Volume 128, Issue 5, Pages 1292-1305 (May 2005) Evidence for Repatterning of the Gastric Fundic Epithelium Associated With Ménétrier’s Disease and TGFα Overexpression  Sachiyo Nomura, Stephen H. Settle, Charles M. Leys, Anna L. Means, Richard M. Peek, Steven D. Leach, Christopher V. Wright, Robert J. Coffey, James R. Goldenring  Gastroenterology  Volume 128, Issue 5, Pages 1292-1305 (May 2005) DOI: 10.1053/j.gastro.2005.03.019 Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 1 Expression of Pdx1 and TFF2 in the normal murine stomach in Pdx1lacZ/+ mice. Serial sections of stomach from Pdx1lacZ/+ mice were cut and dual stained for (left column) β-galactosidase activity and TFF2 immunoreactivity, (middle column) gastrin immunoreactivity with nuclear green counterstain, or (right column) with H&E. The upper panels show low-magnification views of the antrum and incisura with higher-power views of the sections from regions of the incisura (see arrowhead) or antrum (see arrow). Note that in the antral region the last gland expressing gastrin cells is visualized clearly but more proximal glands in the incisura without gastrin cells still are expressing Pdx1 (β-galactosidase-staining nuclei) as well as deep cells expressing TFF2. Magnification is indicated by the bars. Gastroenterology 2005 128, 1292-1305DOI: (10.1053/j.gastro.2005.03.019) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 2 Pdx1 immunostaining in normal C57BL/6 mice. Pdx1 and TFF2 immunostaining were performed in serial sections of gastric mucosa from either the antrum or the fundus. As noted with Pdx1lacZ/+ mice, Pdx1 protein was expressed in the nuclei of antral gland cells. Insets show dual immunohistochemical staining for Pdx1 with TFF2 (middle column) or gastrin (right hand column). Also, we similarly observed a region in the incisura (see arrowhead) where gastrin cells were absent, but glands still were Pdx1 immunoreactive with basally located TFF2-expressing cells. In contrast, no staining for Pdx1 or gastrin was observed in the proximal normal fundus, with staining with TFF2 of mucous neck cells in the midfundic glands. Magnification is indicated by the bars. Gastroenterology 2005 128, 1292-1305DOI: (10.1053/j.gastro.2005.03.019) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 3 Whole-mount assay of β-galactosidase activity in Pdx1lacZ/+/MT-TGFα mice show up-regulation of Pdx1 in response to oral zinc administration. Whole-mount visualization of β-galactosidase activity was performed by incubation of permeabilized stomachs with X-gal substrate. (A) Stomachs from Pdx1lacZ/+/MT-TGFα bigenic mice, MT-TGFα, and Pdx1lacZ/+ single-transgenic mice treated with zinc in drinking water for 2, 8, or 20 weeks were stained. MT-TGFα mice showed only nonspecific staining at the fundic-forestomach border. Pdx1lacZ/+ mice showed β-galactosidase staining sharply confined to the antrum. Pdx1lacZ/+/MT-TGFα mice showed increasing amounts of β-galactosidase staining, first in the distal fundus at 2 weeks of zinc treatment, and then throughout the stomach at greater than 8 weeks. The results are representative of 3 animals in each group. (B) Stomachs from Pdx1lacZ/+ mice treated with DMP777 for 7 or 14 days and from mice infected with H felis for 6 months. The β-galactosidase staining was confined to the antrum. Gastroenterology 2005 128, 1292-1305DOI: (10.1053/j.gastro.2005.03.019) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 4 Pdx1 expression assessed in Pdx1lacZ/+/MT-TGFα mice. Serial sections of fundic mucosa from Pdx1lacZ/+/MT-TGFα mice fed zinc for 2, 8, or 20 weeks were dual stained for β-galactosidase activity with TFF2 immunoreactivity or stained with H&E. After 2 weeks of zinc treatment, Pdx1 staining was observed in aberrant fundic glands usually with basally located TFF2-expressing mucous cells. By 8 weeks of zinc treatment, areas of gastritis cystica profunda (arrow) were evident that showed few TFF2-staining cells. By 20 weeks of zinc treatment large cystic dilatations (arrowhead) were evident in the fundus lined by Pdx1-expressing cells. Magnification is indicated by the bars. Gastroenterology 2005 128, 1292-1305DOI: (10.1053/j.gastro.2005.03.019) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 5 Immunostaining of Pdx1 expression in MT-TGFα mice. Serial sections of gastric fundus from mice fed zinc for 2, 8, or 20 weeks were immunostained for Pdx1, TFF2, or gastrin. Nuclear Pdx1 staining was observed in atrophic fundic glands beginning at 2 weeks. Although Pdx1 expressing glands often were associated with basally located TFF2-expressing mucous cells after 2 weeks of zinc treatment, this was less apparent after 8 weeks of zinc treatment, and most glands lacked TFF2-immunoreactive cells at 20 weeks. Scattered gastrin cells were observed in the fundus of 2- and 8-week treated mice (see insets), but gastrin cells seldom were observed after 20 weeks of zinc treatment. The positions of insets are marked with arrowheads. Magnification is indicated by the bars. Gastroenterology 2005 128, 1292-1305DOI: (10.1053/j.gastro.2005.03.019) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 6 Regulation of Pdx1 expression by TGFα in AGS gastric adenocarcinoma cells. (A) AGS cells were serum-starved overnight and then treated with TGFα for up to 8 hours. Gastrin (□) and Pdx1 (■) mRNA expression were assessed by quantitative PCR (n = 7; mean ± SEM). *P < .01 compared with untreated cells (time 0). (B) Pdx1 and gastrin expression were assessed by quantitative PCR in untreated AGS cells (□) as well as cells transfected for 4 hours with either Pdx1 (■) or scrambled control (▨) siRNA duplexes (mean ± SEM). GAPDH expression levels were similar in all groups (data not shown). *P < .01 compared with untreated cells. Gastroenterology 2005 128, 1292-1305DOI: (10.1053/j.gastro.2005.03.019) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 7 Pdx1 expression in the gastric mucosa of Ménétrier’s disease patients. Sections of gastric mucosa from Ménétrier’s disease patients were stained for Pdx1, TFF2, or gastrin immunoreactivity. In the antrum, a phenotypically normal distribution of Pdx1-expressing cells was observed throughout the antrum with basal TFF2-expressing mucous cells and midglandular gastrin cells. In the gastric fundus of Ménétrier’s patients, Pdx1-staining nuclei were observed throughout the mucosa along with basal TFF2-staining mucous cells. In one patient, gastrin cells were observed at the bases of aberrant fundic glands (gastrin +). In 2 patients, no gastrin cells were observed clearly. Magnification is indicated by the bars. Gastroenterology 2005 128, 1292-1305DOI: (10.1053/j.gastro.2005.03.019) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 8 Expression of Pdx1 mRNA in fundic biopsy specimens from Ménétrier’s disease patients. The expression of mRNAs for Pdx1, TFF2, and gastrin were evaluated in relation to GAPDH expression in fundic biopsy specimens from the greater curvature (gc) or lesser curvature (lc) of 3 Ménétrier’s patients in comparison with fundic and antral biopsy specimens from 3 normal controls. In the normal controls, Pdx1 and gastrin message were not detected in the normal fundus. In contrast, Pdx1 mRNA was detected in all of the Ménétrier’s disease patient fundic biopsy specimens with greater expression in LC biopsy specimens compared with the GC biopsy specimens. Gastrin mRNA was detected in 2 patients. Note the immunohistochemistry in Figure 6 showing gastrin staining is from patient 3, in whom prominent gastrin staining was observed in both GC and LC samples. Gastroenterology 2005 128, 1292-1305DOI: (10.1053/j.gastro.2005.03.019) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 9 Down-regulation of Pdx-1 expression in a Ménétrier’s disease patient after treatment with anti-EGF-receptor antibodies. Biopsy specimens were taken from the gastric fundus of a Ménétrier’s disease patient before and 1 month after treatment with Erbitux. (A) Paraffin sections of biopsy specimens were stained with antibodies against H/K-adenosine triphosphatase, Pdx1, and gastrin. Although staining for H/K-adenosine triphosphatase was absent before Erbitux treatment, proton pump staining, reflecting parietal cells, returned after 1 month of treatment. In contrast, although Pdx1 and gastrin-staining cells were observed before Erbitux treatment, they were reduced markedly after treatment. (B) Pdx1 mRNA expression was evaluated by quantitative PCR in biopsy samples taken from the lesser curvature (■) or greater curvature ( ) before and after treatment (n = 3; mean ± SEM). Pdx1 message levels were reduced significantly after treatment with anti-EGF-receptor antibodies at both sites (*P < .01). Gastrin mRNA levels were detected before treatment in fundic biopsy specimens, but were reduced to nondetectable levels after treatment (data not shown). Gastroenterology 2005 128, 1292-1305DOI: (10.1053/j.gastro.2005.03.019) Copyright © 2005 American Gastroenterological Association Terms and Conditions