Tumour necrosis factor alpha and nuclear factor kappaB inhibit transcription of human TFF3 encoding a gastrointestinal healing peptide.

BACKGROUND: and aims: Tumour necrosis factor alpha (TNF-alpha) induction of nuclear factor kappaB (NFkappaB) activation plays a major role in the pathogenesis of inflammatory bowel disease (IBD). Trefoil factor family peptides TFF1, TFF2, and TFF3 exert protective, curative, and tumour suppressive functions in the gastrointestinal tract. In this study, we investigated effects of the TNF-alpha/NFkappaB regulatory pathway by TNF-alpha on expression of TFFs. METHODS: After TNF-alpha stimulation, expression of TFF genes was analysed by quantitative real time polymerase chain reaction and by reporter gene assays in the gastrointestinal tumour cell lines HT-29 and KATO III. Additionally, NFkappaB subunits and a constitutive repressive form of inhibitory factor kappaB (IkappaB) were transiently coexpressed. In vivo, morphological changes and expression of TFF3, mucins, and NFkappaB were monitored by immunohistochemistry in a rat model of 2,4,6-trinitrobenzene sulphonic acid induced colitis. RESULTS: TNF-alpha stimulation evoked up to 10-fold reduction of TFF3 expression in the colon tumour cell line HT-29. Downregulation of reporter gene transcription of TFF3 was observed with both TNF-alpha and NFkappaB, and was reversible by IkappaB. In vivo, the increase in epithelial expression of NFkappaB coincided with reduced TFF3 expression during the acute phase of experimental colitis. CONCLUSIONS: Downregulation of intestinal trefoil factor TFF3 is caused by repression of transcription through TNF-alpha and NFkappaB activation in vitro. In IBD, perpetual activation of NFkappaB activity may contribute to ulceration and decreased wound healing through reduced TFF3.

Gastroprotective peptide trefoil factor family 2 gene is activated by upstream stimulating factor but not by c-Myc in gastrointestinal cancer cells.

BACKGROUND: Damage to the gastrointestinal mucosa results in the acute up-regulation of the trefoil factor family peptides TFF1, TFF2, and TFF3. They possess protective, healing, and tumour suppressive functions. Little is known about the regulation of TFF gene expression. The promoters of all three TFF genes contain binding sites (E box) for upstream stimulating factor (USF) and Myc/Max/Mad network proteins. AIMS: To determine the nature and function of transcription factors that bind to these E boxes and to understand their role for TFF gene expression. METHODS: TFF promoter activities were determined by reporter gene assays. DNA binding was monitored by electromobility shift assays and by chromatin immunoprecipitation analyses. Expression of endogenous TFF was determined by multiplex RT-PCR. RESULTS: It was observed that the TFF2 promoter is specifically and efficiently activated by USF transcription factors but not by c-Myc. USF displayed comparable binding to a high affinity Myc/Max binding site compared with the three TFF E boxes, while c-Myc exhibited lower affinity to the TFF E boxes. In contrast, pronounced binding differences were observed in cells with a strong preference for USF to interact specifically with the TFF2 E box, while Myc was not above background. Exogenous expression of USF was sufficient to activate the chromosomal TFF2 and to a lesser extent, the TFF1 gene. CONCLUSION: These findings define USF factors as regulators of the TFF2 gene and suggest that promoter specific effects are important for a pronounced gene activation of this cytoprotective peptide.

Aspirin promotes TFF2 gene activation in human gastric cancer cell lines.

Trefoil factor family (TFF) peptides promote cell migration, heal the mucosa and may suppress tumor growth. In reporter gene assays we show that aspirin (1-12 mM) evokes a six-fold up-regulation of TFF2, but not TFF1 and TFF3 transcription in human gastrointestinal cell lines. 6 h after application up-regulation of endogenous TFF2 mRNA was observed. TFF2 transcription was enhanced by indomethacin and arachidonic acid but repressed by staurosporine, suggesting mediation via protein kinase C. We mapped an aspirin responding element -546 to -758 bp upstream of TFF2. Up-regulation of TFF2 by aspirin may partially explain the chemopreventive potential of low dose aspirin in gastrointestinal carcinogenesis.

Transcription factor GATA-6 activates expression of gastroprotective trefoil genes TFF1 and TFF2.

One of the early events in inflammation and epithelial restitution of the gastrointestinal tract is the up-regulation of secretory peptides belonging to the trefoil factor family (TFF) that promote cell migration, protect and heal the mucosa. Their major expression site is stomach (TFF1, TFF2) and intestine (TFF3). Located in the 5'-flanking region of the genes are several consensus sites for members of the GATA transcription factors known to control gut-specific gene expression. By reverse transcription-PCR (RT-PCR), GATA-6 was shown to be expressed in a variety of tumor cell lines of gastric, intestinal and pancreatic origin. In MKN45, KATOIII and LS174T, cotransfection with TFF reporter genes and GATA-6 expression vectors revealed that GATA-6 activates TFF1 and TFF2 4-6-fold, without an effect on TFF3. The functional contribution of GATA binding sequences in the reverse orientation was further characterized by reporter gene assays using TFF2 deletion constructs and by gel shift experiments.