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

Abstract

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.

Cytokine Regulation of the Trefoil Factor Family Binding Protein GKN2 (GDDR/TFIZ1/blottin) in Human Gastrointestinal Epithelial Cells

“…Looking at results obtained from the TRANSFAC database (Figure 1), we notice that in promoters of GKN2 and TFF genes there are several predicted consensus sites for different but, in part, shared transcription factors: GATA-6, NFκB, HNF-3β, C/EBPβ and E-box elements. E-box elements are enhancer elements with the consensus sequence CANNTG that play a regulatory role in the control of transcription, and bind with basic helix-loop-helix type transcription factors [59]. We note that of the 31 total control elements for the GKN2 gene segment that we have analysed, only 12 were proximal to the TATA box, whereas TFF1/2/3 genes have 14, 9 and 12 elements respectively in a similar position.…”

Trefoil Factor 2 (Tff2) Deficiency in Murine Digestive Tract Influences the Immune System

“…(C) Northern Blot analysis of stomach RNA using TFF1 (top) and TFF2 (bottom) exonprobes. TFF2-/-mice show no expression of TFF2 mRNA regulated by both pro-inflammatory [20,21] and antiinflammatory cytokine expression [22,23] acting through diverse transcription factors [21, [24][25][26]. Treatment with exogenous TFF peptides demonstrated both protective and healing functions in stomach and colon [27][28][29].…”

Trefoil factors

“…GATA4 and 5 have been shown to be epigenetically silenced in gastric cancer [55], as is TFF1 [56]. Evidence for GATAregulated trefoil gene transcription has come from experiments in which co-transfection of TFF reporter genes and GATA6 expression vectors in gastric cancer cell lines resulted in TFF1 and 2, but not TFF3, activation [57]. In addition, transient expression of GATA5 in a gastric cancer cell line, AZ521, led to TFF1 protein expression and secretion [55].…”

“…The TFF2 promoter (and to a lesser extent the TFF1 promoter) has been shown to be strongly activated by USF, resulting in augmented TFF2 transcription [57]. USF proteins are associated with growth inhibition [58] as are certain trefoil peptides [59], suggesting that activation of USF binding sites on TFF2 and TFF1 promoters by endogenous stimuli may contribute to inhibition of cell proliferation.…”