Research Article

Bone Morphogenetic Protein Signaling Suppresses Tumorigenesis at Gastric Epithelial Transition Zones in Mice

Sylvia A. Bleuming,1 Xi C. He,3 Liudmila L. Kodach,1 James C. Hardwick,1,4 Frieda A. Koopman,1 Fiebo J. ten Kate,2 Sander J.H. van Deventer,1 Daniel W. Hommes,4 Maikel P. Peppelenbosch,5 G. Johan Offerhaus,6 Linheng Li,3 and Gijs R. van den Brink1,4

1Center for Experimental and Molecular Medicine and 2Department of Pathology, Academic Medical Center, Amsterdam, the Netherlands; 3Stowers Institute for Medical Research, Kansas City, Missouri; 4Department of Gastroenterology and Hepatology, Leiden University Medical Center, Leiden, the Netherlands; 5Department of Biology, Faculty of Medical Sciences, University of Groningen, Groningen, the Netherlands; and 6Department of Pathology, University Medical Center Utrecht, Utrecht, the Netherlands

Abstract We have previously shown that BMPs are expressed in the adult Bone morphogenetic protein (BMP) signaling is known to (4); however, we still know very little about the role of suppress oncogenesis in the small and of BMP signaling in the maintenance of homeostasis of the complex mice and humans. We examined the role of Bmpr1a signaling epithelial system of the stomach. The BMPs are soluble proteins that are part of the transforming growth factor-h (TGF-h) in the stomach. On conditional inactivation of Bmpr1a, mice developed neoplastic lesions specificallyin the squamoco- superfamily. BMP ligands bind to a complex of the BMP receptor lumnar and gastrointestinal transition zones. We hypothe- type II and a BMP receptor type I (Ia or Ib). This leads to the sized that the regulation of epithelial cell fate maybe less phosphorylation of the type I receptor that subsequently phos- well defined in these junctional zones than in the adjacent phorylates the BMP-specific Smads (Smad1, Smad5, and Smad8), and found that the mucosa at the squamocolum- allowing these receptor-associated Smads to form a complex with nar junction in mice shows a lack of differentiated fundic Smad4 and move into the nucleus where the Smad complex binds a gland cell types and that foveolar cells at the gastrointestinal DNA binding protein and acts as a transcriptional enhancer (5). junctional zone lack expression of the foveolar cell marker BMPs regulate apoptosis of intestinal epithelial cells (6) and Muc5ac. Precursor cell proliferation in both transition zones mutations in the BMP receptor 1a (BMPR1A) have been found in f20% of families with juvenile polyposis syndrome whereas was higher than in the surrounding epithelium. Our data show another 20% of cases of juvenile polyposis syndrome are caused by that BMP signaling through Bmpr1a suppresses tumorigenesis h at gastric epithelial junctional zones that are distinct from mutations in SMAD4, the common mediator of BMP and TGF- the adjacent gastric epithelium in both cellular differentiation signaling (7–9). Mice with a conditional inactivation of the Bmpr1a and proliferation. [Cancer Res 2007;67(17):8149–55] (10) and mice that overexpress a BMP antagonist in the intestine (11) have an intestinal phenotype that resembles Juvenile polyposis with the development of multiple hamartomatous polyps in the Introduction intestine. The role of BMP signaling in the adult stomach has not The epithelial layer of the is in a state of been examined. constant renewal. Stem cells in its basal layers generate The glandular mucosa of the murine stomach is divided into a descendants that undergo a period of rapid amplification until proximal zymogenic or fundic region and a distal mucous or antral they withdraw from the cell cycle. Cells are allocated to one of region (12). Stem cells of the are located several cell lineages and mature while they migrate to their final approximately halfway down the tubular units that invaginate into destination where they undergo a process of programmed cell the mucosal surface. From this stem cell zone, cells can migrate in death and/or are shed into the of the gut. Cell fate decisions two ways. Cells that migrate up to the luminal surface of the of epithelial cells are taken in a position-dependent, non–cell- stomach form the pit region or foveola. These pit cells secrete autonomous manner (1). Such decisions are governed by infor- (Muc5ac) and are similar throughout the stomach. Cells mation that is perceived from the cellular environment. During that migrate downward from the stem cell position form the development, morphogens form concentration gradients through specialized glandular region of each unit. In the proximal stomach, tissues to specify cell fate in a position-dependent manner. An these glands are fundic or oxyntic-type glands. Fundic glands are important role for morphogens has now also been established as composed of acid-producing parietal cells, mucous neck cells, regulators of cell fate in the adult gastrointestinal tract. Mutations –producing zymogenic (or chief) cells, and in the Wnt and bone morphogenetic protein (BMP) pathways are endocrine cells. In the distal stomach, the glands are antral-type the cause of inherited polyposis syndromes as well as the initiating glands and they contain a variety of different endocrine cells and event in the development of sporadic gastrointestinal cancer mucinous cells. Here, we examine the role of BMP signaling development (2, 3). through the Bmpr1a using a conditional knockout approach.

Requests for reprints: Gijs R. van den Brink, Department of Gastroenterology and Materials and Methods Hepatology, Leiden University Medical Center, Building 1, C4-P012, P.O. Box 9600, 2300 Mice. To obtain normal tissue of the gastric transition zones, C57BL/6 RC Leiden, the Netherlands. Phone: 31-20-566-9111; E-mail: [email protected]. I2007 American Association for Cancer Research. control mice were given a single i.p. injection of 150 mg/mL bromodeox- doi:10.1158/0008-5472.CAN-06-4659 yuridine (BrdUrd) to label cells in Sphase 1 h before being killed by cervical www.aacrjournals.org 8149 Cancer Res 2007; 67: (17). September 1, 2007

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Figure 1. Neoplastic change at the gastric epithelial transition zones in PolyI-C–treated Mx1-cre-Bmpr1afx/fx mice. A to D, immunohistochemistry for GFP expression in PolyI-C–treated Mx1-cre-Z/EG (A and B) and wild-type (C and D) mice used as negative controls shows efficient Mx1-Cre–mediated recombination in the gastric epithelium (A and B). E to K, H&E section of a neoplastic lesion at the squamocolumnar junction showed an area of carcinoma in situ (F, arrows) adjacent to the squamous epithelial layer (enlarged in G). H to K, H&E section of a small polyp at the gastrointestinal junction (I, intestine; P, pyloric ring). I, intestinal epithelium with normal crypt-villus architecture bordered the distal end of the polyp. J, the epithelium of the polyp was of gastric type and histologically mature, consistent with a hamartomatous polyp. K, the epithelium that borders the proximal end of the polyp showed normal gastric antral glands. L to N, immunohistochemical detection of the intestinal brush border protein villin. L, a small polyp at the gastrointestinal junction (P, pyloric ring). M, epithelial cells in the polyp were villin negative, consistent with the gastric type . N, villin was expressed by in the adjacent intestinal epithelium. A, Â160; B, Â400; C, Â160; D, Â400; E, Â160; F and G, Â400; H, Â80; I to K, Â400; L, Â80; M and N, Â800.

dislocation. To allow optimal orientation of the gastric tissue, flat stomachs the Cre recombinase gene is under control of an IFN-responsive promoter were prepared according to the method described by Lee et al. (12). (15) that can be induced on systemic treatment with polyinosinic- fx/fx To evaluate the role of Bmpr1a signaling in the gastric mucosa, we used a polycytidylic acid (PolyI-C). In the resulting Mx1-cre-Bmpr1a mice, the conditional knockout approach using the Cre/loxP system. We used a floxed Bmpr1a is excised on treatment with PolyI-C. Four-week-old Mx1-cre- fx/fx fx/fx mouse with a conditional null allele of Bmpr1a (Bmpr1a ) that was Bmpr1a mice were injected i.p. with 250-mg PolyI-C. Because we have previously generated and characterized (10, 13, 14). In these mice, LoxP sites previously found that multiple injections with PolyI-C are required for have been introduced in the first and second introns of the Bmpr1a locus. efficient deletion of Bmpr1a, mice were injected with PolyI-C thrice on It was previously shown that when these mice are crossed with CMV-Cre alternate days (14). Animals were sacrificed at 5 months of age. To confirm transgenic mice, the phenotype is identical to the Bmpr1a(À/À) mouse, that the Mx1 promoter–driven Cre efficiently targets the gastric epithelium indicating that deletion of exon 2 is sufficient to completely inactivate as previously described (16), the Mx1-Cre transgenic mouse was crossed fx/fx Bmpr1a function. For our experiment, the Bmpr1a transgenic mouse with the Z/EG reporter mouse, which expresses green fluorescent protein was crossed with the Mx1-Cre transgenic mouse (13) in which expression of (GFP) in cells in which efficient Cre-mediated excision of LoxP sites has

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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2007 American Association for Cancer Research. BMP Signaling at Epithelial Transition Zones occurred (17). Mx1-Cre-Z/EG mice were injected with PolyI-C thrice on Triton and 1% bovine serum albumin. The second day, slides were washed alternate days and sacrificed 18 days after treatment. Stomachs and in PBSand incubated with the appropriate biotinylated secondary antibody duodenums were excised, cut longitudinally, fixed in 4% paraformaldehyde, at room temperature for 1 h in PBSwith 10% normal human serum. Slides and processed following routine histologic procedures. Animal experiments were washed in PBSand incubated with streptavidin-biotin-horseradish were approved by the animal ethics review board. peroxidase (HRP; DAKO) for 1 h. After washing in PBS, peroxidase activity Antibodies and immunohistochemistry. A goat polyclonal anti-villin was detected using the ‘‘Fast DAB’’ system (Sigma) according to the (1:50; C-19) was from Santa Cruz Biotechnology. A mouse monoclonal manufacturer’s instructions. Finally, sections were counterstained with anti-Muc5ac (1:50; 45M1) was from Novocastra. A mouse monoclonal Mayer’s hematoxylin, dehydrated, and mounted in Entellan (Merck) under anti-BrdUrd (1:50) was from Roche. A rabbit polyclonal antibody that coverslips. recognizes the phosphorylated form of the BMP-specific Smads, Smad1, For double staining of H+K+-ATPase and pSmad1,5,8 sections were first Smad5, and Smad8 (pSmad 1,5,8, 1:40) was from Cell Signaling Technology. incubated overnight with anti-pSmad1,5,8. The following day, sections were A rabbit anti–h-catenin (1:1,000) was from Transduction Laboratories. incubated for 60 min with a biotinylated goat anti-rabbit immunoglobulin. A rabbit anti-GFP (1:250) was from Molecular Probes. For all immunohis- Sections were then incubated with streptavidin-biotin-HRP and further tochemistry except for h-catenin (see below), the following protocol was processed as described above to detect pSmad1,5,8 expression. Sections used: sections were dewaxed and rehydrated in graded alcohols. were then incubated overnight with anti–H+K+-ATPase. The following

Endogenous peroxidase activity was quenched with 1.5% H2O2 in methanol. day, an alkaline phosphatase–coupled goat anti-mouse immunoglobulin Antigen retrieval was done by boiling slides for 10 min in 0.01 mol/L was applied. Alkaline phosphatase was detected with the Fast Red detection sodium citrate (pH 6.0). Nonspecific binding sites were blocked with 10% method (DAKO), resulting in a red precipitate. Immunohistochemical normal human serum in PBSfor 30 min. For unmasking of the BrdUrd controls consisted of omission of the primary or secondary antibodies. epitope, slides were incubated in 2 N HCl at 37jC for 60 min and then For detection of h-catenin, a staining protocol was used that has been washed in boric acid (pH 8.5) before the blocking step. Slides were developed at the Clevers laboratory (18). Slides were dewaxed and incubated with the primary antibodies overnight at 4jC in PBSwith 0.1% rehydrated. Then, slides were boiled in a pressure cooker for 10 min and

Figure 2. A and B, compared with adjacent normal epithelium (A), the epithelium of the polyps at the gastroduodenal transition zone showed loss of pSmad1,5,8 staining (B). C, in areas with an underlying inflammatory infiltrate, pSmad1,5,8 staining was absent from the epithelium (long arrows) whereas cells in the inflammatory infiltrate were strongly positive for pSmad1,5,8 (short arrows). D and E, BrdUrd staining showed expansion of BrdUrd-incorporating cells (E, arrows)in S phase in a polyp at the gastroduodenal transition zone. F to H, an early lesion at the gastrointestinal junction. G, the histology showed accumulation of immature epithelial cells with enlarged nuclei and prominent nucleoli (arrows). H, BrdUrd staining of the same area showed an accumulation of BrdUrd- positive cells in the same area. Original magnification: A to C, Â400; D, Â100; E, Â400; F, Â80; G and H, Â800.

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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2007 American Association for Cancer Research. Cancer Research left to cool down very slowly. Slides were blocked with 2-nitro-5- thiobenzoate (TNB) at room temperature and incubated with the anti–h- catenin antibody at 4jC overnight. Slides were washed thrice in TNT [0.1 mol/L Tris (pH 7.5), 0.15 mol/L NaCl, 0.3% Triton X-100] and incubated for 60 min at room temperature with a biotinylated rabbit anti-mouse antibody from the labeled streptavidin-biotin + (LSAB+) kit from DAKO. Slides were washed thrice in TNT and incubated for 30 min at room temperature with streptavidin-HRP from the LSAB+ kit diluted 1:100 in TNB. After washing thrice in TNT, slides were incubated for 10 min at room temperature in BT working solution from the LSAB+ kit (diluted 1:50 in amplification diluent from the same kit). Slides were washed thrice in TNT and incubated for 30 min with streptavidin-HRP diluted 1:100 in TNB at room temperature. Slides were washed thrice in TNT and staining was developed using the DAB substrate-chromogen solution from the LSAB+ kit. BrdUrd-positive nuclei. To assess proliferation of epithelial cells in the gastric mucosa, gastric specimens from BrdUrd-injected control mice were stained with antibodies against BrdUrd. Pictures of each transition zone and the adjacent gastric mucosa were taken at Â100 magnification and positive nuclei counted with the use of an image analysis program (Image- ProPlus, MediaCyberNetics). In each zone, five well-oriented vertical units were counted per mouse. The average number of positive nuclei per vertical unit was compared between groups. To be able to compare the results between animals, it was ensured that all sections visualized the entire axis from the superficial epithelium to the muscularis mucosa. Statistical analysis. Data are presented as mean F SE. Comparisons Figure 3. Expression of h-catenin. A, in normal small intestinal crypts, nuclear between groups of data were made using the Student t test. P < 0.05 was h-catenin can be detected in a fewcells at the base of the crypt ( arrows). h considered statistically significant. B, extensive nuclear accumulation of -catenin can be detected in the small intestinal tumors of conditional Bmpr1a mutant mice (arrows). C and D, no nuclear accumulation is detected in a gastric tumor that was present in the same Results specimen as the intestinal tumor shown in (B). Original magnification: A, Â400; B, Â200; C, Â100; D, Â400. Bmpr1a signaling suppresses tumorigenesis at gastric epithelial junctional zones. To address the role of BMP signaling in the gastric mucosa, we examined mice in which the Bmpr1a can be conditionally inactivated on treatment with PolyI-C. We first Compared with adjacent normal epithelium (Fig. 2A), loss of confirmed the previously reported targeting of gastric epithelial pSmad1,5,8 staining was observed in the epithelium of the polyps cells by the Mx1 promoter–driven Cre (16). Mx1-Cre-Z/EG reporter (Fig. 2B). In parts of the polyps that were rich in inflammatory cells, mice were treated with PolyI-C and the expression of GFP was a hallmark of hamartomas, loss of pSmad1,5,8 staining was detected with an anti-GFP antibody. As can be seen in Fig. 1A to D, observed in the epithelium whereas many cells in the underlying strong staining was detected of epithelial cells throughout inflammatory infiltrate were strongly positive for pSmad1,5,8 the in PolyI-C–treated Mx1-Cre-Z/EG mice (Fig. 1A (Fig. 2C). Similar to the polyps previously described in the intestine and B) whereas no staining was detected in the stomachs of wild- of conditional Bmpr1a mutant mice, polyps at the gastric transition type mice (Fig. 1C and D). This experiment confirmed the efficient zones were characterized by a strong increase in the number of Mx1-Cre–driven recombination in gastric epithelial cells that was BrdUrd-positive cells (Fig. 2D and E). In one mouse, the gastro- fx/fx previously reported by others. PolyI-C–treated Mx1-cre-Bmpr1a intestinal transition zone looked grossly normal but displayed mice showed a phenotype of epithelial hyperplasia in the prox- histologic change with accumulation of immature epithelial cells at imal, fundic region (data not shown). The most remarkable the base of the glands with large nuclei and prominent nucleoli phenotype was observed at the squamocolumnar and gastroin- that were partly BrdUrd positive (Fig. 2F–H), suggesting that the testinal transition zones. Bmpr1a mutant mice developed tumors initiation of tumorigenesis may start with the accumulation of specifically at both gastric epithelial junctional zones (Fig. 1E–K). these immature cells. Five of seven mice showed intraepithelial neoplasia, varying from No nuclear accumulation of B-catenin in gastric tumors of mild dysplasia to carcinoma in situ specifically at the squamo- Bmpr1a mutant mice. Overactivity of the Wnt pathway with columnar junction (Fig. 1E–G). Six of seven mice examined nuclear accumulation of h-catenin plays a critical role in the showed formation of a polyp of variable size specifically at the formation of intestinal tumors. Nuclear accumulation of h-catenin gastrointestinal transition zone (Fig. 1H–K). Histologically, the has been observed in intestinal tumors of mice that overexpress smaller polyps showed normal differentiation of the epithelial the BMP inhibitor Noggin in their intestine and intestinal tumors cells and recruitment of inflammatory cells to the lamina of conditional Bmpr1a mutant mice. These observations suggest propria, consistent with a hamartomatous polyp (Fig. 1H–K). In that BMP signaling negatively regulates Wnt signaling in the the larger polyps, we observed foci of dysplastic epithelial cells, intestine. We examined nuclear expression of h-catenin in normal indicating adenomatous transformation of the hamartomatous small intestinal crypts (Fig. 3A), in intestinal tumors of Bmpr1a polyp (Fig. 1E–G). Histologically, all polyps consisted of gastric mutant mice (Fig. 3B), and in gastric tumors of Bmpr1a mutant type columnar epithelial cells. At the gastrointestinal transition mice (Fig. 3C and D). As expected, we detected nuclear h-catenin zone, the polyps bordered directly on the ; in a few cells at the base of the normal small intestinal crypt epithelial cells in the polyps were negative for the intestinal (Fig. 3A; ref. 18). Similarly, we detected abundant nuclear accu- marker villin (Fig. 1L–N). mulation of h-catenin in intestinal tumors of conditional Bmpr1a

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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2007 American Association for Cancer Research. BMP Signaling at Epithelial Transition Zones mutant mice (Fig. 3B) as described (10). In contrast, no such mice was characterized by one to three gastric glands that are accumulation was observed in gastric tumors of Bmpr1a mutant mainly composed of mucous cells (Fig. 4A and B). The cardiac mice (Fig. 3C and D). A few cells with nuclear h-catenin could be glands lacked the specialized cell types of the fundic glands of the detected in some gastric tumors but most of the tumors were proximal stomach as they contained no zymogenic cells and only negative. The intestinal tumor shown in Fig. 3 was in the same an occasional (Fig. 4A and B). This lack of parietal specimen as the gastric tumor shown in the same figure. The cells was confirmed by immunohistochemistry for H+K+-ATPase intestinal tumor, therefore, served as an internal positive control (Fig. 4C). for the staining. The gastrointestinal transition zone is localized at the pyloric The murine squamocolumnar and gastrointestinal transi- ring. Here, the glandular region of the distal gastric epithelium tion zones. Because differentiating cells at epithelial junctional (antrum) borders the crypt-villus structure of the small intestine zones are exposed to conflicting extrinsic signals from two (Fig. 4D and E). We examined epithelial differentiation of pit cells different epithelial tissues, we hypothesized that the regulation at the gastrointestinal junction using the expression of Muc5ac. of epithelial cell fate may be less well defined in these zones Muc5ac is a that is uniformly expressed by gastric pit cells than in the adjacent epithelium. We have therefore examined in the gastric antrum (Fig. 4F; ref. 20). At the gastrointestinal the squamocolumnar epithelial transition zone of the proximal transition zone, however, expression of Muc5ac was patchy and stomach and the gastrointestinal junction in normal adult mice most pit cells were actually Muc5ac negative (Fig. 4G). These data (n = 7). Similar to the gastric epithelium immediately adjacent to supported the idea that cells at the squamocolumnar and the squamous epithelium of the in humans (the cardia; gastrointestinal junction lack some of the signals that specify cell ref. 19), gastric epithelium at the squamocolumnar epithelium in fate in the adjacent epithelium. Because epithelial cells in the

Figure 4. The gastric junctional zones in the normal mouse. A to C, the squamocolumnar junction. A, H&E section showed pink-staining triangular parietal cells in the fundic mucosa (arrows), whereas the cardiac glands (enlarged in B) at the junction between the squamous epithelium and the fundic mucosa lacked parietal cells and were composed entirely of mucous cells (B, asterisk). C, immunohistochemistry for the parietal cell marker H+K+-ATPase confirmed lack of parietal cells in the cardiac glands (asterisk) in the mouse. D and E, H&E staining of the gastrointestinal junction (arrow, ; P, pyloric ring). E, higher magnification of the gastrointestinal transition zone. F and G, Muc5ac immunohistochemistry. F, uniform expression of Muc5ac in pit cells of the gastric antrum in the mouse. G, at the transition zone, most epithelial cells were Muc5ac negative compared with the pits of the distal antral mucosa (arrows). A, Â100; B and C, Â400; D, Â80; E to G, Â200.

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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2007 American Association for Cancer Research. Cancer Research gastric transition zones may be exposed to conflicting signals similar molecular mechanism in the genesis of gastric polyps in the that regulate their cell fate, we hypothesized that the cell cycle Bmpr1a conditional mutant mouse as we observed very little of epithelial precursor cells in these zones may be less tightly nuclear h-catenin in the gastric polyps. This indicates that the regulated and that these cells may have a higher rate of mechanism of tumor suppression by BMP signaling in the stomach proliferation than those in the surrounding epithelium. We may be distinct from that in the intestine. therefore examined epithelial BrdUrd incorporation in normal Enhanced sensitivity to oncogenesis at the gastrointestinal mice as a marker of the regulation of cell fate at the epithelial junctional zone was previously described in mice. Mice treated junctional zones. Indeed, we found that proliferation was increased with the carcinogen N-nitroso-N-butylurea specifically develop at both gastric epithelial transition zones compared with the polypoid tumors at the junction of the gastric and intestinal adjacent gastric epithelium (Fig. 5A–H). epithelial layers (21). Additionally, mice with a mutation in Smad4 develop tumors specifically at the junction of the stomach and the intestine (22). Enhanced sensitivity to oncogenesis at the junction Discussion of two different epithelial tissues is known to exist in humans but Here, we show that Bmpr1a conditional null mice developed is a poorly understood phenomenon. The best known clinical neoplasia specifically at both gastric junctional zones. A small examples of tumors associated with epithelial transition zones in lesion at the gastrointestinal junctional zone showed that the humans are carcinomas that arise at the esophagogastric junction process of neoplasia seems to start with an accumulation of and at the squamocolumnar junction of the uterine cervix (23, 24). immature epithelial cells that were partly positive for BrdUrd, Other examples are tumors that arise at the anorectal squamoco- indicating that an expansion of the precursor cell compartment is lumnar junction and adenomas and carcinomas of the ampulla of a primary event in the genesis of these polyps. Intestinal tumors of Vater (25–27). conditional Bmpr1a mutant mice (10) and mice that overexpress Epithelial cells that have to make cell fate choices at the junction the BMP antagonist Noggin in the intestine (11) develop intestinal of two distinctive epithelial tissues are prone to receive conflicting tumors that show intense nuclear accumulation of h-catenin. This extrinsic information from the two different epithelial environ- indicates that BMP signaling acts as a break on tumorigenic Wnt ments. At this point, morphogen gradients involved in the signaling activity in the intestine. No evidence was found for a regulation of cell fate of one epithelial tissue type may contrast

Figure 5. Increased number of BrdUrd- positive cells in the gastric junctional zones in normal mice. Immunohistochemistry for BrdUrd. A to C, squamocolumnar junction; arrows, positive cells. Epithelial proliferation was enhanced in the glands in the transition zone (B) compared with those in the adjacent fundic glands (C). D, gastrointestinal transition zone. Compared with normal gastric antral glands (E), BrdUrd incorporation was increased in the transition zone (F). Proliferating cells were found dispersed throughout the units of the transition zone (F, arrows), in contrast to the antrum where proliferating cells localized to a small precursor cell compartment (E). G, number of BrdUrd-positive cells in normal fundic epithelial units (2.0 BrdUrd-positive cells/unit) versus cardiac glands of the transition zone (5.5 BrdUrd-positive cells/unit; P = 0.002; n = 4 control mice). H, number of BrdUrd-positive cells in normal antral epithelial units (3.5 BrdUrd-positive cells/unit) versus glands of the gastrointestinal transition zone (9.6 BrdUrd-positive cells/unit; P = 0.02; n = 4 control mice). Original magnification: A, Â100; B and C, Â400; D, Â100; E and F, Â400.

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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2007 American Association for Cancer Research. BMP Signaling at Epithelial Transition Zones with those that specify another epithelial phenotype. In support of underlying inflammatory infiltrate is strongly positive. In addition, this notion, we found that cellular differentiation in both gastric in the Mx1-Cre Z/EG reporter mice, we observe efficient transition zones is distinct from the surrounding epithelium. This recombination in the gastric epithelium but few GFP-positive cells is clearly seen at the squamocolumnar junction where differenti- in the mesenchyme of the stomach. This is a very interesting ation of epithelial cells in the cardiac glands is less complex than avenue of research, however, and we cannot exclude that Bmpr1a in the adjacent fundic glands. The conflicting signals perceived mutant cells in mesenchyme play a role in our model. Further by cells in a transition zone may not only influence cellular research will have to address this possibility. differentiation but also compromise the usually stringent controls In conclusion, our data show that the squamocolumnar and of the cell cycle and predispose these cells to oncogenesis. We have gastrointestinal junctional zones in mice are epithelial areas that indeed found some preliminary evidence that the regulation of the are distinct from the surrounding epithelium in both cellular cell cycle in the gastric epithelial transition zones may be distinct differentiation and cell cycle regulation. We find that these areas from the adjacent epithelium, but further experiments are needed show an enhanced propensity to oncogenesis that is suppressed by to support this concept. Bmpr1a signaling. It was recently proposed that the juvenile polyposis phenotype in Smad4 mutant mice results from loss of Smad4 in T cells and not in the epithelium (28). Because Smad4 is a common mediator Acknowledgments h for both BMP and TGF- signaling, this suggests that a similar Received 12/27/2006; revised 3/27/2007; accepted 6/7/2007. mechanism may exist for Bmpr1a mutations. We find little Grant support: VENI grant from the Netherlands Organization for Scientific evidence in the current study that tumor formation is mediated Research (NWO) to G. R. van den Brink. Bmp1a The costs of publication of this article were defrayed in part by the payment of page via loss of from mesenchymal cell types as we observe loss charges. This article must therefore be hereby marked advertisement in accordance of pSmad1,5,8 staining in the epithelium of polyps whereas the with 18 U.S.C. Section 1734 solely to indicate this fact.

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Sylvia A. Bleuming, Xi C. He, Liudmila L. Kodach, et al.

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