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The trefoil peptides spasmolytic polypeptide (SP), intestinal trefoil factor (ITF), and pS2 show lineage-specific expression in the normal gut and are strongly induced after mucosal injury. We assessed the relationship between this induction and the development of the regenerative epithelial lineage over time in the rat stomach and verified these observations in the metaplastic and dysplastic human stomach. Antral or colonic ulcers were induced in Wistar rats by application of serosal acetic acid and tissues harvested 2 hours to 125 days later. Human endoscopic biopsies or gastric resection specimens were also assessed. Tissues were examined by radioimmunoassay, immunoblotting, or immunohistochemistry for ITF, SP, and transforming growth factor alpha (rat) or ITF and pS2 (human) expression. ITF and SP mRNA in antral ulcer margins was localized by in situ hybridization. ITF and SP peptide expression rose steadily in ulcer margins after 4 days, with the rise in ITF being more pronounced. By 40 days, several hundred-fold elevations in ITF levels were present, with a field effect in uninvolved mucosa. Hyperproliferative, elongated glands of undifferentiated cells expressing abundant trefoil peptides and acid sulfomucins were present after day 12 and persisted after ulcer healing. ITF mRNA was aberrantly expressed in basal and mid-regions of these regenerative glands. In contrast, transforming growth factor alpha peptide expression rose promptly after injury then fell to baseline levels with healing. Seven months after injury, gastric atrophy, intestinal metaplasia, and severe dysplasia with conserved ITF expression were seen. ITF was also induced in human intestinal metaplasia and conserved in all gastric cancers, whereas expression of the gastric peptide pS2 was progressively reduced in the progression from metaplasia to dysplasia. Persistent, selective overexpression of ITF, possibly acting in an autocrine fashion, is a feature of regeneration after antral ulceration, and may provide insight into the nature of metaplastic phenotypes arising from chronic gastric injury. The loss of pS2 expression in metaplasia and cancer supports a role for this protein in gastric tumor suppression.

Members of the trefoil factor (TFF) family are mucin-associated polypeptides that are expressed along the entire length of the gastrointestinal tract. TFFs have been proposed to play a role in mucosal defence through both protective and reparative mechanisms. The potential relationship between TFFs and mucins in non-gut glycoprotein-secreting epithelia has not been fully explored. In the present study we identified TFF2 and TFF3 mRNA and peptide in rat lymphoid tissues, demonstrated that TFF peptide expression in rat spleen increased 1.5- to 3-fold following experimental induction of the immune response, and showed that hTFF2 and hTFF3 (1-5 mg/ml) stimulated migration of human monocytes. Our data suggest that TFFs may in part be involved in the repair of injury through the modulation of the inflammatory response.

Trefoil peptides are members of a unique family of proteins found predominately throughout the gastrointestinal tract, whose proposed functions include mucus stabilization, stimulation and/or differentiation of epithelial cells during wound repair. Recent trefoil knockout studies have reported delays in epithelial cell migration or maturation pathways together with almost a complete lack of mucus. In order to fully explore the role of trefoil peptides in gastrointestinal maturation, these studies were undertaken to accurately characterize the expression of trefoil peptides in the developing rat gut. The results of RPA suggest that trefoil mRNA's are expressed as early as 15 days post coitus (dpc) in the intestine and stomach. Proteins are detected at 17 dpc by radioimmunoassay and immunohistochemical studies, which localize trefoil peptide expression to the lumenal surface of epithelial cells. At 17 dpc the gut is lined by pseudo-stratified, undifferentiated epithelial cells. Polarized, columnar cells are not detected until at least 18 dpc, with sparse mucus staining and parietal cell markers not being detected until 18 and 19 dpc respectively. This data demonstrates that trefoil peptides are early markers of epithelial cell maturation in the developing rat gut. The time course of their expression, well before the mucus cell type is specified, suggests a potential role in epithelial cell differentiation.

Intestinal trefoil factor (ITF) gene expression was detected in five colon cancer cell lines. ITF was synthesized by mucous cells of LIM 1215 and LIM 1863 lines, from which it is secreted constitutively. The ITF mRNA transcript was estimated to be 0.6 kb. In LIM 1215 cells, the expression of ITF was potently and dose-dependently inhibited by short-chain fatty acids (butyrate > propionate > acetate) within 8 h of application. The inhibitory effect of butyrate was ablated by actinomycin D and preceded its effects on differentiation of LIM 1215 cells as indicated by induction of alkaline phosphatase activity and counting of periodic acid-Schiff-positive cells. The human ITF promoter contained an 11-residue consensus sequence with high homology to the butyrate response element of the cyclin D1 gene. Mobility shift assays show specific binding of this response element to nuclear protein extracts of LIM 1215 cells. We conclude that butyrate inhibits ITF expression in colon cancer cells and that this effect may be mediated transcriptionally and independently of its effects on differentiation.