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Conversion of gastric mucosa to intestinal metaplasia in Cdx2-expressing transgenic mice
BBRC Biochemical and Biophysical Research Communications 294 (2002) 470–479 www.academicpress.com
Conversion of gastric mucosa to intestinal metaplasia in Cdx2-expressing transgenic mice Hiroyuki Mutoh,a,* Yoji Hakamata,b Kiichi Sato,a Akashi Eda,a Ichiro Yanaka,a Sayaka Honda,a Hiroyuki Osawa,a Yoshinari Kaneko,a and Kentaro Suganoa a b
Department of Gastroenterology, Jichi Medical School, Yakushiji 3311-1, Minamikawachimachi, Kawachigun, Tochigi 329-0498, Japan Center for Molecular Medicine, Jichi Medical School, Yakushiji 3311-1, Minamikawachimachi, Kawachigun, Tochigi 329-0498, Japan Received 22 April 2002
Abstract Gastric intestinal metaplasia occurs as a pathological condition in the gastric mucosa. To clarify how an intestine-specific homeobox gene, Cdx2, affects the morphogenesis of gastric mucosa, we generated transgenic mice expressing Cdx2 in parietal cells. Until Day 18 after birth, the number of parietal cells inthegastric mucosa of transgenic mice was the same as for their normal littermates. However, at Day 19, we detected several glands in which parietal cells disappeared and the proliferating zone moved from the isthmus to the base of the glands. Thereafter, parietal cells decreased gradually and disappeared at Day 37. All of the gastric mucosal cells, except for enterochromaffin-like (ECL) cells, were completely replaced by intestinal metaplasia, consisting of goblet cells, enteroendocrine cells, and absorptive cells expressing alkaline phosphatase. Pseudopyloric gland metaplasia was also formed. The transgenic mouse is a very useful model for clarifying physiological differentiation of gastric and intestinal cell lineages and analyzing the molecular events from intestinal metaplasia to adenocarcinoma. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Intestinal metaplasia; Transcription factor; Cdx2; Transgenic mouse
Glandular stomach in the zymogenic zone contains mucus-producing pit and neck cells, Hþ -producing parietal cells, a variety of enteroendocrine cell subpopulations, and zymogenic (chief) cells that elaborate pepsinogen. In the normal pathway of cell renewal in gastric units of the zymogenic zone of adult mice, these cells are derived from stem cells located in the isthmus. On the other hand, intestinal metaplasia occurs as a pathological condition. Gastric adenocarcinoma of the intestinal type is assumed to arise in a stepwise fashion from chronic gastritis, atrophic gastritis, intestinal metaplasia, dysplasia, and carcinoma [1]. Intestinal metaplasia has been reported in Helicobactor pylori-infected humans and Mongolian gerbils, in rats receivingcarcinogen [2–6] or local X-ray irradiation treatment [7], and in mice after injection of xenogeneic stomach antigen [8]. However, little is known about the molec*
Corresponding author. Fax: +81-285-44-8297. E-mail address: [email protected] (H. Mutoh).
ular mechanisms in the progression from normal gastric epithelium to intestinal metaplasia. Normal stomach does not express transcription factors Cdx1 and Cdx2, however, intestinal metaplastic tissue in human stomach stainsintensely for Cdx1 [9]. We have also observed expression of Cdx1 and Cdx2 in human intestinal metaplastic mucosa [10], implicating their role in the development of intestinal metaplasia. Transcription factors Cdx1 and Cdx2 are members of the caudal-related homeobox family. In the mouse embryo, the expression of Cdx1 and Cdx2 is limited to the developing intestinal epithelium from day 14 and 12.5 days post-coitum, respectively [11,12] and remains limited to the small intestine and colon through adulthood [11,13,14]. In humans, expression of Cdx1 and Cdx2 genes is also limited to the intestine and colon in the adult [15,16]. Furthermore, functional studies have shown that Cdx2 is important in the regulation of intestinal gene transcription [14] and differentiation of intestinal cells [17].
0006-291X/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S 0 0 0 6 - 2 9 1 X ( 0 2 ) 0 0 4 8 0 - 1
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We reasoned that insight into themechanisms leading fromnormal gastric mucosa to intestinal metaplasia might be gained by expressing the intestine-specific transcription factor Cdx2 in the stomach. Administration of the antiherpetic drug ganciclovir to transgenic mice, in which herpes simplex virus 1 thymidine kinase (HSV-1-tk) was targeted to parietal cells, caused a rapid and specific ablation of parietal cells [18]. Parietal cell ablation also led to the loss of other gastric epithelial cells that were not expression sites of the HSV-1-tk suicide gene [18]. In this study, we used the Hþ =Kþ ATPase promoter to express Cdx2 specifically in the gastric mucosa and examined how the expression of Cdx2 in the parietal cells affects the normal morphogenesis of oxyntic glands.
Methods Generation of transgenic mice. Cdx2 cDNA was inserted into the EcoRI site of pBS/HKATPase, yielding pBS/HKATP/Cdx2. pBS/ HKATPase contains nucleotides )1417 to +15 of the rat Hþ =Kþ ATPase b-subunit gene in pBluescript II SK(+). TheHþ =Kþ -ATPase/ Cdx2 insert in pBS/HKATP/Cdx2 was released, purified, and then used for pronuclear injection of 500 C57BL/6 oocytes. Injected eggs were transferred to pseudopregnant Swiss Webster females using standard techniques [19]. Eighty live-born mice were screened for the presence of the Cdx2 transgene by a PCR method and three transgenic founders were identified. Three lines were established (pedigrees #17, #26, and #53). Histopathology. Stomach tissue specimens were fixed in neutral buffered 10% formalin for 12–24 h, washed in 70% ethanol, processed by standard methods, embedded in paraffin, sectioned at 5 lm, and stained with hematoxylin and eosin (H&E) (for histopathologic assessment) and Alcian blue at pH 2.5. For characterization of the mucus, Alcian blue (pH 2.5) with high-iron diamine (AB-HID) stain was applied. Immunohistochemistry. Five-micron thick sections were cut, deparaffinized, rehydrated in PBS, placed in 10 mmol/L citrate buffer (pH 6.0), and heated in an 850-W microwave for 10 min. Slides were preincubated with blocking buffer (Vector Laboratories) for 15 min at room temperature. Primary antisera were diluted in PBS and incubated overnight at 4 °C. Slides were then washed in PBS and incubated with Envision (DAKO). After developmentwith 3,30 -diaminobenzidine tetrahydrochloride (Vector Laboratories), slides were counterstained with hematoxylin and viewed under a light microscope. Our panel of primary antisera included: rabbit anti-pepsinogen C (1:100, Biodesign), mouse anti-Hþ =Kþ -ATPase (1:100, developed in our laboratory), anti-PCNA (1:2000, Sigama), anti-MUC5AC (1:30, Novocastra), anti-MUC6 (1:50, Kanto Chemical), anti-serotonin (1:100, Novocastra), anti-histidine decarboxylase (HDC) (1:2000, Progen), and anti-Cdx2 (1:100, BioGenex). Serum gastrin concentrations. Sera were obtained at the time of death and stored at )20 °C until gastrin determination. Serum gastrin was quantified in duplicate by radioimmunoassay [20]. Measurement of gastric acidity and acid secretion. Control and transgenic mice (7 wk old) were fasted overnight and then anesthetized with ether. After the abdominal wall was incised, the pylorus was ligated and the incision was sutured. The gastric fluid in the stomach was collected 1 h after the pylorus ligation. For maximal acid output, secretion was stimulated by injecting pentagastrin (250 lg=kg body wt), carbachol (60 lg=kg body wt), and histamine (0.1 mg/kg body wt)
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at the pylorus ligation. The gastric fluid secreted during 1 h was titrated with 0.1 N NaOH to pH 7.0 using a microtitrator.
Results A chronological analysis of the change from gastric mucosa to intestinal metaplasia To investigate whetherthe intestine-specific transcription factor Cdx2 can promote the development of intestinal metaplasia in the stomach, we generated transgenic mice with stomach-specific expression of Cdx2. The promoter of the noncatalytic b-subunit gene of rat Hþ =Kþ -ATPase was used to direct expression of Cdx2 in the parietal cell lineage. Cdx2 transgenic mice werefertile and were indistinguishable from their wildtype littermates in behavior, outward appearance, and weight. Until Day 18 after birth, the structure and number of parietal cells in thegastric mucosa of transgenic mice were the same as fortheir normal littermates (data not shown). However, staining with proliferating cell nuclear antigen (PCNA) indicates that the proliferative zone is located at the base of some glands of the gastric mucosa from Day 19 transgenic mice, while PCNA-positive cells of the normal gastric mucosa are located in the isthmus (Fig. 1A). Furthermore, an antibody for Hþ =Kþ -ATPase did not stain the PCNA-positive glands at all (Fig. 1B), while an antibody for Cdx2 stained the PCNA-positive glands (Fig. 1C). These results indicate that in some glands expressing Cdx2, the proliferating zone moved from the isthmus to the base of the glands. Thereafter, the number of parietal cells decreased and PCNA-positive intestinal type crypts increased very slowly until around Day 33 when dramatic changes were observed. By Day 37, most of the parietal cells haddisappeare (Fig. 1D), while almost all of the epithelial cells expressed Cdx2 (Fig. 1E). In the gastric mucosa of Day 244 transgenic mice, the parietal cells were completely depleted (Fig. 1F) and almost all of the epithelial cells expressed Cdx2 (Fig. 1G). To ascertain the functional consequence of parietal cell loss, we determined the pH of the stomach from transgenic mice at Day 37. The pH was 7:8 0:2, compared with 2:0 0:1 innormal mice. Plasma gastrin levels from the normal control mice averaged 245 31 pg/mL and those from transgenic mice averaged 1120 89 pg/mL. Consistent with hypergastrinemia, a marked increase of G cells secreting gastrin in the antrum was confirmed in both Day 37 and Day 244 transgenic mice, using antibody for gastrin (data not shown). To further clarify the acid secretion, we measured acid secretory capacity in control and transgenic mice at Day 40–60. Acid production was stimulated by pentagastrin (250 lg=kg body wt), histamine (0.1 mg/kg
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Fig. 1. PCNA staining (Day 19 (A)), Hþ =Kþ -ATPase staining (Day 19 (B), Day 37 (D), and Day 244 (B)), and Cdx2 staining (Day 19 (C), Day 37 (E), and Day 244 (G)) for the stomach specimen from transgenic mice. (A) The gland with PCNA-positive cells in the base appeared at Day 19 (indicated by arrow), while PCNA-positive cells of the normal gastric mucosa around the gland were located in the isthmus. (B) Antibody for Hþ =Kþ -ATPase did not stain the gland with PCNA-positive cells in the base (indicated by arrow). (C) Antibody for Cdx2 stained the gland with PCNA-positive cells in the base (indicated by arrow). (D) The Hþ =Kþ -ATPase-positive glandular regions were extremely rare in the zymogenic zones of Day 37 transgenic mice. (E) Antibody for Cdx2 stained almost all of the epithelial cells in the zymogenic zones of Day 37 transgenic mice. (F) In Day 244 transgenic mice, Hþ =Kþ -ATPase-positive parietal cells completely disappeared. (G) Antibody for Cdx2 stained almost all of the epithelial cells in the zymogenic zones of Day 244 transgenic mice.
body wt), or carbachol (60 lg=kg body wt) injection. Neither basal nor stimulated acid outputs could be detected in the transgenic mice, indicating the complete
loss of parietal cells in the gastric mucosa. In contrast, both basal and stimulated acid outputs could be detected in the normal mice.
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Characterizations of intestinal metaplasia Hematoxylin- and eosin-stained sections of the forestomach disclosed no histopathologic changes in transgenic animals. Normal gastric units located in the
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zymogenic zone (corpus) contain five epithelial lineages (foveolar, mucous neck, parietal, chief cells, and enteroendocrine cells) (Fig. 2A). These normal mucosal architectures of the zymogenic and mucoparietal zones were completely lost and replaced by intestinal meta-
Fig. 2. Fundic mucosa of the stomach of the normal (A) and transgenic mice at Day 37 (B and C) and Day 244 (D and E) (hematoxylin and eosin staining). (A) The normal mucosal architecture in the zymogenic zone (corpus) contains four epithelial lineages (foveolar, mucous neck, parietal, and chief cells). (B) Parietal and chief cells were completely lost at Day 37. The mucosa was completely replaced by intestinal metaplasia. The submucosa (bottom) is not affected. There are numerous goblet cells and absorptive cells with microvillous brush borders. Paneth cells are not seen in the intestinal metaplastic mucosa. (C) Magnified view of metaplastic gland with microvillous brush border at Day 37. (D) The pseudopyloric gland metaplasia is observed beneath the intestinal metaplastic mucosa at Day 244. Intestinal metaplastic glands show cystic dilatations with flattening of their lining epithelium. (E) High power view of metaplastic gland demonstrates brush border at Day 244.
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plasia in Day 37 transgenic mouse stomach (Fig. 2B). These metaplastic glands were composed of a large number of goblet cells (Fig. 2B) and columnar intestinal-type epithelial cells with microvilli (Fig. 2C). The stomachs prepared from Day 244 transgenic mice were also replaced by intestinal metaplastic mucosa (Fig. 2D). Pseudopyloric gland metaplasia was formed on the base of the intestinal metaplastic mucosa (Fig. 2D). Intestinal metaplastic glands showed cystic dilatations with flattening of their lining epithelium on Day 244 (Fig. 2D). However, Paneth cells were not seen in the intestinal metaplastic mucosa (Fig. 2B and D). The prominent brush (striated) border was observed on the surface of the intestinal-type cells by high power view of metaplastic glands (Fig. 2C and E). To clarify the character of the goblet cells, we stained the intestinal metaplastic mucosa with Alcian blue. No Alcian blue staining at pH 2.5 was observed in the gastric mucosa from normal mice (data not shown). In the gastric mucosa of both Day 37 and Day 244 transgenic mice, Alcian blue at pH 2.5 stained a number of goblet cells in the zymogenic zone (Fig. 3A and B). HID-Alcian blue staining at pH 2.5 showed that the metaplastic glands in the mucosa were composed of goblet cells secreting both sulfomucin (black) and sialomucin (blue) (Fig. 3C and D).
To further clarify the character and function of the intestinal metaplasia, the sections were stained for alkaline phosphatase (ALP). ALP activity was observed on the surface of the intestinal metaplastic cells (Fig. 4A and B). We also examined the appearance of intestine-specific enteroendocrine cells. Serotonin-expressing cells were not observed in the normal gastric oxyntic mucosa (data not shown). However, a number of serotonin-expressing enteroendocrine cells appeared in the metaplastic mucosa of transgenic mice at Day 37 and Day 244 (Fig. 4G and H). The density of serotonin-expressing enteroendocrine cells was similar to that of the normal intestine. Alteration of the gastric glandular epithelium The antrum of transgenic mice was normal except for a marked increase of gastrin-secreting G cells (data not shown), indicating that the Hþ =Kþ -ATPase promoter that we used was functional in the parietal cell lineage in the zymogenic zones. Cdx2 proteins were expressed only in the zymogenic zone, not in the antrum, as determined by staining with antibody for Cdx2 (Fig. 1E and G)). To examine whether the cells in the transgenic gastric fundus have gastric epithelial characteristics, we stained transgenic gastric specimens with several gastric muco-
Fig. 3. Alcian blue staining at pH 2.5 (A and B) and HID-Alcian blue staining at pH 2.5 (C and D). (A and B) The intestinal type epithelium contains numerous goblet cells stained blue by Alcian blue staining pH 2.5 (A: Day 37, B: Day 244). The deep compartment contains glands showing the features of pseudopyloric metaplasia at Day 244 (B). (C and D) The metaplastic glands in the mucosa are covered by intestinal metaplasia composed of sulfomucin (black) and sialomucin (blue) containing goblet cells (C: Day 37, D: Day 244).
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Fig. 4. Alkaline phosphatase activity (A: Day 37, B: Day 244) and serotonin (C: Day 37, D: Day 244). (A and B) Phase microscopy of a cytochemically reacted stomach demonstrates alkaline phosphatase activity by a red reaction product. (C and D) Serotonin-containing enteroendocrine cells were recognized in the intestinal metaplastic mucosa.
sal markers in addition to Hþ =Kþ -ATPase of the parietal cell lineage (Fig. 1B, D, and F). The antibody for gastric mucin, MUC5AC, which is characteristic of gastric foveolar cells, diffusely stained the normal gastric mucosa (Fig. 5A). However, we did not observe any obvious MUC5AC staining in the foveolar regions in the intestinal metaplastic mucosa (Fig. 5B and C). We then stained the gastric specimens of transgenic mice with antibody for MUC6 mucin, which is specific for both mucous neck and pyloric gland cells [21,22]. In the normal gastric mucosa, mucous neck cells were strongly stained (Fig. 5D) and pyloric gland was relatively weakly stained (data not shown). Strongly staining mucous neck cells were very rare in the gastric mucosa of Day 37 and Day 244 transgenic mice (Fig. 5E and F). However, the pseudopyloric gland cells in the base of the intestinal metaplastic mucosa of Day 37 and Day 244 transgenic mice were stained weakly compared to the strongly positive mucous neck cells (Fig. 5E and F). Pepsinogen C is expressed in the chief cells in normal
mouse stomach (Fig. 5G). However, pepsinogen C immunoreactivity was not detected in the transgenic mice (Fig. 5H and I). Finally, we stained the histamine-producing enterochromaffin-like (ECL) cells in the oxyntic mucosa using the antibody for HDC. The number of ECL cells in the normal oxyntic mucosa was small (Fig. 6A). However, a marked increase in ECL cell density was observed in the oxyntic mucosa of transgenic mice on Day 37 and Day 244 (Fig. 6B and C). These ECL cells were mainly located in the basal portion of the intestinal metaplasia.
Discussion We have established transgenic mice expressing Cdx2 in the gastric mucosa as a model for analyzing the relationship between Cdx2 protein expression and intestinal metaplastic change. Remarkably, expression of a single gene, Cdx2, completely changed gastric mucosa to
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Fig. 5. Immunohistochemical staining for MUC5AC (A, B, C), MUC6 (D, E, F), and pepsinogen C (G, H, I). (A) Foveolar-type gastric mucin (MUC5AC) is diffusely positive in the normal gastric foveolar cells. (B) In contrast, MUC5AC is rare in gastric mucosa of Day 37 transgenic mice. (C) In Day 244 transgenic mice, MUC5AC-positive foveolar cells completely disappeared. (D) Fundic mucosa of the normal stomach was stained with antibody specific for MUC6 mucin. MUC6-strongly positive mucous neck cells were distributed around the neck region of the fundic gland. (E) In the gastric specimens of Day 37 transgenic mice, the weak staining for MUC6 could be observed in the pseudopyloric gland cells located in the base of the intestinal metaplastic mucosa. (F) The pseudopyloric gland cells of Day 244 transgenic mice were stained slightly weakly in the base of gland of the intestinal metaplastic mucosa. MUC6 mucin is expressed extensively in the cytoplasmic portion of pseudopyloric glands. (G) Pepsinogen C is expressed in the chief cells in normal mouse stomach. (H and I) However, pepsinogen C was not stained in the transgenic mouse (H: Day 37, I: Day 244).
intestinal metaplasia, indicating that Cdx2 has an essential role as a transcription factor for intestinal differentiation. The mouse intestinal epithelium contains four principal terminally differentiated cell types: absorptive enterocyte, goblet, enteroendocrine, and Paneth cells. Three lineages (enterocyte-, goblet-, and enteroendocrine-like cells) were recognized in the intestinal metaplastic mucosa of the Cdx2 transgenic mice. Enterocytes, goblet, and enteroendocrine cells differentiate and mature during an ordinary migration that takes them from the crypt to the apex of the villus. Paneth cells differentiate during their descent to the base of the crypt. Both Cdx1 and Cdx2 have gradients of expression in the crypt-villus axis, with Cdx1 primarily in the crypt and Cdx2 primarily in the villus. In the transgenic mice, Cdx2 changed gastric mucosal cells to
enterocyte-, goblet-, and enteroendocrine-like cells, indicating that Cdx2 is an essential transcription factor for the differentiation of these three cell lineages. In heterozygous Cdx2-knockout mice in which the bacterial lacZ gene is under control of the Cdx2 promoter, a marked lacZ expression was recognized in goblet cells while Paneth cells in the base were not stained [23]. These results correspond with our observation in transgenic mice, in which Paneth cells were not recognized despite the presence of goblet cells in the intestinal metaplastic mucosa. Though Cdx2 expression induced goblet cell differentiation, other factors that are expressed in the crypt, such as Cdx1, would be needed for the formation of Paneth cells. Serotonin-producing enteroendocrine cells were also expressed in the intestinal metaplastic mucosa. We have reported that secretin- and cholecystokinin-expressing
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Fig. 6. Immunostaining for HDC in the gastric fundic mucosa. (A) Antibody for HDC stained a small number of ECL cells in gastric fundic mucosa of normal mice. (B: Day 37 and C: Day 244) Diffuse ECL cell hyperplasia was recognized in the basal portion of the intestinal metaplasia.
enteroendocrine cells expressed the transcription factor BETA2 (NeuroD) which is essential for the transcription of these two genes [24,25]. Serotonin-producing cells also express BETA2 [25]. In the intestinal metaplastic mucosa, Cdx2 might induce not only intestine-specific genes such as sucrase–isomaltase, but also other transcription factors, such as BETA2, which may themselves induce expression of enteroendocrine-specific genes. Heterozygous Cdx2 knockout mice developed multiple intestinal polyp-like lesions [26,27]. The polyps did not express Cdx2 and contained areas of squamous metaplasia in the form of keratinizing stratified squamous epithelium, similar to that occurring in the mouse esophagus and forestomach, gastric cardia containing columnar mucus-secreting cells, and gastric corpus
containing clearly identifiable parietal cells [27]. Cdx2 transforms gastric epithelial cells into intestinal type cells in our Cdx2 transgenic mice, while intestinal polyplike lesions that did not express Cdx2 in Cdx2 knockout mice contained gastric epithelial cells [27]. These findings suggest that Cdx2 may play a fundamental role in generating intestinal mucosa during development in vivo. Foveolar, mucous neck, parietal, and chief cells were completely replaced by intestinal metaplastic mucosa in Cdx2-transgenic mice. However, ECL cells increased in the intestinal metaplastic mucosa. The increase in the number of ECL cells in transgenic mice indicated that the origin of ECL cells is different from that of other gastric mucosal cells because the other gastric type cells were completely lost in transgenic mice. In humans,
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hyperplasia distributed as micronodular nests composed of ECL cells is thought to be related to chronic gastritis with mucosal atrophy and intestinal metaplasia. In Cdx2-transgenic mice, the expanded ECL cells are located in the basal portion of the glands in the area with intestinal metaplasia. These observations indicate that intestinal metaplastic change affects gastric mucosal cells except for ECL cells. In humans, chronic gastritis commonly leads to loss of parietal and chief cells, a diminution of the gastric glands, and thus atrophy or thinning of the mucosa. In addition, chronic atrophic gastritis often leads to the development of intestinal-type metaplasia. Pseudopyloric metaplasia is also a common feature of atrophic gastritis. Goblet cells, Paneth cells, intestinal types of endocrine cells, and tall columnar cells with prominent microvilli may all be present in the intestinal metaplasia. In this study, Cdx2 transgenic mice showed a histopathology that bears close resemblance to intestinal-type metaplasia and pseudopyloric gland metaplasia in humans. Our results suggest that Cdx2 may have a regulatory role in the development of intestinal metaplasia and pseudopyloric gland metaplasia. Moreover, we have shown the expression of Cdx2 may be the early event triggering the development of intestinal metaplasia of human gastric mucosa [10]. In conclusion, the Cdx2 transgenic mouse is a good model for investigating the differentiation of gastric and intestinal mucosal cell lineages. It also represents an extremely useful model for both analyzing the molecular events from intestinal metaplasia to adenocarcinoma and generating mucosa with an absorptive and enzymatic function for the development of molecular therapies.
Acknowledgments We are grateful to Dr. T. Takeuchi (Gunma University) for providing the pBS/HKATPase plasmid, Dr. K. Miyamoto (Tokushima University) for antibodies for sucrase and PepT1, and P. Traber (University of Pennsylvania) for the mouse Cdx2 expression vector pRc/CMV-Cdx-2. The expert technical assistance of Ms. M. Nozawa, S. Terauchi, and K. Sasaki is much appreciated.
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Conversion of gastric mucosa to intestinal metaplasia in Cdx2-expressing transgenic mice Hiroyuki Mutoh,a,* Yoji Hakamata,b Kiichi Sato,a Akashi Eda,a Ichiro Yanaka,a Sayaka Honda,a Hiroyuki Osawa,a Yoshinari Kaneko,a and Kentaro Suganoa a b
Department of Gastroenterology, Jichi Medical School, Yakushiji 3311-1, Minamikawachimachi, Kawachigun, Tochigi 329-0498, Japan Center for Molecular Medicine, Jichi Medical School, Yakushiji 3311-1, Minamikawachimachi, Kawachigun, Tochigi 329-0498, Japan Received 22 April 2002
Abstract Gastric intestinal metaplasia occurs as a pathological condition in the gastric mucosa. To clarify how an intestine-specific homeobox gene, Cdx2, affects the morphogenesis of gastric mucosa, we generated transgenic mice expressing Cdx2 in parietal cells. Until Day 18 after birth, the number of parietal cells inthegastric mucosa of transgenic mice was the same as for their normal littermates. However, at Day 19, we detected several glands in which parietal cells disappeared and the proliferating zone moved from the isthmus to the base of the glands. Thereafter, parietal cells decreased gradually and disappeared at Day 37. All of the gastric mucosal cells, except for enterochromaffin-like (ECL) cells, were completely replaced by intestinal metaplasia, consisting of goblet cells, enteroendocrine cells, and absorptive cells expressing alkaline phosphatase. Pseudopyloric gland metaplasia was also formed. The transgenic mouse is a very useful model for clarifying physiological differentiation of gastric and intestinal cell lineages and analyzing the molecular events from intestinal metaplasia to adenocarcinoma. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Intestinal metaplasia; Transcription factor; Cdx2; Transgenic mouse
Glandular stomach in the zymogenic zone contains mucus-producing pit and neck cells, Hþ -producing parietal cells, a variety of enteroendocrine cell subpopulations, and zymogenic (chief) cells that elaborate pepsinogen. In the normal pathway of cell renewal in gastric units of the zymogenic zone of adult mice, these cells are derived from stem cells located in the isthmus. On the other hand, intestinal metaplasia occurs as a pathological condition. Gastric adenocarcinoma of the intestinal type is assumed to arise in a stepwise fashion from chronic gastritis, atrophic gastritis, intestinal metaplasia, dysplasia, and carcinoma [1]. Intestinal metaplasia has been reported in Helicobactor pylori-infected humans and Mongolian gerbils, in rats receivingcarcinogen [2–6] or local X-ray irradiation treatment [7], and in mice after injection of xenogeneic stomach antigen [8]. However, little is known about the molec*
Corresponding author. Fax: +81-285-44-8297. E-mail address: [email protected] (H. Mutoh).
ular mechanisms in the progression from normal gastric epithelium to intestinal metaplasia. Normal stomach does not express transcription factors Cdx1 and Cdx2, however, intestinal metaplastic tissue in human stomach stainsintensely for Cdx1 [9]. We have also observed expression of Cdx1 and Cdx2 in human intestinal metaplastic mucosa [10], implicating their role in the development of intestinal metaplasia. Transcription factors Cdx1 and Cdx2 are members of the caudal-related homeobox family. In the mouse embryo, the expression of Cdx1 and Cdx2 is limited to the developing intestinal epithelium from day 14 and 12.5 days post-coitum, respectively [11,12] and remains limited to the small intestine and colon through adulthood [11,13,14]. In humans, expression of Cdx1 and Cdx2 genes is also limited to the intestine and colon in the adult [15,16]. Furthermore, functional studies have shown that Cdx2 is important in the regulation of intestinal gene transcription [14] and differentiation of intestinal cells [17].
0006-291X/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S 0 0 0 6 - 2 9 1 X ( 0 2 ) 0 0 4 8 0 - 1
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We reasoned that insight into themechanisms leading fromnormal gastric mucosa to intestinal metaplasia might be gained by expressing the intestine-specific transcription factor Cdx2 in the stomach. Administration of the antiherpetic drug ganciclovir to transgenic mice, in which herpes simplex virus 1 thymidine kinase (HSV-1-tk) was targeted to parietal cells, caused a rapid and specific ablation of parietal cells [18]. Parietal cell ablation also led to the loss of other gastric epithelial cells that were not expression sites of the HSV-1-tk suicide gene [18]. In this study, we used the Hþ =Kþ ATPase promoter to express Cdx2 specifically in the gastric mucosa and examined how the expression of Cdx2 in the parietal cells affects the normal morphogenesis of oxyntic glands.
Methods Generation of transgenic mice. Cdx2 cDNA was inserted into the EcoRI site of pBS/HKATPase, yielding pBS/HKATP/Cdx2. pBS/ HKATPase contains nucleotides )1417 to +15 of the rat Hþ =Kþ ATPase b-subunit gene in pBluescript II SK(+). TheHþ =Kþ -ATPase/ Cdx2 insert in pBS/HKATP/Cdx2 was released, purified, and then used for pronuclear injection of 500 C57BL/6 oocytes. Injected eggs were transferred to pseudopregnant Swiss Webster females using standard techniques [19]. Eighty live-born mice were screened for the presence of the Cdx2 transgene by a PCR method and three transgenic founders were identified. Three lines were established (pedigrees #17, #26, and #53). Histopathology. Stomach tissue specimens were fixed in neutral buffered 10% formalin for 12–24 h, washed in 70% ethanol, processed by standard methods, embedded in paraffin, sectioned at 5 lm, and stained with hematoxylin and eosin (H&E) (for histopathologic assessment) and Alcian blue at pH 2.5. For characterization of the mucus, Alcian blue (pH 2.5) with high-iron diamine (AB-HID) stain was applied. Immunohistochemistry. Five-micron thick sections were cut, deparaffinized, rehydrated in PBS, placed in 10 mmol/L citrate buffer (pH 6.0), and heated in an 850-W microwave for 10 min. Slides were preincubated with blocking buffer (Vector Laboratories) for 15 min at room temperature. Primary antisera were diluted in PBS and incubated overnight at 4 °C. Slides were then washed in PBS and incubated with Envision (DAKO). After developmentwith 3,30 -diaminobenzidine tetrahydrochloride (Vector Laboratories), slides were counterstained with hematoxylin and viewed under a light microscope. Our panel of primary antisera included: rabbit anti-pepsinogen C (1:100, Biodesign), mouse anti-Hþ =Kþ -ATPase (1:100, developed in our laboratory), anti-PCNA (1:2000, Sigama), anti-MUC5AC (1:30, Novocastra), anti-MUC6 (1:50, Kanto Chemical), anti-serotonin (1:100, Novocastra), anti-histidine decarboxylase (HDC) (1:2000, Progen), and anti-Cdx2 (1:100, BioGenex). Serum gastrin concentrations. Sera were obtained at the time of death and stored at )20 °C until gastrin determination. Serum gastrin was quantified in duplicate by radioimmunoassay [20]. Measurement of gastric acidity and acid secretion. Control and transgenic mice (7 wk old) were fasted overnight and then anesthetized with ether. After the abdominal wall was incised, the pylorus was ligated and the incision was sutured. The gastric fluid in the stomach was collected 1 h after the pylorus ligation. For maximal acid output, secretion was stimulated by injecting pentagastrin (250 lg=kg body wt), carbachol (60 lg=kg body wt), and histamine (0.1 mg/kg body wt)
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at the pylorus ligation. The gastric fluid secreted during 1 h was titrated with 0.1 N NaOH to pH 7.0 using a microtitrator.
Results A chronological analysis of the change from gastric mucosa to intestinal metaplasia To investigate whetherthe intestine-specific transcription factor Cdx2 can promote the development of intestinal metaplasia in the stomach, we generated transgenic mice with stomach-specific expression of Cdx2. The promoter of the noncatalytic b-subunit gene of rat Hþ =Kþ -ATPase was used to direct expression of Cdx2 in the parietal cell lineage. Cdx2 transgenic mice werefertile and were indistinguishable from their wildtype littermates in behavior, outward appearance, and weight. Until Day 18 after birth, the structure and number of parietal cells in thegastric mucosa of transgenic mice were the same as fortheir normal littermates (data not shown). However, staining with proliferating cell nuclear antigen (PCNA) indicates that the proliferative zone is located at the base of some glands of the gastric mucosa from Day 19 transgenic mice, while PCNA-positive cells of the normal gastric mucosa are located in the isthmus (Fig. 1A). Furthermore, an antibody for Hþ =Kþ -ATPase did not stain the PCNA-positive glands at all (Fig. 1B), while an antibody for Cdx2 stained the PCNA-positive glands (Fig. 1C). These results indicate that in some glands expressing Cdx2, the proliferating zone moved from the isthmus to the base of the glands. Thereafter, the number of parietal cells decreased and PCNA-positive intestinal type crypts increased very slowly until around Day 33 when dramatic changes were observed. By Day 37, most of the parietal cells haddisappeare (Fig. 1D), while almost all of the epithelial cells expressed Cdx2 (Fig. 1E). In the gastric mucosa of Day 244 transgenic mice, the parietal cells were completely depleted (Fig. 1F) and almost all of the epithelial cells expressed Cdx2 (Fig. 1G). To ascertain the functional consequence of parietal cell loss, we determined the pH of the stomach from transgenic mice at Day 37. The pH was 7:8 0:2, compared with 2:0 0:1 innormal mice. Plasma gastrin levels from the normal control mice averaged 245 31 pg/mL and those from transgenic mice averaged 1120 89 pg/mL. Consistent with hypergastrinemia, a marked increase of G cells secreting gastrin in the antrum was confirmed in both Day 37 and Day 244 transgenic mice, using antibody for gastrin (data not shown). To further clarify the acid secretion, we measured acid secretory capacity in control and transgenic mice at Day 40–60. Acid production was stimulated by pentagastrin (250 lg=kg body wt), histamine (0.1 mg/kg
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Fig. 1. PCNA staining (Day 19 (A)), Hþ =Kþ -ATPase staining (Day 19 (B), Day 37 (D), and Day 244 (B)), and Cdx2 staining (Day 19 (C), Day 37 (E), and Day 244 (G)) for the stomach specimen from transgenic mice. (A) The gland with PCNA-positive cells in the base appeared at Day 19 (indicated by arrow), while PCNA-positive cells of the normal gastric mucosa around the gland were located in the isthmus. (B) Antibody for Hþ =Kþ -ATPase did not stain the gland with PCNA-positive cells in the base (indicated by arrow). (C) Antibody for Cdx2 stained the gland with PCNA-positive cells in the base (indicated by arrow). (D) The Hþ =Kþ -ATPase-positive glandular regions were extremely rare in the zymogenic zones of Day 37 transgenic mice. (E) Antibody for Cdx2 stained almost all of the epithelial cells in the zymogenic zones of Day 37 transgenic mice. (F) In Day 244 transgenic mice, Hþ =Kþ -ATPase-positive parietal cells completely disappeared. (G) Antibody for Cdx2 stained almost all of the epithelial cells in the zymogenic zones of Day 244 transgenic mice.
body wt), or carbachol (60 lg=kg body wt) injection. Neither basal nor stimulated acid outputs could be detected in the transgenic mice, indicating the complete
loss of parietal cells in the gastric mucosa. In contrast, both basal and stimulated acid outputs could be detected in the normal mice.
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Characterizations of intestinal metaplasia Hematoxylin- and eosin-stained sections of the forestomach disclosed no histopathologic changes in transgenic animals. Normal gastric units located in the
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zymogenic zone (corpus) contain five epithelial lineages (foveolar, mucous neck, parietal, chief cells, and enteroendocrine cells) (Fig. 2A). These normal mucosal architectures of the zymogenic and mucoparietal zones were completely lost and replaced by intestinal meta-
Fig. 2. Fundic mucosa of the stomach of the normal (A) and transgenic mice at Day 37 (B and C) and Day 244 (D and E) (hematoxylin and eosin staining). (A) The normal mucosal architecture in the zymogenic zone (corpus) contains four epithelial lineages (foveolar, mucous neck, parietal, and chief cells). (B) Parietal and chief cells were completely lost at Day 37. The mucosa was completely replaced by intestinal metaplasia. The submucosa (bottom) is not affected. There are numerous goblet cells and absorptive cells with microvillous brush borders. Paneth cells are not seen in the intestinal metaplastic mucosa. (C) Magnified view of metaplastic gland with microvillous brush border at Day 37. (D) The pseudopyloric gland metaplasia is observed beneath the intestinal metaplastic mucosa at Day 244. Intestinal metaplastic glands show cystic dilatations with flattening of their lining epithelium. (E) High power view of metaplastic gland demonstrates brush border at Day 244.
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plasia in Day 37 transgenic mouse stomach (Fig. 2B). These metaplastic glands were composed of a large number of goblet cells (Fig. 2B) and columnar intestinal-type epithelial cells with microvilli (Fig. 2C). The stomachs prepared from Day 244 transgenic mice were also replaced by intestinal metaplastic mucosa (Fig. 2D). Pseudopyloric gland metaplasia was formed on the base of the intestinal metaplastic mucosa (Fig. 2D). Intestinal metaplastic glands showed cystic dilatations with flattening of their lining epithelium on Day 244 (Fig. 2D). However, Paneth cells were not seen in the intestinal metaplastic mucosa (Fig. 2B and D). The prominent brush (striated) border was observed on the surface of the intestinal-type cells by high power view of metaplastic glands (Fig. 2C and E). To clarify the character of the goblet cells, we stained the intestinal metaplastic mucosa with Alcian blue. No Alcian blue staining at pH 2.5 was observed in the gastric mucosa from normal mice (data not shown). In the gastric mucosa of both Day 37 and Day 244 transgenic mice, Alcian blue at pH 2.5 stained a number of goblet cells in the zymogenic zone (Fig. 3A and B). HID-Alcian blue staining at pH 2.5 showed that the metaplastic glands in the mucosa were composed of goblet cells secreting both sulfomucin (black) and sialomucin (blue) (Fig. 3C and D).
To further clarify the character and function of the intestinal metaplasia, the sections were stained for alkaline phosphatase (ALP). ALP activity was observed on the surface of the intestinal metaplastic cells (Fig. 4A and B). We also examined the appearance of intestine-specific enteroendocrine cells. Serotonin-expressing cells were not observed in the normal gastric oxyntic mucosa (data not shown). However, a number of serotonin-expressing enteroendocrine cells appeared in the metaplastic mucosa of transgenic mice at Day 37 and Day 244 (Fig. 4G and H). The density of serotonin-expressing enteroendocrine cells was similar to that of the normal intestine. Alteration of the gastric glandular epithelium The antrum of transgenic mice was normal except for a marked increase of gastrin-secreting G cells (data not shown), indicating that the Hþ =Kþ -ATPase promoter that we used was functional in the parietal cell lineage in the zymogenic zones. Cdx2 proteins were expressed only in the zymogenic zone, not in the antrum, as determined by staining with antibody for Cdx2 (Fig. 1E and G)). To examine whether the cells in the transgenic gastric fundus have gastric epithelial characteristics, we stained transgenic gastric specimens with several gastric muco-
Fig. 3. Alcian blue staining at pH 2.5 (A and B) and HID-Alcian blue staining at pH 2.5 (C and D). (A and B) The intestinal type epithelium contains numerous goblet cells stained blue by Alcian blue staining pH 2.5 (A: Day 37, B: Day 244). The deep compartment contains glands showing the features of pseudopyloric metaplasia at Day 244 (B). (C and D) The metaplastic glands in the mucosa are covered by intestinal metaplasia composed of sulfomucin (black) and sialomucin (blue) containing goblet cells (C: Day 37, D: Day 244).
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Fig. 4. Alkaline phosphatase activity (A: Day 37, B: Day 244) and serotonin (C: Day 37, D: Day 244). (A and B) Phase microscopy of a cytochemically reacted stomach demonstrates alkaline phosphatase activity by a red reaction product. (C and D) Serotonin-containing enteroendocrine cells were recognized in the intestinal metaplastic mucosa.
sal markers in addition to Hþ =Kþ -ATPase of the parietal cell lineage (Fig. 1B, D, and F). The antibody for gastric mucin, MUC5AC, which is characteristic of gastric foveolar cells, diffusely stained the normal gastric mucosa (Fig. 5A). However, we did not observe any obvious MUC5AC staining in the foveolar regions in the intestinal metaplastic mucosa (Fig. 5B and C). We then stained the gastric specimens of transgenic mice with antibody for MUC6 mucin, which is specific for both mucous neck and pyloric gland cells [21,22]. In the normal gastric mucosa, mucous neck cells were strongly stained (Fig. 5D) and pyloric gland was relatively weakly stained (data not shown). Strongly staining mucous neck cells were very rare in the gastric mucosa of Day 37 and Day 244 transgenic mice (Fig. 5E and F). However, the pseudopyloric gland cells in the base of the intestinal metaplastic mucosa of Day 37 and Day 244 transgenic mice were stained weakly compared to the strongly positive mucous neck cells (Fig. 5E and F). Pepsinogen C is expressed in the chief cells in normal
mouse stomach (Fig. 5G). However, pepsinogen C immunoreactivity was not detected in the transgenic mice (Fig. 5H and I). Finally, we stained the histamine-producing enterochromaffin-like (ECL) cells in the oxyntic mucosa using the antibody for HDC. The number of ECL cells in the normal oxyntic mucosa was small (Fig. 6A). However, a marked increase in ECL cell density was observed in the oxyntic mucosa of transgenic mice on Day 37 and Day 244 (Fig. 6B and C). These ECL cells were mainly located in the basal portion of the intestinal metaplasia.
Discussion We have established transgenic mice expressing Cdx2 in the gastric mucosa as a model for analyzing the relationship between Cdx2 protein expression and intestinal metaplastic change. Remarkably, expression of a single gene, Cdx2, completely changed gastric mucosa to
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Fig. 5. Immunohistochemical staining for MUC5AC (A, B, C), MUC6 (D, E, F), and pepsinogen C (G, H, I). (A) Foveolar-type gastric mucin (MUC5AC) is diffusely positive in the normal gastric foveolar cells. (B) In contrast, MUC5AC is rare in gastric mucosa of Day 37 transgenic mice. (C) In Day 244 transgenic mice, MUC5AC-positive foveolar cells completely disappeared. (D) Fundic mucosa of the normal stomach was stained with antibody specific for MUC6 mucin. MUC6-strongly positive mucous neck cells were distributed around the neck region of the fundic gland. (E) In the gastric specimens of Day 37 transgenic mice, the weak staining for MUC6 could be observed in the pseudopyloric gland cells located in the base of the intestinal metaplastic mucosa. (F) The pseudopyloric gland cells of Day 244 transgenic mice were stained slightly weakly in the base of gland of the intestinal metaplastic mucosa. MUC6 mucin is expressed extensively in the cytoplasmic portion of pseudopyloric glands. (G) Pepsinogen C is expressed in the chief cells in normal mouse stomach. (H and I) However, pepsinogen C was not stained in the transgenic mouse (H: Day 37, I: Day 244).
intestinal metaplasia, indicating that Cdx2 has an essential role as a transcription factor for intestinal differentiation. The mouse intestinal epithelium contains four principal terminally differentiated cell types: absorptive enterocyte, goblet, enteroendocrine, and Paneth cells. Three lineages (enterocyte-, goblet-, and enteroendocrine-like cells) were recognized in the intestinal metaplastic mucosa of the Cdx2 transgenic mice. Enterocytes, goblet, and enteroendocrine cells differentiate and mature during an ordinary migration that takes them from the crypt to the apex of the villus. Paneth cells differentiate during their descent to the base of the crypt. Both Cdx1 and Cdx2 have gradients of expression in the crypt-villus axis, with Cdx1 primarily in the crypt and Cdx2 primarily in the villus. In the transgenic mice, Cdx2 changed gastric mucosal cells to
enterocyte-, goblet-, and enteroendocrine-like cells, indicating that Cdx2 is an essential transcription factor for the differentiation of these three cell lineages. In heterozygous Cdx2-knockout mice in which the bacterial lacZ gene is under control of the Cdx2 promoter, a marked lacZ expression was recognized in goblet cells while Paneth cells in the base were not stained [23]. These results correspond with our observation in transgenic mice, in which Paneth cells were not recognized despite the presence of goblet cells in the intestinal metaplastic mucosa. Though Cdx2 expression induced goblet cell differentiation, other factors that are expressed in the crypt, such as Cdx1, would be needed for the formation of Paneth cells. Serotonin-producing enteroendocrine cells were also expressed in the intestinal metaplastic mucosa. We have reported that secretin- and cholecystokinin-expressing
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Fig. 6. Immunostaining for HDC in the gastric fundic mucosa. (A) Antibody for HDC stained a small number of ECL cells in gastric fundic mucosa of normal mice. (B: Day 37 and C: Day 244) Diffuse ECL cell hyperplasia was recognized in the basal portion of the intestinal metaplasia.
enteroendocrine cells expressed the transcription factor BETA2 (NeuroD) which is essential for the transcription of these two genes [24,25]. Serotonin-producing cells also express BETA2 [25]. In the intestinal metaplastic mucosa, Cdx2 might induce not only intestine-specific genes such as sucrase–isomaltase, but also other transcription factors, such as BETA2, which may themselves induce expression of enteroendocrine-specific genes. Heterozygous Cdx2 knockout mice developed multiple intestinal polyp-like lesions [26,27]. The polyps did not express Cdx2 and contained areas of squamous metaplasia in the form of keratinizing stratified squamous epithelium, similar to that occurring in the mouse esophagus and forestomach, gastric cardia containing columnar mucus-secreting cells, and gastric corpus
containing clearly identifiable parietal cells [27]. Cdx2 transforms gastric epithelial cells into intestinal type cells in our Cdx2 transgenic mice, while intestinal polyplike lesions that did not express Cdx2 in Cdx2 knockout mice contained gastric epithelial cells [27]. These findings suggest that Cdx2 may play a fundamental role in generating intestinal mucosa during development in vivo. Foveolar, mucous neck, parietal, and chief cells were completely replaced by intestinal metaplastic mucosa in Cdx2-transgenic mice. However, ECL cells increased in the intestinal metaplastic mucosa. The increase in the number of ECL cells in transgenic mice indicated that the origin of ECL cells is different from that of other gastric mucosal cells because the other gastric type cells were completely lost in transgenic mice. In humans,
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hyperplasia distributed as micronodular nests composed of ECL cells is thought to be related to chronic gastritis with mucosal atrophy and intestinal metaplasia. In Cdx2-transgenic mice, the expanded ECL cells are located in the basal portion of the glands in the area with intestinal metaplasia. These observations indicate that intestinal metaplastic change affects gastric mucosal cells except for ECL cells. In humans, chronic gastritis commonly leads to loss of parietal and chief cells, a diminution of the gastric glands, and thus atrophy or thinning of the mucosa. In addition, chronic atrophic gastritis often leads to the development of intestinal-type metaplasia. Pseudopyloric metaplasia is also a common feature of atrophic gastritis. Goblet cells, Paneth cells, intestinal types of endocrine cells, and tall columnar cells with prominent microvilli may all be present in the intestinal metaplasia. In this study, Cdx2 transgenic mice showed a histopathology that bears close resemblance to intestinal-type metaplasia and pseudopyloric gland metaplasia in humans. Our results suggest that Cdx2 may have a regulatory role in the development of intestinal metaplasia and pseudopyloric gland metaplasia. Moreover, we have shown the expression of Cdx2 may be the early event triggering the development of intestinal metaplasia of human gastric mucosa [10]. In conclusion, the Cdx2 transgenic mouse is a good model for investigating the differentiation of gastric and intestinal mucosal cell lineages. It also represents an extremely useful model for both analyzing the molecular events from intestinal metaplasia to adenocarcinoma and generating mucosa with an absorptive and enzymatic function for the development of molecular therapies.
Acknowledgments We are grateful to Dr. T. Takeuchi (Gunma University) for providing the pBS/HKATPase plasmid, Dr. K. Miyamoto (Tokushima University) for antibodies for sucrase and PepT1, and P. Traber (University of Pennsylvania) for the mouse Cdx2 expression vector pRc/CMV-Cdx-2. The expert technical assistance of Ms. M. Nozawa, S. Terauchi, and K. Sasaki is much appreciated.
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