Gli2 expression is not increased in the <i>Gast^−/−</i> corpus.

<p>Representative X-gal staining of corpi of <i>Gast^+/+</i> and <i>Gast^−/−</i> mice harboring the <i>LacZ</i> reporter for Sonic hedgehog (<i>Shh</i>) (<b>A</b> and <b>B</b>), <i>Gli1</i> (<b>C</b> and <b>D</b>) and <i>Gli2</i> (<b>E</b> and <b>F</b>). Bars are 100 µm.</p

Epithelial expression of Gli2 in the <i>Gast^−/−</i> antrum.

<p>The antral expression of Hedgehog pathway molecules was determined in 9–13 month-old littermate controls (<i>Gast^+/+</i>) (panels <b>A</b>, <b>C</b> and <b>E</b>) and <i>Gast^−/−</i> (panels <b>B</b>, <b>D</b> and <b>F</b>) mice by X-gal staining of LacZ reporter mice for Sonic hedgehog (<i>Shh</i>) (<b>A</b> and <b>B</b>), <i>Gli1</i> (<b>C</b> and <b>D</b>) and <i>Gli2</i> (<b>E</b> and <b>F</b>). A high power field of Gli2-LacZ staining is shown in F, where nuclear (yellow arrows) and perinuclear (black arrows) staining was observed along with cytoplasmic reporter accumulation. Whole stomachs from <i>Gast^+/+</i> and <i>Gast^−/−</i> were analyzed for gene expression of <i>Shh</i> (<b>G</b>), <i>Gli1</i> (<b>H</b>), and <i>Gli2</i> (<b>I</b>). Bars in panels <b>A</b> to <b>F</b> are 100 µm. Data presented as mean±SEM. N = 8 per group. *P≤0.05.</p

Inflammation and Gli2 Suppress Gastrin Gene Expression in a Murine Model of Antral Hyperplasia

<div><p>Chronic inflammation in the stomach can lead to gastric cancer. We previously reported that gastrin-deficient (<em>Gast^−/−</em>) mice develop bacterial overgrowth, inflammatory infiltrate, increased Il-1β expression, antral hyperplasia and eventually antral tumors. Since Hedgehog (Hh) signaling is active in gastric cancers but its role in precursor lesions is poorly understood, we examined the role of inflammation and Hh signaling in antral hyperplasia. <em>LacZ</em> reporter mice for <em>Sonic hedgehog</em> (<em>Shh</em>), <em>Gli1</em>, and <em>Gli2</em> expression bred onto the <em>Gast^−/−</em> background revealed reduced <em>Shh</em> and <em>Gli1</em> expression in the antra compared to wild type controls (WT). <em>Gli2</em> expression in the <em>Gast^−/−</em> corpus was unchanged. However in the hyperplastic <em>Gast^−/−</em> antra, <em>Gli2</em> expression increased in both the mesenchyme and epithelium, whereas expression in WT mice remained exclusively mesenchymal. These observations suggested that <em>Gli2</em> is differentially regulated in the hyperplastic <em>Gast^−/−</em> antrum versus the corpus and by a Shh ligand-independent mechanism. Moreover, the proinflammatory cytokines Il-1β and Il-11, which promote gastric epithelial proliferation, were increased in the <em>Gast^−/−</em> stomach along with Infγ. To test if inflammation could account for elevated epithelial <em>Gli2</em> expression in the <em>Gast^−/−</em> antra, the human gastric cell line AGS was treated with IL-1β and was found to increase <em>GLI2</em> but decrease <em>GLI1</em> levels. IL-1β also repressed human <em>GAST</em> gene expression. Indeed, GLI2 but not GLI1 or GLI3 expression repressed gastrin luciferase reporter activity by ∼50 percent. Moreover, chromatin immunoprecipitation of GLI2 in AGS cells confirmed that GLI2 directly binds to the <em>GAST</em> promoter. Using a mouse model of constitutively active epithelial GLI2 expression, we found that activated GLI2 repressed <em>Gast</em> expression but induced <em>Il-1β</em> gene expression and proliferation in the gastric antrum, along with a reduction of the number of G-cells. In summary, epithelial Gli2 expression was sufficient to stimulate <em>Il-1β</em> expression, repress <em>Gast</em> gene expression and increase proliferation, leading to antral hyperplasia.</p> </div

Epithelial activation of Gli2 induced Il-1β and reduced <i>Gast</i> expression and G-cell number.

<p><b>A</b>) <i>Gast</i> gene expression is reduced after Gli2 activation in <i>Shh-Cre;R26-LSL-rtTA;tetO-GLI2</i>ΔN (GLI2ΔN) mice after 3 days of treatment with doxycycline. <b>B</b>) <i>Il-1β</i> was induced in the antra of GLI2ΔN mice antra. <b>C</b>) Representative images of hematoxylin-eosin staining (top panels), MYC staining to detect epitope-tagged GLI2?N (red), Gast staining (green) and merged images (lower panel) of the antrum of control and GLI2ΔN mice after 3 days of doxycycline. <b>D</b>) Representative images of proliferation marker Ki-67 staining in control and GLI2ΔN mice after 3 days of doxycycline. Data presented as mean±SEM. N = 2 mice per group per time. Bars are 100 µm in panel <b>C)</b> and 50 µm in panel <b>D)</b>.</p