Importance of the alternative NF-κB activation pathway in inflammation-associated gastrointestinal carcinogenesis.

Chronic inflammation is a common factor in the development of many gastrointestinal malignancies. Examples include inflammatory bowel disease predisposing to colorectal cancer, Barrett's esophagus as a precursor of esophageal adenocarcinoma, and Helicobacter pylori-induced gastric cancer. The classical activation pathway of NF-κB signaling has been identified as regulating several sporadic and inflammation-associated gastrointestinal tract malignancies. Emerging evidence suggests that the alternative NF-κB signaling pathway also exerts a distinct influence on these processes. This review brings together current knowledge of the role of the alternative NF-κB signaling pathway in the gastrointestinal tract, with a particular emphasis on inflammation-associated cancer development.

Microarray analysis identifies matrix metalloproteinases (MMPs) as key genes whose expression is up-regulated in human adipocytes by macrophage-conditioned medium.

White adipose tissue exhibits inflammation as tissue mass expands in obesity, involving macrophage infiltration and a direct inflammatory response by adipocytes. DNA microarrays and conditioned medium have been used to examine the effects of macrophages on global gene expression in human adipocytes. SGBS adipocytes, differentiated in culture, were treated with macrophage-conditioned medium (U937 cells) for 4 or 24 h; control cells received unconditioned medium. Agilent arrays comprising 44,000 probes were used to analyse gene expression. Microarray analysis identified 1,088 genes differentially expressed in response to the conditioned medium at both 4 and 24 h (754 up-regulated, 334 down-regulated at 24 h); these included genes associated with inflammation and macrophage infiltration. A cluster of matrix metalloproteinase genes were highly up-regulated at both time-points, including MMP1, MMP3, MMP9, MMP10, MMP12 and MMP19. At 4 and 24 h, MMP1 was the most highly up-regulated gene (>2,400-fold increase in mRNA at 24 h). ELISA measurements indicated that substantial quantities of MMP1 and MMP3 were released from adipocytes incubated with conditioned medium, with little release by control adipocytes. Treatment with TNFalpha induced substantial increases in MMP1 (>100-fold) and MMP3 (27-fold) mRNA level and MMP1 and MMP3 release in adipocytes, suggesting that this cytokine could contribute to the stimulation of MMP expression by macrophages. In conclusion, macrophage-secreted factors induce a major inflammatory response in human adipocytes, with expression of MMP family members being strongly up-regulated. The induction of MMP1 and other MMPs suggests that macrophages stimulate tissue remodelling during adipose tissue expansion in obesity.

Gastrin-induced miR-222 promotes gastric tumor development by suppressing p27kip1.

BACKGROUND AND AIMS: Elevated circulating concentrations of the hormone gastrin contribute to the development of gastric adenocarcinoma and types-1 and 2 gastric neuroendocrine tumors (NETs). MicroRNAs (miRNAs) are small non-coding RNAs that post-transcriptionally regulate proteins which in turn influence various biological processes. We hypothesised that gastrin induces the expression of specific gastric miRNAs within CCK2 receptor (CCK2R) expressing cells and that these mediate functionally important actions of gastrin. RESULTS: Gastrin increased miR-222 expression in AGSGR cells, with maximum changes observed at 10 nM G17 for 24 h. Signalling occurred via CCK2R and the PKC and PI3K pathways. miR-222 expression was increased in the serum and gastric corpus mucosa of hypergastrinemic INS-GAS mice and hypergastrinemic patients with autoimmune atrophic gastritis and type 1 gastric NETs; it decreased in patients following treatment with the CCK2R antagonist netazepide (YF476). Gastrin-induced miR-222 overexpression resulted in reduced expression and cytoplasmic mislocalisation of p27kip1, which in turn caused actin remodelling and increased migration in AGSGR cells. MATERIALS AND METHODS: miRNA PCR arrays were used to identify changes in miRNA expression following G17 treatment of human gastric adenocarcinoma cells stably transfected with CCK2R (AGSGR). miR-222 was further investigated using primer assays and samples from hypergastrinemic mice and humans. Chemically synthesised mimics and inhibitors were used to assess cellular phenotypical changes associated with miR-222 dysregulation. CONCLUSIONS: These data indicate a novel mechanism contributing to gastrin-associated gastric tumor development. miR-222 may also be a promising biomarker for monitoring gastrin induced premalignant changes in the stomach.

The role of proteasome beta subunits in gastrin-mediated transcription of plasminogen activator inhibitor-2 and regenerating protein1.

The hormone gastrin physiologically regulates gastric acid secretion and also contributes to maintaining gastric epithelial architecture by regulating expression of genes such as plasminogen activator inhibitor 2 (PAI-2) and regenerating protein 1 (Reg1). Here we examine the role of proteasome subunit PSMB1 in the transcriptional regulation of PAI-2 and Reg1 by gastrin, and its subcellular distribution during gastrin stimulation. We used the gastric cancer cell line AGS, permanently transfected with the CCK2 receptor (AGS-GR) to study gastrin stimulated expression of PAI-2 and Reg1 reporter constructs when PSMB1 was knocked down by siRNA. Binding of PSMB1 to the PAI-2 and Reg1 promoters was assessed by chromatin immunoprecipitation (ChIP) assay. Subcellular distribution of PSMB1 was determined by immunocytochemistry and Western Blot. Gastrin robustly increased expression of PAI-2 and Reg1 in AGS-GR cells, but when PSMB1 was knocked down the responses were dramatically reduced. In ChIP assays, following immunoprecipitation of chromatin with a PSMB1 antibody there was a substantial enrichment of DNA from the gastrin responsive regions of the PAI-2 and Reg1 promoters compared with chromatin precipitated with control IgG. In AGS-GR cells stimulated with gastrin there was a significant increase in the ratio of nuclear:cytoplasmic PSMB1 over the same timescale as recruitment of PSMB1 to the PAI-2 and Reg1 promoters seen in ChIP assays. We conclude that PSMB1 is part of the transcriptional machinery required for gastrin stimulated expression of PAI-2 and Reg1, and that its change in subcellular distribution in response to gastrin is consistent with this role.

A microarray analysis of the hypoxia-induced modulation of gene expression in human adipocytes.

The effect of hypoxia on global gene expression in human adipocytes has been examined using DNA microarrays. Adipocytes (Zen-Bio, day 12 post-differentiation) were exposed to hypoxia (1% O(2)) or 'normoxia' (21% O(2)) for 24 h and extracted RNA probed with Agilent arrays containing 41,152 probes. A total of 1346 probes were differentially expressed (>2.0-fold change, P < 0.01) in response to hypoxia; 650 genes were up-regulated (including LEP, IL6, VEGF, ANGPTL4) and 650 down-regulated (including ADIPOQ, UCP2). Major genes not previously identified as hypoxia-sensitive in adipocytes include AQP3, FABP3, FABP5 and PPARGC1A. Ingenuity analysis indicated that several pathways and functions were modulated by hypoxia, including glucose utilization, lipid oxidation and cell death. Network analysis indicated a down-regulation of p38/MAPK and PGC-1α signalling in the adipocytes. It is concluded that hypoxia has extensive effects on human adipocyte gene expression, consistent with low O(2) tension underlying adipose tissue dysfunction in obesity.

Stimulation of inflammatory gene expression in human preadipocytes by macrophage-conditioned medium: upregulation of IL-6 production by macrophage-derived IL-1ß.

The aim of this study was to examine the effects of macrophage secretions on global gene expression in human preadipocytes using microarrays. Preadipocytes were cultured with unconditioned or conditioned medium from U937 macrophages, and gene expression examined with Agilent arrays (43,000 probes). 472 transcripts were differentially regulated (>2-fold difference; P<0.05) between preadipocytes in the conditioned medium compared to the unconditioned; 401 were upregulated and 71 downregulated. The upregulated transcripts were particularly linked to inflammation, including IL-1ß, IL-6, and CCL20 (16.8-, 10.0-, and 8.9-fold increases, respectively) together with matrix metalloproteinases (MMP3, MMP9 and MMP12). Major pathways regulated by the conditioned medium were linked to inflammation, macrophage infiltration and lipid accumulation. Network analysis identified NFkB and IL-1ß as central nodes in the upregulation of multiple inflammation-related genes. Treatment with an IL-1ß neutralising antibody abolished the stimulation of IL-6 secretion by conditioned medium, indicating that IL-1ß is a key regulator of preadipocyte IL-6 production. Macrophages evoke extensive changes in preadipocyte gene expression.