Loss of parietal cell expression of Sonic hedgehog induces hypergastrinemia and hyperproliferation of surface mucous cells

Abstract

Background & aims: Sonic Hedgehog (Shh) is expressed in the adult stomach, but its role as a gastric morphogen is unclear. We sought to identify mechanisms by which Shh might regulate gastric epithelial cell function and differentiation.

Methods: Mice with a parietal cell-specific deletion of Shh (HKCre/Shh(KO)) were created. Gastric morphology and function were studied in control and HKCre/Shh(KO) mice between 1 and 8 months of age.

Results: In contrast to control mice, HKCre/Shh(KO) mice developed gastric hypochlorhydria, hypergastrinemia, and a phenotype that resembled foveolar hyperplasia. The fundic mucosa of HKCre/Shh(KO) mice had an expanded surface pit cell lineage that was documented by increased incorporation of bromodeoxyuridine and was attributed to the hypergastrinemia. Compared with controls, numbers of total mucous neck and zymogen cells were significantly decreased in stomachs of HKCre/Shh(KO) mice. In addition, zymogen and neck cell markers were coexpressed in the same cell populations, indicating disrupted differentiation of the zymogen cell lineage from the mucous neck cells in the stomachs of HKCre/Shh(KO) mice. Laser capture microdissection of the surface epithelium, followed by quantitative reverse-transcription polymerase chain reaction, revealed a significant increase in expression of Indian Hedgehog, glioma-associated oncogene homolog 1, Wnt, and cyclin D1. Laser capture microdissection analysis also showed a significant increase in Snail with a concomitant decrease in E-cadherin.

Conclusions: In the stomachs of adult mice, loss of Shh from parietal cells results in hypochlorhydria and hypergastrinemia. Hypergastrinemia might subsequently induce increased Hedgehog and Wnt signaling in the surface pit epithelium, resulting in hyperproliferation.

Figure 1

Expression pattern of Shh and cell lineage markers in the gastric mucosa. (A) Average fold change in gene expression of MUC5AC, Atp4α, MUC6, PgC, Shh, and Ihh in RNA collected from the neck region of both control and HKCre/Shh^KO mouse stomachs. Immunofluorescence of (B) control and (C) HKCre/Shh^KO mouse groups stained for Shh (red) together with H⁺,K⁺-ATPase (green). Merged images are shown (inset). Colocalization is indicated in yellow. Representative of n = 8. Original magnification 40×. (D) Average fold change in gene expression of MUC5AC, Atp4α, MUC6, PgC, Shh, and Ihh in RNA collected from the pit region of both control and HKCre/Shh^KO mouse stomachs. Immunofluorescence of (E) control and (F) HKCre/Shh^KO mouse groups stained for Shh (red) together with UEAI (green). Merged images are shown (inset). Colocalization is indicated in yellow. Representative of n = 8. Data are presented as the mean ± SEM where *P < .05 compared with either MUC5AC, Atp4α, MUC6, or PgC gene expression; #P < .05 compared with the control group; n = 3 mice in each group.

Figure 2

Histologic evaluation of control and HKCre/Shh^KO mice. H&E staining of gastric tissue collected from 8-month-old (A) control and (B) HKCre/Shh^KO mice (original magnification 10×). Sections from (C) control and (D) HKCre/Shh^KO mice were stained with periodic acid–Schiff/Alcian blue. Original magnification 10×. Gastric sections collected from (E) control or (F) HKCre/Shh^KO groups were graded on the level of foveolar hyperplasia as detailed in Materials and Methods. Histologic score for tissues collected from 1-, 2-, 3-, 4-, and 8-month-old mice were recorded. *P < .05 compared with control, n = 8 mice per group.

Figure 3

Quantification of parietal, mucous neck, and zymogen cells in control and HKCre/Shh^KO mice. (A) The number of parietal cells (H⁺,K⁺-ATPase β subunit), mucous neck cells (GSII), zymogen cells (IF), and dual-labeled GSII/IF cells in 8-month-old control and HKCre/Shh^KO mice was counted. Data are presented as the mean ± SEM where *P < .05 compared with the control animals, n = 8 mice in each group. Immunofluorescence of (B) control and (C) HKCre/Shh^KO mouse groups stained for IF (red) together with GSII (green). Merged images are shown. Colocalization is indicated in yellow. Representative of n = 8. Original magnification 40×.

Figure 4

Expression of surface mucous pit cells and BrdU-labeled nuclei in control and HKCre/Shh^KO mice. Immunofluorescence for UEAI (green) and BrdU (red) in 8-month-old (A) control and (B) HKCre/Shh^KO mice. Morphometric analysis of (C) UEAI-positive cells and (D) BrdU-labeled nuclei was counted from both control and HKCre/Shh^KO mice at 1, 2, 3, 4, and 8 months of age and expressed as UEAI or BrdU-positive cells per gland. (E) Immunoblots of cytoplasmic and nuclear β-catenin using protein extracts of 8-month-old control and HKCre/Shh^KO mice. (F) Cytoplasmic and nuclear β-catenin expression was quantified and normalized for either glyceraldehyde-3-phosphate dehydrogenase (GAPDH; cytoplasmic protein) or Lamin B1 (nuclear protein). Data are expressed as the mean ± SEM, n = 4–8 mice per group where *P < .05 compared with control mice.

Figure 5

Snail and E-cadherin expression in control and HKCre/Shh^KO mice. (A) Average fold change in gene expression of Shh, Ihh, Gli1,Wnt3a, Wnt5A, and cyclin D1 in RNA collected from the surface pit region of control and HKCre/Shh^KO mouse stomachs. Immunofluorescence of (B–D) control and (E–G) HKCre/Shh^KO stomachs immunostained for (B, C and E, F) Snail and nuclear marker TPORO or (D and G) Snail and UEAI. Snail staining is indicated (arrow). (H) Average fold change in gene expression of Snail and E-cadherin in RNA collected from the surface pit region of control and HKCre/Shh^KO mouse stomachs. Immunofluorescence of (I and J) control and (K and L) HKCre/Shh^KO stomachs immunostained for E-cadherin. Membrane expressed E-cadherin (arrow) in the control mice and cytoplasmic expression (arrow) of E-cadherin in HKCre/Shh^KO mice. Original magnification: I and K, 20×; J and L, 40×. Representative figures of n = 8 per group at 4 months of age. Data are expressed as the mean ± SEM where *P < .05 compared with the control animals, n = 3 mice in each group.

Figure 6

Changes in gastric acidity and gastrin and somatostatin cell number in control and HKCre/Shh^KO mice. Gastric acid measurements (microequivalents H⁺/Kg) in PBS- or histamine (HIST)-treated (A) control and (B) HKCre/Shh^KO mice at 1–8 months of age. Morphometric analysis of (C) gastrin (G)- and (D) somatostatin (SOM)-expressing cells in antrums of control and HKCre/Shh^KO mice is shown. Electron micrographs of 4-month-old (E) control and (F) HKCre/Shh^KO mouse glandular stomach. cn, canalicular membrane; n, nucleus; m, mitochondria. Scale bar = 2 µm. Representative micrograph of 6 individual animals. Data are expressed as the mean ± SEM where *P < .05 compared with control or PBS-treated group, n = 6–8 animals per group.

Figure 7

Changes in plasma gastrin, proliferation, and surface pit, mucous neck, and zymogen cell numbers in control and HKCre/Shh^KO mice. (A) Plasma was collected from untreated and octreotide (Oct)-treated control and HKCre/Shh^KO mice and circulating gastrin concentrations measured by radioimmunoassay. (B) Morphometric analysis of UEAI-positive cells and BrdU-labeled nuclei was counted using gastric sections collected from untreated- and Oct-treated control and HKCre/Shh^KO mice and expressed as UEAI- or BrdU-positive cells per gland. (C) Average fold change gene expression of Shh and Ihh in RNA collected from the surface pit region of control and HKCre/Shh^KO mice without Oct (W/O Oct) or with Oct (W/ Oct) treatment. (D) Average fold change in gene expression of Gli1, Wnt3a, Wnt5A, and cyclin D1 in RNA collected from the surface pit region of control and HKCre/Shh^KO mice W/O Oct or W/ Oct treatment. Quantification of mucous neck cells (GSII), zymogen cells (IF), and dual-labeled GSII/IF cells in control and HKCre/Shh^KO mice (E) W/O Oct or (F) W/ Oct treatment. Data are shown as the mean ± SEM where *P < .05 compared with the control animals, #P < .05 compared with HKCre/Shh^KO mice W/O Oct, n = 4 mice in each group.

Figure 8

Proposed mechanism for the development of hyperproliferation in the surface mucous cells with loss of Shh. (A) In the epithelium, loss of Shh disrupts the inhibitory pathway between somatostatin and gastrin, thus inducing hypergastrinemia. Hypergastrinemia up-regulates the expression of Ihh within the epithelial surface mucous cells. (B) Upon binding to Ptch receptor in the mesenchyme, Ihh removes the inhibitory effect of Ptch on Smo and causes the translocation of Gli1 to the nucleus, where it induces expression of target gene Snail and canonical Wnt. (C) Activation of the Wnt pathway within the epithelium targets cyclin D1, which promotes cell proliferation.