Molecular Alterations in the Stomach of Tff1-Deficient Mice: Early Steps in Antral Carcinogenesis

Abstract

TFF1 is a peptide of the gastric mucosa co-secreted with the mucin MUC5AC. It plays a key role in gastric mucosal protection and repair. Tff1-deficient (Tff1^KO) mice obligatorily develop antropyloric adenoma and about 30% progress to carcinomas. Thus, these mice represent a model for gastric tumorigenesis. Here, we compared the expression of selected genes in Tff1^KO mice and the corresponding wild-type animals (RT-PCR analyses). Furthermore, we systematically investigated the different molecular forms of Tff1 and its heterodimer partner gastrokine-2 (Gkn2) in the stomach (Western blot analyses). As a hallmark, a large portion of murine Tff1 occurs in a monomeric form. This is unexpected because of its odd number of seven cysteine residues. Probably the three conserved acid amino acid residues (EEE) flanking the 7th cysteine residue allow monomeric secretion. As a consequence, the free thiol of monomeric Tff1 could have a protective scavenger function, e.g., for reactive oxygen/nitrogen species. Furthermore, a minor subset of Tff1 forms a disulfide-linked heterodimer with IgG Fc binding protein (Fcgbp). Of special note, in Tff1^KO animals a homodimeric form of Gkn2 was observed. In addition, Tff1^KO animals showed strongly reduced Tff2 transcript and protein levels, which might explain their increased sensitivity to Helicobacter pylori infection.

Keywords: FCGBP; ROS; TFF1; TFF2; carcinogenesis; gastric cancer; inflammation; innate immunity; oxidative stress; trefoil factor.

Figure 1

Semi-quantitative RT-PCR analyses. Tff1 (21x), Tff2 (20x), Tff3 (30x), Gkn1 (21x), Gkn2 (22x), Gkn3 (24x), Fcgbp (31x), Muc5ac (25x), Muc6 (27x), Pdia3 (25x), Agr2 (24x), Gast (26x), Pdx1 (33x), Mist1 (35x), Troy (35x), Cckbr (33x), Irx3 (35x), Epdr1 (33x), Cxcl1 (32x), and Cxcl5 (32x) expression was monitored in the corpus and antrum of 10 male wild-type (WT) and 10 male Tff1^KO mice. The relative gene expression levels were normalized against β-actin (Actb, 24x). The number of amplification cycles is given in parentheses. Significances are indicated by asterisks (*, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001). WT animals: black bars; Tff1^KO animals: white bars.

Figure 2

Western blot analyses of gastric corpus and antrum extracts of wild-type (WT) and Tff1^KO animals. (A) Extracts of two male animals of each group were separated on a 15% SDS-PAGE under reducing conditions, and analyzed for Gkn2 and Tff1, respectively. Left: molecular mass standard. Loading control: reactivity for ß-actin (Actb). (B) Semi-quantitative analysis of the relative Gkn2 content (10 male animals per group). (C) Analysis of the same samples as in (A), but under non-reducing conditions. Marked are the positions of the Gkn2 homodimer ((Gkn2)₂; indicated by an asterisk), the Tff1-Gkn2 heterodimer, the Gkn2 monomer, possible Tff1-X homo/heterodimers (indicated by asterisks), and the Tff1 monomer. (D) Semi-quantitative analysis of relative monomeric Gkn2, heterodimeric Tff1-Gkn2, and homodimeric (Gkn2)₂ contents, respectively (10 male animals per group). (E) Left: Non-reducing SDS-PAGE (Coomassie-stained) of antral extracts pooled from three female WT and three female Tff1^KO animals (55 µg protein per lane) and elution of the regions 1 and 2; M, molecular mass standard. Right: Reducing Western blot analysis of the eluates concerning Gkn2. (F) Left: Non-reducing SDS-PAGE (Coomassie-stained) of a corpus extract of a single male WT mouse and elution of the regions 1–3. Right: Reducing Western blot analysis of the eluates concerning Tff1. Significances are indicated by asterisks (*, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001). WT animals: black bars; Tff1^KO animals: white bars.

Figure 3

Western blot analyses of gastric corpus and antrum extracts of female wild-type (WT) and female Tff1^KO animals. Extracts of two animals per group were separated by 1% agarose gel electrophoresis. Shown are the reactivities for Tff1 and Fcgbp, respectively. As a positive control for Fcgbp, two murine colon extracts were analyzed (C1, C2). The start and the dye bromophenol blue (BPB) are marked on the left.

Figure 4

Western blot analyses of gastric corpus and antrum extracts of wild-type (WT) and Tff1^KO animals. (A) Extracts of three male animals per group were separated on a 15% SDS-PAGE under reducing conditions and analyzed for their Tff2 reactivity. Left: molecular mass standard. Loading control: reactivity for ß-actin (Actb). (B) Semi-quantitative analysis of the relative Tff2 content (10 male animals per group). Significances are indicated by asterisks (***, p ≤ 0.001). WT animals: black bars; Tff1^KO animals: white bars. (C) Analysis of the same samples as in (A), but under non-reducing conditions (post-in-gel reduction). (D) Left: Non-reducing SDS-PAGE (Coomassie-stained) of an antral extract of a single male WT animal (75 µg) followed by elution of the high-(H) and low-molecular mass regions (L1, L2). Additionally, the remaining high-molecular-mass samples not entering the gel were removed from the gel pockets (P). Right: Western blot analysis (reducing conditions) concerning Tff2 of L1, L2, H, P, and an antral extract as a control (c) and semi-quantitative analysis of the relative Tff2 content (six male WT animals; 60–75 µg/lane).