Bone Morphogenetic Protein Signaling Regulates Gastric Epithelial Cell Development and Proliferation in Mice

Abstract

BACKGROUND & AIMS We investigated the role of bone morphogenetic protein (BMP) signaling in the regulation of gastric epithelial cell growth and differentiation by generating transgenic mice that express the BMP inhibitor noggin in the stomach. METHODS The promoter of the mouse H+/K+-ATPase β-subunit gene, which is specifically expressed in parietal cells, was used to regulate expression of noggin in the gastric epithelium of mice. The transgenic mice were analyzed for noggin expression, tissue morphology, cellular composition of the gastric mucosa, gastric acid content, and plasma levels of gastrin. Tissues were analyzed by immunohistochemical, quantitative real-time polymerase chain reaction, immunoblot, microtitration, and radioimmunoassay analyses. RESULTS In the stomachs of the transgenic mice, phosphorylation of Smad1, 5, and 8 decreased, indicating inhibition of BMP signaling. Mucosa were of increased height, with dilated glands, cystic structures, reduced numbers of parietal cells, and increased numbers of cells that coexpressed intrinsic factor, trefoil factor 2, and griffonia simplicifolialectin II, compared with wild-type mice. In the transgenic mice, levels of the H+/K+-ATPase α-subunit protein and messenger RNA were reduced, whereas those of intrinsic factor increased. The transgenic mice were hypochloridric and had an increased number of Ki67- and proliferating cell nuclear antigen-positive cells; increased levels of plasma gastrin; increased expression of transforming growth factor-α, amphiregulin, and gastrin; and activation of extracellular signal-regulated kinase 2. CONCLUSIONS Inhibiting BMP signaling in the stomachs of mice by expression of noggin causes loss of parietal cells, development of transitional cells that express markers of mucus neck and zymogenic lineages, and activation of proliferation. BMPs are therefore important regulators of gastric epithelial cell homeostasis.

Regulation of Gastric Epithelial Cell Homeostasis by Gastrin and Bone Morphogenetic Protein Signaling

“…Mice H/K-noggin transgenic mice and gastrin knockout mice were previously described (Shinohara et al 2010;Takabayashi et al 2014). Animals were housed in the animal maintenance facility at the University of Michigan.…”

“…Gastric samples obtained from the corpus of WT, NogTG, GKO and NogTG;GasKO mice aged 3 months, were homogenized on ice in 1-mL microdounce homogenizers (~15 strokes) using 500 lL of lysis buffer (Shinohara et al 2010). The lysates were spun at 16,000 9 g for 5 min at 4°C.…”

“…RNA was isolated from the corpus of WT, NogTG, GKO, and NogTG;GasKO mice aged 3 months, using TRIzol reagent (Invitrogen, Carlsbad, CA), followed by DNase treatment and purification with the RNeasy Mini kit (Qiagen, Valencia, CA). RT reactions were conducted according to previously published reports (Shinohara et al 2010;Takabayashi et al 2014). Reactions were performed using the Icycler (Bio-Rad, Hercules, CA) with a 20 lL reaction in PCR buffer (Invitrogen) containing 2 lL of cDNA (RT product), 5.5 mmol/L MgCl 2 , 100 nmol/L primers 200 nmol/L dNTPs, 0.19 SYBR Green, 10 nmol/L fluorescein, and 0.025 U Platinum Taq (Invitrogen).…”

“…The lysates were spun at 16,000 9 g for 5 min at 4°C. Equal volumes of lysates containing 80 lg of proteins were mixed with 59 electrophoresis buffer (Shinohara et al 2010), boiled for 5 min (except for the H + /K + -ATPase a-subunit western blots, in which the samples were not boiled) and loaded on 10% SDS-polyacrylamide mini-gels, which were run at 200 volts for 1 h. After transfer and blocking ) the membranes were incubated for 16-18 h at 4°C in 10 mL of TBST, 5% dry milk, containing specific antibodies recognizing IF (1:3000) (gift from Dr. David Alpers, Washington University, St. Louis, MO), and the H + /K + -ATPase a-subunit (1:1000) (Medical and Biological Laboratories, Nagoya, Japan). Control blots were performed using antibodies recognizing GAPDH (1:1000) (Chemicon International, Temecula, CA).…”

“…In a series of recent in vivo investigations, we demonstrated that inhibition of BMP signaling in the gastric epithelium causes profound aberrations in the normal mechanisms that regulate the proliferation, maturation and differentiation of several lineages of gastric epithelial cells (Shinohara et al 2010;Takabayashi et al 2014). In particular, we reported that the mucosa of transgenic mice expressing noggin in the fundic epithelium, exhibits the presence of increased epithelial cell proliferation, enhanced MAP/ERK kinase activation, diminished parietal-cell (PC) number, and expansion of populations of transitional cells expressing both the mucus neck cell marker GSII and the zymogenic cell (ZC) marker IF.…”