Transcriptional regulation of the human trefoil factor, TFF1, by gastrin1   Zara E Khan, Timothy C Wang, Guanglin Cui, Alfred L Chi, Rod Dimaline  Gastroenterology  Volume 125, Issue 2, Pages 510-521 (August 2003) DOI: 10.1016/S0016-5085(03)00908-9

Figure 1 TFF1 mRNA abundance in gastric fundus mucosa of transgenic mice. (A ) Representative blots from gastrin-deficient mice (GAS-KO) and their background controls, C57/BL6 (wild-type). (B) Representative blots from hypergastrinemic INS-GAS transgenic mice (IG) and their background controls, FVB/N (wild-type). (C ) TFF1 mRNA abundance in gastric fundus mucosa of transgenic mice versus their respective wild-type controls. Values are mean ± SEM (n = 5) and are corrected against GAPDH. ∗P < 0.05 (t test). Gastroenterology 2003 125, 510-521DOI: (10.1016/S0016-5085(03)00908-9)

Figure 2 TFF1-like immunoreactivity in mouse gastric fundus. TFF1-like immunoreactivity was localized in sections from the gastric fundus from 2-month-old male animals. (A ) INS-GAS mouse. (B) Background strain for INS-GAS (FVB/N). (C) GAS-KO mouse. (D) Background strain for GAS-KO (C57/Bl6). Sections were counterstained with hematoxylin. (Original magnification 100×.) Gastroenterology 2003 125, 510-521DOI: (10.1016/S0016-5085(03)00908-9)

Figure 3 Acute up-regulation of TFF1 by gastrin. (A ) Northern blot of TFF1 in AGS-GR cells treated with 10−8 mol/L gastrin (+) or vehicle (−) for 3, 6, 9, or 15 hours. (B) Mean increase in TFF1 mRNA abundance in AGS-GR cells following gastrin treatment. Values are mean ± SEM (n = 3) and are corrected against GAPDH. ∗P < 0.02; ∗∗P < 0.005 (t test). (C ) Western blot of TFF1 (right panel) and GAPDH (left panel) in stomach from a gastrin- (G17) or saline- (Sal) treated GAS-KO mouse. Size markers are indicated in kilodaltons. Gastroenterology 2003 125, 510-521DOI: (10.1016/S0016-5085(03)00908-9)

Figure 4 Activity of TFF1-luciferase reporter vectors in AGS-GR cells. (A ) 5′ flanking region of the human TFF1 gene. Potential cis-regulatory regions are underlined; bases −428 to −332 encompass a complex enhancer region.33 Arrows indicate the starting point of deletional mutants. Numbering is relative to the start of transcription (+1). (B) Constitutive activity of TFF1-luciferase reporter constructs in unstimulated AGS-GR cells. Cells (5 × 10−6) were harvested 24 hours after transfection with 1 μg of TFF1-luciferase constructs as indicated. Luciferase (firefly) activity is expressed relative to the activity of the constitutively active Renilla luciferase construct included in each experiment. Values are mean ± SEM and are derived from a minimum of 3 separate experiments. (C ) Activity of the same constructs in AGS-GR cells stimulated with 10−9 mol/L gastrin for 8 hours. Gastroenterology 2003 125, 510-521DOI: (10.1016/S0016-5085(03)00908-9)

Figure 5 Specificity of the gastrin response. AGS-GR (open bars) or parental AGS (filled bars) cells were transfected with a luciferase promoter-reporter construct containing 1396 base pairs of the TFF1 promoter as outlined in the legend to Figure 4. Cells were stimulated for 8 hours with gastrin (10−9 mol/L) in the presence or absence of the specific gastrin/CCKB-receptor antagonist L740093 (10−7 mol/L). Values are mean ± SEM of 3 separate experiments. ∗Significantly different from control, unstimulated activity, P < 0.002, t test. Gastroenterology 2003 125, 510-521DOI: (10.1016/S0016-5085(03)00908-9)

Figure 6 Mutation of the region −303 to −294. AGS-GR cells were transfected with a 303—base pair TFF1-luciferase construct and stimulated with gastrin (10−9 mol/L) or vehicle as outlined in the legend to Figure 4. Promoter sequences were either wild type or contained mutations in the SP1 motif, the MAZ motif, or both. Values (mean ± SEM) are derived from a minimum of 3 separate experiments and are expressed as percent of wild-type response. ∗Significantly different from wild type, P < 0.005; ∗∗P < 0.0001, ANOVA. Gastroenterology 2003 125, 510-521DOI: (10.1016/S0016-5085(03)00908-9)

Figure 7 Nuclear extracts from AGS-GR cells bind the human TFF1 promoter. Electrophoretic mobility shift assays were performed using nuclear extracts from vehicle-treated or gastrin-stimulated AGS-GR cells and a radiolabeled probe incorporating bases −285 to −314 of the human TFF1 promoter. (A ) Lane 1, radiolabeled probe only; lanes 2–5, radiolabeled probe plus nuclear extract from gastrin-stimulated cells; lanes 6–9, radiolabeled probe plus nuclear extract from vehicle-treated cells. Lanes 3 and 7, 50× excess unlabeled wild-type probe; lanes 4 and 8, 20× excess unlabeled wild-type probe; lanes 5 and 9, 10× excess unlabeled wild-type probe. (B) Lane 1, radiolabeled probe only; lanes 2–5, radiolabeled probe plus nuclear extract from gastrin-stimulated cells. Lane 3, 50× excess unlabeled wild-type probe; lane 4, 50× excess unlabeled probe with SP1 site mutated; lane 5, 50× excess unlabeled probe with MAZ site mutated. (C ) Effect of transcription factor antibodies on binding of nuclear extract from gastrin-stimulated cells. Lane 1, radiolabeled probe only; lanes 2–7, radiolabeled probe plus nuclear extract. Lane 3, SP1 antibody; lane 4, SP2 antibody; lane 5, SP3 antibody; lane 6, SP4 antibody; lane 7, MAZ antibody. Gastroenterology 2003 125, 510-521DOI: (10.1016/S0016-5085(03)00908-9)

Figure 8 The effect of EGF receptor inhibition. AGS-GR cells were transfected with the TFF1-luciferase construct as outlined in the legend to Figure 4. Cells were stimulated for 8 hours by gastrin (10−9 mol/L) or EGF (2 × 10−9 mol/L) in the presence of the tyrphostin AG1478 (3 × 10−6 mol/L) or vehicle. Values (mean ± SEM, n = 3) are expressed as fold increase over unstimulated control. Gastroenterology 2003 125, 510-521DOI: (10.1016/S0016-5085(03)00908-9)

Figure 9 Gastrin-stimulated signaling pathways. AGS-GR cells were transfected with 0.5 μg TFF1-332 luciferase reporter construct and then stimulated with gastrin (10−9 mol/L) for 8 hours or vehicle in the presence or absence of the protein kinase C inhibitor Ro320432 (10−6 mol/L) or the MEK inhibitor PD098059 (2 × 10−5 mol/L). In some experiments, TFF1-332 was transfected together with plasmids encoding dominant negative Ras, Raf, or ERKs26 or control DNA before stimulation with gastrin (10−9 mol/L) for 8 hours or vehicle. Values (mean ± SEM) are expressed as percent of gastrin stimulation in the presence of control (pBSK−) plasmid DNA or vehicle and are derived from a minimum of 3 separate experiments. ∗P < 0.05, ANOVA. Gastroenterology 2003 125, 510-521DOI: (10.1016/S0016-5085(03)00908-9)

Figure 10 Indirect transactivation of TFF1 by gastrin. Parental AGS cells (3 × 105) were transfected with TFF1-1397 or TFF1-332 and then cocultured with equal numbers of AGS-GR cells as described in Materials and Methods. Cocultures were treated with gastrin (10−8 mol/L) for 8 hours. Values (mean ± SEM) are expressed as percent of vehicle-treated control and are from a minimum of 3 separate experiments. ∗P < 0.0005, t test. Gastroenterology 2003 125, 510-521DOI: (10.1016/S0016-5085(03)00908-9)

Figure 11 Expression of TFF1 and CCKBR in response to mucosal injury. TFF1- and CCKBR-like immunoreactivities were visualized 2 days after cryoinjury of mouse gastric fundus mucosa. Expression of both CCKBR and TFF1 was seen at the ulcer margin (A and C, respectively) compared with control animals (B and D). Occasional cells showed colocalization of CCKBR and TFF1 (E). A–D are counterstained with hematoxylin (original magnification 100×). E has no counterstain (original magnification 400×). Gastroenterology 2003 125, 510-521DOI: (10.1016/S0016-5085(03)00908-9)