Trefoil factor family (TFF) peptides are major secretory products of mucous epithelia and play a multifunctional role in cytoprotection, apoptosis, and immune response. Recently, a TFF2-binding protein was discovered in mice and named blottin. It is down-regulated in gastric cancer (GDDR), abundant in human gastric surface (TFIZ1) and its similarity to gastrokine-1 led to the gene’s name GKN2. To investigate the mode of GKN2 regulation activity of a luciferase reporter gene, controlled by the GKN2 promoter, was monitored upon treatment with various pro-inflammatory (TNF-α, IL-1β, IL-6, IFN-γ) and anti-inflammatory (TGF-β1) cytokines using gastric (AGS, KATO III) and colonic (HT-29) cell lines. To assess the direct role of transcription factors (NFĸB, HNF-3β, hGATA6) in regulating GKN2 we performed transient co-transfection of their expression plasmids and the reporter gene construct. GKN2 gene was down-regulated by pro-inflammatory cytokines in all tested cell lines while up-regulated by TGF-β1 only in the colonic cell line. GKN2 expression was significantly reduced in both gastric adenocarcinoma cell lines by the active form of NFĸB transcription factor, whereas in the colonic cell line an up-regulation was noticed. Down-regulation by IL-6 was mediated by C/EBPβ transcription factor in case of HT-29 but not of KATO III cells. We conclude that the regulation of GKN2 parallels that of TFF genes, indicating that together they may play an important role in maintaining the homeostasis of the gastrointestinal tract.

Tff peptides are secreted mainly by the gastrointestinal epithelial cells and their primary role is maintaining normal structure and function of mucous epithelia. Ongoing studies on their expression pattern have disclosed other sites of their synthesis thus revealing additional physiological functions in the organism. Here we present new data about Tff3 expression in the cochlea of the rodent inner ear. On the basis of RT-PCR we describe the presence of Tff3 transcripts in both, a mouse cDNA library isolated from whole cochleae from postnatal days 3-15 (P3-P15), and also in cochlear tissue. By using a riboprobe for the fragment containing exon 1, 2 and 3 of Tff3, in situ hybridization, localized Tff3 signals in neurons of spiral ganglion and vestibular organ. We did not observe any abnormalities in the middle ear of Tff3 knock-out mice, neither did histological examination of the inner ear indicate any gross morphological changes in the cochlea. However, ABR (auditory evoked brain stem responses) audiograms revealed that the Tff3 knock-out animals show an accelerated presbyacusis and a hearing loss of about 15 dB at low frequencies increasing to 25 dB loss at higher frequencies. These findings suggest that Tff3 could play a role in neurosensory signaling. Further studies are needed to clarify this new function in the auditory system.

The 3 members of the mammalian trefoil factor family (TFF) are expressed and secreted as cytoprotective peptides along the entire length of the normal gastrointestinal tract. More recently, they were shown to display multifunctional properties. Goblet cells of the small and large intestine constitute a major source for the synthesis of the third family member, TFF3 (formerly intestinal trefoil factor, ITF). TFF3, like the other family members, is rapidly up-regulated in response to physical wounding of the digestive tract. In addition, Tff3 was also detected in the posterior pituitary gland. Apart from this Tff3 function as a neuropeptide, also presence of Tff3 in the mouse cochlea was noted and Tff3-deficient animals display hearing impairment and accelerated presbyacusis. To elucidate Tff3's mode of function and its unexpected contribution to the hearing process, we strived to determine Tff3's interacting partners and to establish the functional network. To this end, we used a protein-protein binding assay based on a specific transcriptional regulation in yeast cells (the yeast-two-hybrid assay). We looked for interacting partners of Tff3 in a mouse cochlea cDNA library (from donors aged 3-15 days, P3-P15). Our data show that several binding candidates exist and that they could contribute to the known involvement of the trefoil peptides to apoptosis.