Indian Hedgehog mediates gastrin-induced proliferation in stomach of adult mice

Abstract

Background & aims: Loss of expression of Sonic Hedgehog (Shh) from parietal cells results in hypergastrinemia in mice, accompanied by increased expression of Indian Hedgehog (Ihh) and hyperproliferation of surface mucous cells. We investigated whether hypergastrinemia induces gastric epithelial proliferation by activating Ihh signaling in mice.

Methods: We studied mice with parietal cell-specific deletion of Shh (PC-Shh(KO)) and hypergastrinemia, crossed with gastrin-deficient (GKO) mice (PC-Shh(KO)/GKO). When mice were 3-4 months old, gastric tissues were collected and analyzed by histology, for incorporation of bromodeoxyuridine, and for expression of the surface mucous cell marker Ulex europaeus. PC-Shh(KO)/GKO mice were given gastrin infusions for 7 days; gastric surface epithelium was collected and expression of Ihh was quantified by laser capture microdissection followed by quantitative reverse transcriptase polymerase chain reaction. Mouse stomach-derived organoids were incubated with or without inhibitors of WNT (DKK1) or Smoothened (vismodegib) and then cocultured with immortalized stomach mesenchymal cells, to assess proliferative responses to gastrin.

Results: Gastric tissues from PC-Shh(KO)/GKO mice with hypergastrinemia had an expanded surface pit epithelium, indicated by a significant increase in numbers of bromodeoxyuridine- and Ulex europaeus-positive cells, but there was no evidence for hyperproliferation. Gastrin infusion of PC PC-Shh(KO)/GKO mice increased expression of Ihh and proliferation within the surface epithelium compared with mice given infusions of saline. In gastric organoids cocultured with immortalized stomach mesenchymal cells, antagonists of WNT and Smoothened inhibited gastrin-induced proliferation and WNT activity. Activity of WNT in media collected from immortalized stomach mesenchymal cells correlated with increased expression of glioma-associated oncogene homolog 1, and was inhibited by DKK1 or vismodegib.

Conclusions: Ihh signaling mediates gastrin-induced proliferation of epithelial cells in stomachs of adult mice.

Keywords: Development; Gastric Epithelium; Signal Transduction; Tissue Regeneration.

Figure 1. Quantification of epithelial cells and surface pit mucous-proliferating cells in control, GKO, PC-Shh^KO and PC-Shh^KO/GKO mouse stomachs

(A) Stomach sections collected from BrdU-injected mice were immunostained for UEAI (green) and BrdU (red) in 3–4 month old control, GKO, PC-Shh^KO and PC-Shh^KO/GKO mice. (B) BrdU-labeled nuclei were counted and expressed as BrdU positive cells/gland. (C) Stomach sections collected from control, GKO, PC-Shh^KO and PC-Shh^KO/GKO were immunostained for surface mucous pit cells (UEAI, green), parietal cells (HK, blue) and chromagranin A (Cg A, red). (D) The number of UEAI+, HK+ and CgA+ cells were quantified. Representative of n = 4 mice per group. Data is expressed as the mean ± SEM. *P < 0.05 compared to control mice, #P<0.05 compared to PC-Shh^KO mice as analyzed by one way ANOVA.

Figure 2. Expression pattern of Shh and Ihh in fundic gastric mucosa

(A) Fundic sections were collected from stomachs of Shh::GFP mice were immunostained using antibodies specific for UEAI (blue), GFP (Shh-expressing cells, green) and Ihh (red). Representative of n=4 mice. Higher magnification is shown in (B). Quantitative RT-PCR was performed on RNA prepared from total epithelium, surface pit epithelium, neck, and base collected from control, GKO, PC-Shh^KO and PC-Shh^KO/GKO mice by LCM. Shown is the expression of Shh (C) and Ihh (D) mRNA relative to total epithelium collected from the control group. Data is expressed as the mean ± SEM. *P < 0.05 compared to control total epithelium, #P<0.05 compared to control neck or pit as analyzed by one way ANOVA.

Figure 3. Epithelial proliferation in response to gastrin and cyclopamine treatments

(A) Circulating gastrin concentrations were measured in plasma collected from PC-Shh^KO/GKO mice infused with PBS, gastrin (GAS) alone or GAS + cyclopamine (Cyc). Each data point represents the concentration of plasma gastrin (pM) from an individual animal. (B) Quantitative RT-PCR was performed on stomach RNA prepared from gastric mucosa collected from PC-Shh^KO/GKO mice infused with PBS, GAS alone or GAS + Cyc. Shown is the expression of Ihh and Gli1 mRNA relative to PBS infused mice. (C) Stomach sections were immunostained for UEAI (green) and BrdU (red) in PC-Shh^KO/GKO mice infused with PBS, gastrin (GAS) alone or GAS + cyclopamine (Cyc). (D) BrdU-labeled nuclei were counted from PC-Shh^KO/GKO mouse stomachs and expressed as BrdU positive cells/gland. (E) The number of surface pit cells were counted and expressed as UEAI positive cells/gland. Data is expressed as the mean ± SEM. *P < 0.05 compared to PBS treated group as analyzed by one way ANOVA, #P<0.05 compared to gastrin infused group. ND: not detected, n = 3–6 animals/group.

Figure 4. Epithelial and non-epithelial Ihh and Gli1 expression

(A) Dissection of total stomach tissue (T), epithelium (EP) and non-epithelium (NEP) from PC-Shh^KO/GKO mouse stomach. Quantitative RT-PCR analysis of known (B) mesenchymal (vimentin, MAdCAM1 and Actg2) and (C) epithelial (keratin 20 and Muc5ac) markers. Representative of three independently isolated fractions from total stomach. Quantitative RT-PCR was performed on stomach RNA prepared from total, epithelial and non-epithelial fractions collected from untreated (NT), gastrin infused and L-365, 260 + gastrin infused mice. Shown is the expression of (D) Ihh and (E) Gli1 mRNA relative to untreated total stomach tissue. Data were normalized to HPRT expression and presented as the mean ± SEM. * P<0.05 vs. NT group, # P<0.05 vs. gastrin infused group, n = 4 mice/group.

Figure 5. Gastrin-induced proliferation in gastric organoids/ISMCs co-cultures

(A) Fundic gastric organoids derived from PC-Shh^KO/GKO or CCK-BRKO mouse stomachs were co-cultured on transwell membranes with immortalized stomach mesenchymal cells (ISMcs) and treated with either 0nM gastrin, 100 nM gastrin with or without Wnt antagonist (recombinant Dkk1) or smoothened inhibitor (Vismodgib). (B) Organoids were immunostained for EdU (red) and nucleus (blue). Representative of n = 4 mice individual organoid cultures per group. Total cell number and EdU-labeled nuclei were counted from the four groups and expressed as EdU positive cells/total cells ratio. (C) Wnt activity was measured by a TOPflash assay using medium collected from the lower chamber of ISMC cultures. (D) qRT-PCR was used to measure Gli1 expression using RNA collected from cultured ISMCs. (E) Medium was collected from the top chamber of organoids cultures, immunoprecipitated and changes in Ihh protein expression measured by western blot. (F) Ihh expression was measured in organoids collected from co-cultured by qRT-PCR. *P<0.05 compared to 0nM gastrin group. #P<0.05 compared to 100nM gastrin group. n = 4 individual cultures per group.

Figure 6. Organoid proliferation in response to ISMC conditioned medium

(A) ISMCs were transduced to express empty vector (ISMC^EV) or a knockdown of smoothened (ISMC^SmoKD) that was confirmed by western blot analysis. ISMC^EV and ISMC^SmoKD cells were treated with either PBS (treatments 1 and 3), recombinant Ihh (rIhh, treatments 2 and 4) or recombinant Ihh plus Dkk1 (treatments 5 and 6). (B) qRT-PCR was used to measure Gli1 expression using RNA collected from cultured ISMCs. (C) Wnt activity was measured by a TOPflash assay using medium collected from the conditioned medium of ISMC cultures. (D) Organoids were immunostained for EdU (red) and nucleus (blue). Representative of n = 4 mice individual organoid cultures per group. (E) Total cell number and EdU-labeled nuclei were counted from the six groups and expressed as EdU positive cells/total cells ratio. *P<0.05 compared to PBS treated group. n = 4 individual cultures per group.

Figure 7. Proposed model of gastrin-induced epithelial proliferation

We propose a signaling pathway involving the crosstalk between epithelial Ihh signaling to Gli1 within the mesenchyme, then relaying to an intermediate mediator, such as Wnt, to induce proliferation. Gli1 activation within the mesenchyme may signal via Wnt to induce proliferation within the surface epithelium.