tg mice were found to be homozygous for the resistant Mom1r allele, while Apc+/Min mice were homozygous for the susceptible Mom1s allele (data not shown). Thus, F1 animals obtained from these crossings were 100% heterozygous Mom1r/s (data not shown), providing an explanation for the overall low polyp number. Our findings are in contrast to the unchanged number of intestinal polyps after EGF administration to Apc+/Min mice [16]. However, these authors administered EGF over a rather short period (4 weeks). In addition, the signaling pathways initiated by EGF or BTC are considerably distinct, with BTC having a much more pronounced anti-apoptotic activity [26].
M. Dahlhoff et al. / FEBS Letters 582 (2008) 2911–2915
More importantly, our results are in line with the significant reduction in polyp multiplicity, but not in tumor size and quality due to decreased EGFR activity, as described in Apc+/Min mice homozygous for the hypomorphic allele Egfrwa2 [15] or carrying the antimorphic allele Egfrwa5 [20]. In the former report [15], the authors proposed the concept that nascent tumors – which are highly susceptible to being lost – require a threshold level of EGFR activity to become established. Once a certain growth stage has been reached, tumor survival can be achieved by the already initiated downstream activity or by parallel, EGFR-independent tumorigenic events. Our results confirm for the first time an important prediction of this model, namely that increasing EGFR activity due to greater ligand availability during this initial stage would increase final polyp number. 3.5. BTC has anti-apoptotic effects on intestinal epithelial cells Western blots carried out employing protein extracts from the small intestine epithelium revealed markedly lower levels of activated caspase-3 in samples from BTC-tg mice, as compared to control mice (Fig. 3D). This result confirms previous observations indicating that BTC has stronger anti-apoptotic actions as compared to EGF [26]. Furthermore, this finding suggests that BTC overexpression creates an anti-apoptotic environment in the gut epithelium, supporting the survival of newly formed adenomas. In summary, the present study uncovers a remarkable, EGFR-dependent mitogenic effect of BTC in the intestinal epithelium. An additional important finding is the absence of overt pathological (and specially neoplastic) alterations in
Fig. 3. BTC overexpression in Apc+/Min mice increases adenoma multiplicity. (A) Percentage and absolute numbers of mice in both groups showing at least one adenoma. (B) Polyp number in the small intestine of Apc+/Min mice with or without the BTC transgene. Each dot represents the total polyp number of an individual animal. The horizontal line represents the mean value of each genotype. (C) The mean polyp size in each single mouse is represented by a dot, while the horizontal line shows the mean for each genotype. (D) Western blots showing levels of caspase-3, cleaved caspase-3, or GAPDH and quantitative analysis indicating a significant decrease in the cleaved caspase-3/caspase-3 ratio in BTC-tg mice as compared to control littermates.
M. Dahlhoff et al. / FEBS Letters 582 (2008) 2911–2915
the intestinal epithelium after long-term BTC excess in this tissue, which suggest a potential therapeutical application of these growth factor (or derivatives) for supporting cell proliferation after gut resection or reparative processes after tissue injury. The observed increase in polyp number after introduction of the BTC transgene into the Apc+/Min background is most probably a general effect of the EGFR as a ‘‘survival factor’’ for nascent adenomas. Acknowledgements: The authors would like to thank Dr. Ingrid Renner-Mu¨ller, Petra Renner and Tanja Hndawy (animal facility) as well as Joseph Millauer and Stephanie Riesemann (genotyping and expression studies) for their expert assistance. We are indebted to the MRC and to Dr. David Threadgill for providing us with the Egfrwa5 mutant mice. Note added in proof: While this manuscript was being considered, a report describing an increase in the number of polyps (but unaltered polyp size or progression) in the small intestine of transgenic Apc+/Min mice overexpressing the EGFR ligand TGFA was published (Bilger et al., Carcinogenesis, in press).
References [1] Amit, I., Wides, R. and Yarden, Y. (2007) Evolvable signaling networks of receptor tyrosine kinases: relevance of robustness to malignancy and to cancer therapy. Mol. Syst. Biol. 3, 151. [2] Harris, R.C., Chung, E. and Coffey, R.J. (2003) EGF receptor ligands. Exp. Cell Res. 284, 2–13. [3] Barnard, J.A., Beauchamp, R.D., Russell, W.E., Dubois, R.N. and Coffey, R.J. (1995) Epidermal growth factor-related peptides and their relevance to gastrointestinal pathophysiology. Gastroenterology 108, 564–580. [4] Threadgill, D.W., Dlugosz, A.A., Hansen, L.A., Tennenbaum, T., Lichti, U., Yee, D., LaMantia, C., Mourton, T., Herrup, K. and Harris, R.C. (1995) Targeted disruption of mouse EGF receptor: effect of genetic background on mutant phenotype. Science 269, 230–234. [5] Miettinen, P.J., Berger, J.E., Meneses, J., Phung, Y., Pedersen, R.A., Werb, Z. and Derynck, R. (1995) Epithelial immaturity and multiorgan failure in mice lacking epidermal growth factor receptor. Nature 376, 337–341. [6] Egger, B., Procaccino, F., Lakshmanan, J., Reinshagen, M., Hoffmann, P., Patel, A., Reuben, W., Gnanakkan, S., Liu, L., Barajas, L. and Eysselein, V.E. (1997) Mice lacking transforming growth factor alpha have an increased susceptibility to dextran sulfate-induced colitis. Gastroenterology 113, 825–832. [7] Lee, D., Pearsall, R.S., Das, S., Dey, S.K., Godfrey, V.L. and Threadgill, D.W. (2004) Epiregulin is not essential for development of intestinal tumors but is required for protection from intestinal damage. Mol. Cell Biol. 24, 8907–8916. [8] Troyer, K.L., Luetteke, N.C., Saxon, M.L., Qiu, T.H., Xian, C.J. and Lee, D.C. (2001) Growth retardation, duodenal lesions, and aberrant ileum architecture in triple null mice lacking EGF, amphiregulin, and TGF-alpha. Gastroenterology 121, 68–78. [9] Erwin, C.R., Helmrath, M.A., Shin, C.E., Falcone Jr., R.A., Stern, L.E. and Warner, B.W. (1999) Intestinal overexpression of EGF in transgenic mice enhances adaptation after small bowel resection. Am. J. Physiol. 277, G533–G540. [10] Acra, S.A., Bulus, N., Bogatcheva, G., Coffey, R.J. and Barnard, J.A. (1998) Increased intestinal epithelial proliferation in metallothioneine-transforming growth factor alpha transgenic mice. Regul. Pept. 74, 105–112.
2915 [11] Egger, B., Carey, H.V., Procaccino, F., Chai, N.N., Sandgren, E.P., Lakshmanan, J., Buslon, V.S., French, S.W., Buchler, M.W. and Eysselein, V.E. (1998) Reduced susceptibility of mice overexpressing transforming growth factor alpha to dextran sodium sulphate induced colitis. Gut 43, 64–70. [12] Messa, C., Russo, F., Caruso, M.G. and Di Leo, A. (1998) EGF, TGF-alpha, and EGF-R in human colorectal adenocarcinoma. Acta Oncol. 37, 285–289. [13] Normanno, N., De Luca, A., Bianco, C., Strizzi, L., Mancino, M., Maiello, M.R., Carotenuto, A., De Feo, G., Caponigro, F. and Salomon, D.S. (2006) Epidermal growth factor receptor (EGFR) signaling in cancer. Gene 366, 2–16. [14] Zandi, R., Larsen, A.B., Andersen, P., Stockhausen, M.T. and Poulsen, H.S. (2007) Mechanisms for oncogenic activation of the epidermal growth factor receptor. Cell Signal. 19, 2013–2023. [15] Roberts, R.B., Min, L., Washington, M.K., Olsen, S.J., Settle, S.H., Coffey, R.J. and Threadgill, D.W. (2002) Importance of epidermal growth factor receptor signaling in establishment of adenomas and maintenance of carcinomas during intestinal tumorigenesis. Proc. Natl. Acad. Sci. USA 99, 1521–1526. [16] Bashir, O., FitzGerald, A.J., Berlanga-Acosta, J., Playford, R.J. and Goodlad, R.A. (2003) Effect of epidermal growth factor administration on intestinal cell proliferation, crypt fission and polyp formation in multiple intestinal neoplasia (Min) mice. Clin. Sci. (London) 105, 323–330. [17] Kallincos, N.C., Xian, C.J., Dunbar, A.J., Couper, R.T. and Read, L.C. (2000) Cloning of rat betacellulin and characterization of its expression in the gastrointestinal tract. Growth Factors 18, 203–213. [18] Howarth, G.S., Bastian, S.E., Dunbar, A.J. and Goddard, C. (2003) Betacellulin promotes growth of the gastrointestinal organs and effects a diuresis in normal rats. Growth Factors 21, 79–86. [19] Schneider, M.R., Dahlhoff, M., Herbach, N., Renner-Mueller, I., Dalke, C., Puk, O., Graw, J., Wanke, R. and Wolf, E. (2005) Betacellulin overexpression in transgenic mice causes disproportionate growth, pulmonary hemorrhage syndrome, and complex eye pathology. Endocrinology 146, 5237–5246. [20] Lee, D., Cross, S.H., Strunk, K.E., Morgan, J.E., Bailey, C.L., Jackson, I.J. and Threadgill, D.W. (2004) Wa5 is a novel ENUinduced antimorphic allele of the epidermal growth factor receptor. Mamm. Genome 15, 525–536. [21] Moser, A.R., Pitot, H.C. and Dove, W.F. (1990) A dominant mutation that predisposes to multiple intestinal neoplasia in the mouse. Science 247, 322–324. [22] Moolenbeek, C. and Ruitenberg, E.J. (1981) The ‘‘Swiss roll’’: a simple technique for histological studies of the rodent intestine. Lab. Anim. 15, 57–59. [23] Gregorieff, A., Grosschedl, R. and Clevers, H. (2004) Hindgut defects and transformation of the gastro-intestinal tract in Tcf4( / )/Tcf1( / ) embryos. EMBO J. 23, 1825–1833. [24] Riese, D.J., Bermingham, Y., van Raaij, T.M., Buckley, S., Plowman, G.D. and Stern, D.F. (1996) Betacellulin activates the epidermal growth factor receptor and erbB-4, and induces cellular response patterns distinct from those stimulated by epidermal growth factor or neuregulin-beta. Oncogene 12, 345–353. [25] Gould, K.A., Dietrich, W.F., Borenstein, N., Lander, E.S. and Dove, W.F. (1996) Mom1 is a semi-dominant modifier of intestinal adenoma size and multiplicity in Min/+ mice. Genetics 144, 1769–1776. [26] Saito, T., Okada, S., Ohshima, K., Yamada, E., Sato, M., Uehara, Y., Shimizu, H., Pessin, J.E. and Mori, M. (2004) Differential activation of epidermal growth factor (EGF) receptor downstream signaling pathways by betacellulin and EGF. Endocrinology 145, 4232–4243.