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 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

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

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

Maintenance of Taste Organs Is Strictly Dependent on Epithelial Hedgehog/GLI Signaling

<div><p>For homeostasis, lingual taste papilla organs require regulation of epithelial cell survival and renewal, with sustained innervation and stromal interactions. To investigate a role for Hedgehog/GLI signaling in adult taste organs we used a panel of conditional mouse models to manipulate GLI activity within epithelial cells of the fungiform and circumvallate papillae. Hedgehog signaling suppression rapidly led to taste bud loss, papilla disruption, and decreased proliferation in domains of papilla epithelium that contribute to taste cells. Hedgehog responding cells were eliminated from the epithelium but retained in the papilla stromal core. Despite papilla disruption and loss of taste buds that are a major source of Hedgehog ligand, innervation to taste papillae was maintained, and not misdirected, even after prolonged GLI blockade. Further, vimentin-positive fibroblasts remained in the papilla core. However, retained innervation and stromal cells were not sufficient to maintain taste bud cells in the context of compromised epithelial Hedgehog signaling. Importantly taste organ disruption after GLI blockade was reversible in papillae that retained some taste bud cell remnants where reactivation of Hedgehog signaling led to regeneration of papilla epithelium and taste buds. Therefore, taste bud progenitors were either retained during epithelial GLI blockade or readily repopulated during recovery, and were poised to regenerate taste buds once Hedgehog signaling was restored, with innervation and papilla connective tissue elements in place. Our data argue that Hedgehog signaling is essential for adult tongue tissue maintenance and that taste papilla epithelial cells represent the key targets for physiologic Hedgehog-dependent regulation of taste organ homeostasis. Because disruption of GLI transcriptional activity in taste papilla epithelium is sufficient to drive taste organ loss, similar to pharmacologic Hedgehog pathway inhibition, the findings suggest that taste alterations in cancer patients using systemic Hedgehog pathway inhibitors result principally from interruption of signaling activity in taste papillae.</p></div

Innervation to fungiform papillae and taste buds is retained in epithelial HH/GLI suppression models.

<p><b>A,C,E,G. Chorda/Lingual</b>: K8 immunostaining to label taste bud cells and neurofilament (NF) to label fibers of the chorda tympani/lingual nerve innervation to FP and TB. <b>A</b>. In Control the Chorda/lingual innervation is throughout the FP core and forms a dense ‘basket’ under the TB at the apex of the papilla core. With HH/GLI suppression at specified time points in <i>K5GliR</i> (<b>C</b>), <i>epiGliR</i> (<b>E</b>), and <i>Gli2cKO</i> (<b>G</b>) mice, NF-positive fibers are retained in the FP even though papillae are of atypical morphology and TBs are lost (E) or much reduced (G). At the illustrated time points in all models there are substantial morphological effects, as seen in Figs <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006442#pgen.1006442.g001" target="_blank">1</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006442#pgen.1006442.g002" target="_blank">2</a>. <b>B,D,F,H</b>. <b>Chorda tympani</b>: K8 immunostaining to label taste bud cells and P2X3 to label fibers of the chorda tympani nerve innervation to FP and TB. <b>B</b>. In Control for Chorda tympani innervation the P2X3-positive label is within fibers in the papilla core (arrow) and within the TB. With HH/GLI suppression at specified time points in <i>K5GliR</i> (<b>D</b>), <i>epiGliR</i> (<b>F</b>), and <i>Gli2cKO</i> (<b>H</b>) mice, P2X3-positive fibers (arrows) are retained in the FP and TB cells even though papillae are of atypical morphology and TBs are lost (F) or much reduced (H). Scale bar in H applies to all panels. * in H indicate areas of nonspecific staining in the surface epithelial cells.</p

Circumvallate papilla structure is retained but taste buds are lost with conditional Gli2 deletion.

<p>Hematoxylin and eosin sections of the circumvallate papilla from Control (A,B) and conditional epithelial gene deletion, <i>Gli2cKO</i> (C,D) tongues. Papilla structure was measured from the number of serial sections that encompassed the papilla (depth) and length of each ‘wall’ marked by two bars in A. Whereas papilla shape is retained (A,C) numbers of taste buds are much reduced after gene deletion (B,D). Taste bud remnants (dotted ovals) or with complete pores (arrows) are numerous in Control papilla (B, enlarged from Box in A) but few are found with HH/GLI suppression (D, enlarged from Box in C). One scale bar applies for A and C; or B and D. <b>E,F</b>. Graphs for circumvallate structure (E) and numbers of taste bud profiles/remnants and pores (F) at increasing durations after <i>Gli2</i> gene deletion. CV depth and wall length did not change across time points in <i>Gli2cKO</i> tongues (E). Profile measures, used to indicate cell collections, were decreased by 28 days after <i>Gli2</i> gene deletion (F) and TB pore measures, to indicate complete TBs, also decreased (F). Taste buds with pores are essentially eliminated after 16 days gene deletion (F). Numbers of tongues studied at each time point after gene deletion are in parentheses in the legends. Full statistics for ANOVAs are presented in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006442#pgen.1006442.s001" target="_blank">S1E and S1F Fig</a>.</p

Inhibition of epithelial HH/GLI signaling leads to alterations in fungiform papillae and epithelium and loss of taste buds.

<p><b>A</b>. Fungiform papilla and taste bud diagrams to illustrate cells types and location of HH pathway signaling elements. Canonical HH signaling is restricted to cells expressing both <i>Gli1</i> and <i>Ptch1</i> (Control Papilla and Taste bud, left). Center and right panel diagrams illustrate cell/tissue areas (blue) of dominant-negative transgene expression in doxycycline-inducible <i>K5GliR</i> and <i>epiGliR</i> mouse models. <b>B</b>. Three types of Fungiform Papilla and Taste Bud (FP/TB) used for quantifying effects of HH repression: I, Typical FP/TB: Typical papilla morphology with intact apical taste bud; II, Atypical FP/TB: Atypical, mis-shapen papilla morphology and taste bud remnant or cluster of distinctly staining cells; III, Atypical FP/No TB: Atypical papilla morphology with keratinized apical point and no discernable taste bud cells. <b>C</b>. H&E sections illustrate FP/TB morphology for Control and <i>K5GliR</i> and <i>epiGliR</i> mouse tongues at indicated time points after transgene induction. Scale bar applies to all micrographs except low power and insets. Low power image for <i>epiGliR</i> Control illustrates overall tongue morphology. Inset at Day 11 indicates disruption of fungiform and filiform papillae but at Day 35 <i>K5GliR</i>, inset illustrates intact filiform. Graphs present quantification of TYPE I, II and III taste organs at increasing durations of transgene activation. TYPE I (Typical) FP/TB are substantially reduced (<i>K5GliR</i>) or eliminated (<i>epiGliR</i>) whereas TYPE III (Atypical/No TB) organs accumulate. Numbers of tongues at each time point are included in parentheses in graph legends. Full statistics for ANOVA are presented in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006442#pgen.1006442.s001" target="_blank">S1A and S1B Fig</a>.</p

HH-responding cells are lost from the papilla epithelium but retained in connective tissue, and taste bud size, and SHH expression, decrease with duration of HH/GLI repression.

<p><b>TOP ROW</b>: X-Gal staining to detect β-gal-positive, HH-responding cells in Control <i>Gli1</i>^{<i>lacZ/+</i>} mice is typically in perigemmal cells (P), basal epithelial cells of the FP wall (W), and cells of the stromal core (ST). After <i>epiGliR</i> transgene activation for 5 days, and continuing at 11 days, there is loss of detectable HH signaling in epithelium with <i>lacZ</i>-positive cells in the FP stroma only. <b>BOTTOM ROW</b>: SHH is expressed principally within taste buds cells (K8) in Control FP. During HH/GLI repression in <i>epiGliR</i> tongues, after 5 days remaining taste buds are progressively reduced in size and SHH ligand is reduced in association with taste cell loss. After 11 days very few TB remnants are observed (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006442#pgen.1006442.g001" target="_blank">Fig 1</a> data) and these are very small cell collections. Inset at Day 11 illustrates the nature of the cell remnants that remain in a very small percentage of FP.</p

HH-responding cells are lost from circumvallate papilla epithelium but remain in stroma after epithelial Gli2 deletion, and taste bud size, and SHH expression, decrease with duration of HH/GLI repression.

<p><b>A,B</b>. <i>Gli1lacZ</i>-positive cells in Control and <b>C,D;E,F</b> after gene deletion, in <i>Gli2cKO</i> CVs, at 5 and 35 days. B,D,F are enlargements for boxed regions in paired panels. <b>A,B</b>. In Control CV, HH- responding cells are in epithelial cells (perigemmal) surrounding the taste buds and in stroma of the papilla surround, including just under the basal lamina. <b>C,D; E,F</b>. From 5 days of gene deletion and continuing through 35 days, <i>Gli1lacZ</i>-positive cells are lost from the papilla epithelium but remain in stroma just under the taste bud- bearing epithelium. Dotted ovals are used to denote taste buds (B) or taste bud profiles/remnants (D,F). <b>G,I,K</b>. SHH and K8 Immunoreactions in Control, and at Day 5 and Day 35 after gene deletion illustrate reduction in K8-positive taste bud cells and associated loss of SHH expression. <b>H,J,L</b>. Same sections as those in merged panels (G,I,K) to illustrate SHH ligand alone in taste bud cells and remnants. After 35 days SHH expression is retained in reduced cell collections.</p