Negative regulation of gastrin-induced NR4A2 expression.

<p><b>A</b>: AGS-G_R cells transfected with NR4A2-luc and ICER expression plasmids or empty vector. Cells were treated with gastrin for 6 h prior to measurement of NR4A2 activity. Data shown represent mean ± SEM of five biological replicas (** p<0.03, * p = 0.06). <b>B</b>: AGS-G_R cells transfected with NBRE-luc and ICER expression plasmid or empty vector and treated with gastrin for 4 h prior to measurement of NBRE activity. Data shown represent mean ± SEM of four biological replicas (** p<0.03). <b>C</b>: AGS-G_R cells were transfected with pZfp36l1 expression plasmid or empty vector and treated with gastrin (5 nM) NR4A2 mRNA expression was measured by qRT-PCR. Data shown represent one of three biological replicas; mean ± SD of three technical replicas is shown. <b>D</b>: Cyclin L1 represents one of three control genes examined.</p

NR4A2 suppresses gastrin-induced migration and invasion.

<p><b>A</b>: Real-time cell migration monitored (0-24 h) in AGS-G_R cells transfected with siNR4A2 or siCtr, with or without gastrin treatment (10 nM). Results show one representative of three biological replicas; mean ±SD of three technical replicas. <b>B</b>: Invasion assay with AGS-G_R cells transfected with pCMX-NR4A2 or pCMX (control) was performed in 24-well plates containing 8-µm pore Matrigel-coated inserts (with or without 0.3 nM gastrin). Cells invading the lower surface of the membrane were stained with Reastain Quick-Diff reagents and total numbers of cells in 5 fields per membrane were counted. The mean of three independent experiments is shown.</p

Immunostaining of NR4A2 in gastric adenocarcinoma.

<p><b>A-B</b>: NR4A2 immunoreactivity in normal oxyntic mucosa showing strong intensity in scattered single cells (neuroendocrine cells) and weaker staining intensity in the other epithelial cells. <b>C-F</b>: NR4A2 immunoreactivity in gastric adenocarcinomas of intestinal (C-D) and diffuse (E-F) type, showing a general staining in tumor cells with mixed nuclear or cytoplasmic localization and variable intensities. (A, C, E at x200 magnification, with boxes representing B, D and F at x400 magnification).</p

NR4A2 activates NBRE promoter elements.

<p><b>A</b>: Gastrin-induced NBRE-luc activation. Data represent one of two biological replicas. <b>B</b>: The effect of NR4A2 siRNA on gastrin-induced NBRE activation. Data represent mean ± SEM of four biological replicas (** p<0.01, * p=0.1). <b>C-D</b>: Effect of specific inhibitors of PKA (H-89, 10µM), PI3K (LY 294002, 10µM) or PKC (GF 109203x, 3.5µM) on (<b>C</b>) gastrin-induced NR4A2 gene expression and (<b>D</b>) gastrin-induced NBRE activation. Data represent one of three biological replicas; mean ± SD of six technical replicas.</p

The cytoprotective protein clusterin is overexpressed in hypergastrinemic rodent models of oxyntic preneoplasia and promotes gastric cancer cell survival

<div><p>The cytoprotective protein clusterin is often dysregulated during tumorigenesis, and in the stomach, upregulation of clusterin marks emergence of the oxyntic atrophy (loss of acid-producing parietal cells)-associated spasmolytic polypeptide-expressing metaplasia (SPEM). The hormone gastrin is important for normal function and maturation of the gastric oxyntic mucosa and hypergastrinemia might be involved in gastric carcinogenesis. Gastrin induces expression of clusterin in adenocarcinoma cells. In the present study, we examined the expression patterns and gastrin-mediated regulation of clusterin in gastric tissue from: humans; rats treated with proton pump (H+/K+-ATPase) inhibitors and/or a gastrin receptor (CCK2R) antagonist; H+/K+-ATPase β-subunit knockout (H/K-β KO) mice; and Mongolian gerbils infected with <i>Helicobacter pylori</i> and given a CCK2R antagonist. Biological function of secretory clusterin was studied in human gastric cancer cells. Clusterin was highly expressed in neuroendocrine cells in normal oxyntic mucosa of humans and rodents. In response to hypergastrinemia, expression of clusterin increased significantly and its localization shifted to basal groups of proliferative cells in the mucous neck cell-chief cell lineage in all animal models. That shift was partially inhibited by antagonizing the CCK2R in rats and gerbils. The oxyntic mucosa of H/K-β KO mice contained areas with clusterin-positive mucous cells resembling SPEM. In gastric adenocarcinomas, clusterin mRNA expression was higher in diffuse tumors containing signet ring cells compared with diffuse tumors without signet ring cells, and clusterin seemed to be secreted by tumor cells. In gastric cancer cell lines, gastrin increased secretion of clusterin, and both gastrin and secretory clusterin promoted survival after starvation- and chemotherapy-induced stress. Overall, our results indicate that clusterin is overexpressed in hypergastrinemic rodent models of oxyntic preneoplasia and stimulates gastric cancer cell survival.</p></div

The guanylate cyclase-C signaling pathway is down-regulated in inflammatory bowel disease

<p><b><i>Objective.</i></b> Activation of membrane receptor guanylate cyclase-C (GC-C) is implicated in gastrointestinal fluid and electrolyte balance, preservation of intestinal barrier integrity, anti-trophic effects and inhibition of pain sensation. To evaluate GC-C signaling, we examined the regulation of GC-C (<i>GUCY2C</i>/<i>Gucy2c</i>) and its endogenous ligands guanylin (GN/<i>GUCA2A</i>/<i>Guca2a</i>) and uroguanylin (UGN/<i>GUCA2B</i>/<i>Guca2b</i>) in colonic Crohn’s disease (CD), ulcerative colitis (UC) and in rats with 2,4,6-Trinitrobenzene sulphonic acid (TNBS) colitis. Correlation analyses between expression of <i>GUCA2A</i> and <i>GUCY2C</i> and expression of inflammatory cytokines (<i>IL1A,</i> <i>IL1B,</i> <i>TNFA</i> and <i>IFNG</i>) were conducted. Additionally, expression of transcription factors for <i>GUCA2A</i> and <i>GUCY2C,</i> and the GC-C signaling pathway, were examined. <b><i>Material and methods.</i></b> Biopsies from active UC/CD, un-inflamed UC/CD and healthy controls, and inflamed and healthy rat colon were investigated with gene expression microarray, immunohistochemistry (IHC) and <i>in situ</i> hybridization (ISH). <b><i>Results.</i></b> <i>GUCA2A</i>/<i>Guca2a,</i> <i>GUCA2B,</i> <i>GUCY2C</i>/<i>Gucy2c,</i> transcription factors, as well as several cyclic guanosine-3′,5′-monophosphate downstream mediators were all significantly down-regulated in both inflamed colonic inflammatory bowel disease (IBD) mucosa and TNBS colitis. Expression of <i>GUCA2A</i> and <i>GUCY2C</i> negatively correlated to expression of inflammatory cytokines. IHC and ISH confirmed microarray results for <i>GUCA2A</i>/<i>Guca2a</i> and <i>GUCY2C</i>/<i>Gucy2c</i> in inflamed samples. We identified a highly significant positive correlation between the expression of the transcription factor caudal type homeobox 2 (<i>CDX2</i>) and the expression of the downstream target gene <i>GUCY2C.</i> <b><i>Conclusions.</i> GUCA2A,</b> <i>GUCA2B</i> and <i>GUCY2C</i> as well as several steps of the GC-C signaling pathway are down-regulated in IBD. This may have implications in IBD pathogenesis.</p

Clusterin and TFF2 expression in oxyntic mucosa of Mongolian gerbils increase after <i>H</i>. <i>pylori</i> infection and normalize when antagonizing the CCK2R.

<p>(A, B) Number per oxyntic area (0.44 mm²) of (A) CLU-positive/VMAT2(neuroendocrine (NE))-positive cells and CLU-positive/VMAT2(NE)-negative cells and (B) dual CLU-positive/Ki67-positive cells from uninfected control (n = 4), <i>H</i>. <i>pylori</i>-infected (<i>H</i>. <i>pylori</i>) (n = 7), and CCK2R-antagonized <i>H</i>. <i>pylori</i>-infected gerbils (<i>H</i>. <i>pylori</i>+CCK2Ra) (n = 4). (C, D) Oxyntic expression of CLU (brown) and dual CLU-positive (green)/VMAT2-positive (red) ECL cells in (C) uninfected control (n = 4) and (D) CCK2R-antagonized <i>H</i>. <i>pylori</i>-infected gerbils (n = 4). (E) ISH and IHC showing expression of clusterin in oxyntic mucosa of <i>H</i>. <i>pylori</i>-infected gerbils (n = 7). (F) Double immunofluorescence staining showing co-expression of CLU (green) and TFF2 (red, upper figure) and CLU (green) and Ki67 (red, lower figure) in oxyntic mucosa of <i>H</i>. <i>pylori</i>-infected gerbils. (G) IHC showing TFF2 expression (brown) in oxyntic mucosa of uninfected controls and <i>H</i>. <i>pylori</i>-infected gerbils untreated and treated with a CCK2R antagonist. Data presented as means ± SEM. *ANOVA with Tukey-adjusted <i>p</i> value < 0.05. Nuclei were counterstained with hematoxylin (blue) or DAPI (blue). The basal zone (~100 μm from the gland bottom) is highlighted with a dotted line. Scale bars = (C, D left column, E left column, G) 200 μm; (E right column, F upper figure) 100 μm; (D right column, F lower figure) 50 μm.</p

Clusterin expression in oxyntic mucosa increases and localization shifts from neuroendocrine cells to chief cells after sustained hypoacidity and hypergastrinemia.

<p>(A) IHC and ISH showing similar expression patterns for clusterin (brown) protein and mRNA, examined in control (n = 8) and hypergastrinemic PPI-rats (n = 6). (B) Number of CLU-positive/VMAT2(neuroendocrine (NE))-positive cells and CLU-positive/VMAT2(NE)-negative cells per oxyntic gland (n = 4 rats per group). (C) Double immunofluorescence staining of oxyntic mucosa from hypergastrinemic PPI-rats showing scarce CLU (green) expression in ECL cells (VMAT2-positive (red)). (D) Double immunofluorescence staining of oxyntic mucosa from hypergastrinemic PPI-rats showing CLU (green) expression in groups of chief cells (MIST1-positive (purple)) and triple immunofluorescence staining showing CLU (green) expression in proliferating (PCNA-positive (light blue)) chief cells (PGA5 (red)). Ctr = control; PPI = PPI-induced hypergastrinemia; CCK2Ra = CCK2R antagonist; PPI+CCK2Ra = PPI-induced hypergastrinemia+CCK2R antagonist. Data presented as means ± SEM. *, ** and # = ANOVA with Bonferroni-adjusted <i>p</i> value < 0.05. (*comparison of CLU+/NE+ cells per gland in control vs other groups individually; **comparison of CLU+/NE- cells per gland in PPI vs control or CCK2Ra; #comparison of CLU+/NE- cells per gland in PPI+CCK2Ra vs CCK2Ra.) Nuclei were counterstained with hematoxylin (blue) or DAPI (blue). The basal zone (~100 μm from the gland bottom) is highlighted with a dotted line. Scale bars = (A) 100 μm; (C, D) 50 μm.</p

Clusterin and mucous neck cell markers are co-localized in oxyntic mucosa of H/K-β KO mice.

<p>(A, B) ISH and IHC showing overexpression of clusterin (brown) in oxyntic mucosa of H/K-β KO mice aged 8 months (n = 4) (middle column) and 14 months (n = 4) (right column) compared with wild-type control mice (n = 4) (left column). (C) Immunofluorescence staining showing increased GSII (green) expression in oxyntic mucosa of H/K-β KO mice aged 8 months and 14 months compared with wild-type control mice, particularly apparent in cells located in the basal half of glands. (D) Double immunofluorescence staining showing CLU (green) expression in TFF2-positive cells (red) in oxyntic mucosa from wild-type control mice and H/K-β KO mice aged 8 and 14 months. (E) Double immunofluorescence staining showing co-expression of GSII (green) and PEPII (red) in oxyntic mucosa from H/K-β KO mice aged 3 months. (F) Number of dual CLU-positive/Ki67-positive cells per area (0.44 mm²) of oxyntic mucosa (n = 4 mice per group). Data presented as means ± SEM. The basal zone (~100 μm from the gland bottom) is highlighted with a dotted line. *ANOVA with Tukey-adjusted <i>p</i> value < 0.05. Nuclei were counterstained with hematoxylin (blue) or DAPI (blue). Scale bars = 100 μm.</p

Clusterin expression in neuroendocrine cells.

<p>(A) Double immunofluorescence staining of oxyntic mucosa from control rats showing CLU (green) expression in ECL cells (HDC-positive (red)) and A-like cells (ghrelin-positive (red)). A split image of the green (CLU) and red (HDC) fluorescences separately, and a higher magnification of a few dual positive cells (inset), are shown to clearly illustrate the co-expression of CLU in neuroendocrine cells, representative for all species. (B) Double immunofluorescence staining of oxyntic mucosa from control rats showing no CLU (green) expression in parietal cells (H+/K+-ATPase β (HK)-positive (red)). (C) Double immunofluorescence staining of oxyntic mucosa from wild-type control mice showing CLU (red) expression in ECL cells (HDC-positive (green)). (D) Double immunofluorescence staining of oxyntic mucosa from wild-type control mice showing CLU (red) expression in mucous neck cells (GSII-positive (green)). (E) Double immunofluorescence staining of oxyntic mucosa from control Mongolian gerbils showing CLU (green) expression in ECL cells (VMAT2-positive (red)). (F) ISH and IHC of human oxyntic mucosa showing clusterin (brown and green) expression in scattered single cells partially overlapping with the neuroendocrine marker CgA (red). Inset shows high power view of single CLU-positive cells. Nuclei were counterstained with hematoxylin (blue) or DAPI (blue). The basal zone (~100 μm from the gland bottom) is highlighted with a dotted line. Scale bars = (B, D, F middle column) 100 μm; (A, C, E, F left and right column) 50 μm; (F inset) 20 μm.</p