SUMMARY:It has been advanced that the trefoil factor (TFF) 1 gene is a candidate tumor-suppressor gene and may be involved in the development and/or progression of human gastric cancer. We aimed to clarify the putative role of TFF1 in gastric carcinogenesis. Ninety gastric carcinomas and eight gastric carcinoma-derived cell lines were screened for TFF1 mutations; subsets of the primary tumors and of the cell lines were subjected to loss of heterozygosity (LOH), immunohistochemistry, and promoter methylation analyses. TFF1 mutations were not detected in any of 90 gastric carcinomas. Eight (28%) of 28 informative cases displayed LOH at the TFF1 locus and absence of TFF1 staining by immunohistochemistry. These results indicate a frequent loss of TFF1 expression in gastric carcinomas through a mutation-independent mechanism. Extensive TFF1 promoter methylation was observed in nonexpressing gastric carcinoma-derived cell lines and tissues. Expressing cell lines, as well as normal gastric mucosa, presented little or no methylation of the promoter. Gastric carcinoma DNA presented de novo methylation of the promoter. These results point to the involvement of promoter methylation in the shutting down of TFF1. We conclude that TFF1 point mutations seem to be a rare event in gastric carcinogenesis. The loss of expression of TFF1 in a proportion of gastric carcinomas may be explained by LOH and methylation of the TFF1 promoter region. Our results further support the role of TFF1 inactivation in gastric carcinogenesis, in agreement with the results obtained in the Tff1-knockout mice model. (Lab Invest 2002, 82:1319 -1326.

Background and Aims: Trefoil peptides (TFF1, TFF2 and TFF3) are acute phase proteins up-regulated in response to gastrointestinal mucosal damage. They promote cell migration, protect and heal the mucosa and may function as tumorsuppressors. We assumed them to be regulated by the proinflammatory cytokines interleukin-1β (IL1β) and interleukin-6 (IL6), which trigger the transcriptional factors NF-ĸB and C/EBPβ. Methods: Following IL1β and IL6 stimulation, expression of TFF genes was analyzed in gastrointestinal cell lines HT-29 and KATO III by reporter gene assays using TFF promoter constructs and by quantitative real-time PCR. NF-ĸB and C/EBPβ were transiently co-expressed. Results: We have functionally identified transcription factors NF-ĸB and C/EBPβ to inhibit transcription of human TFF genes. Down-regulation of TFF transcription is also observed by IL1β and IL6, suggesting crosstalk with or in response to the immune system. IL1β and IL6 caused a 3- to 11-fold reduction in TFF mRNA expression, displayed in real-time PCR. Conclusions: Down-regulation of intestinal trefoil factor TFF3 due to transcriptional repression by IL1β through NF-ĸB as well as by IL6 through C/EBPβ activation in vitro may reflect the situation in vivo and may contribute to ulceration and decreased wound healing during inflammatory bowel disease. Additionally, IL1 and IL6 over-expression in chronic gastritis may lead to mucosal damage and gastric carcinogenesis through transcriptional repression of TFF1 and TFF2.

The trefoil factor family (TFF) peptides are important in gastro-intestinal mucosal protection and repair. Their mechanism of action remains unclear and receptors are sought. We aimed to identify and characterise proteins binding to TFF2. A fusion protein of mouse TFF2 with alkaline phosphatase was generated and used to probe 2-D protein blots of mouse stomach. The resulting spots were analysed by MS. The protein identified was characterised by bioinformatics, rapid amplification of cDNA ends, in situ hybridisation (ISH) and immunohistochemistry (IHC). Functional assays were performed in gastrointestinal cell lines. A single major murine protein was identified and named blottin. It was previously unknown as a translated product. Blottin is also present in rat and human; the latter gene is also known as GDDR. The predicted full-length proteins are 184 amino acids long (20 kDa), reducing to 164 amino acids (18 kDa) after signal peptide cleavage. ISH of gastrointestinal tissues shows abundant blottin mRNA in gastric surface and foveolar epithelium. IHC shows cytoplasmic staining for blottin protein, and by immunoelectron microscopy in mucus granules and Golgi stacks. Previous work showed that blottin is down-regulated in gastric cancers. Blottin contains a BRICHOS domain, and has 56% similarity with gastrokine-1. Cultured HT-29 cells express blottin and show increased DNA synthesis with antiblottin antibody; however, this effect is reversed by the immunising peptide. We have identified and characterised a TFF2-binding protein produced by gastric epithelium. Blottin may play a role in gastrointestinal mucosal protection and modulate gut epithelial cell proliferation.

On an mRNA level we found 128 differentially expressed genes. We observed modulation of a number of crucial genes involved in innate and adaptive immunity in the TFF2-/- mice. Expression of proteasomal subunits genes (LMP2, LMP7 and PSMB5) involved in the MHC class I presentation pathway were modulated indicating the formation of immunoproteasomes improving antigen presentation. Expression of one subunit of a transporter (TAP1) responsible for importing degraded antigens into ER was increased, similarly to the BAG2 gene that modulates chaperone activity in ER helping proper loading on MHC class I molecules. Several mouse defensin (cryptdin) genes coding important intestinal microbicidal proteins were up-regulated as a consequence of TFF2 deficiency. Normally moderate expression of TFF3 was highly increased in stomach.

The protective effect of Trefoil Factor Family (TFF) proteins in the gastrointestinal tract by promoting the healing of injured mucosa is well known. An increasing body of evidence connects TFFs, especially, TFF2 and TFF3, with a possible role in immune regulation. TFF2 is able to inhibit lipopolysaccharide-induced nitric oxide production in monocytes and can potently limit leukocyte recruitment at the site of injury. An analysis of gene expression in gastrointestinal tissue of TFF2-deficient mice reveals some new aspects of TFF2's role in the immune response.