Transcriptional regulation of the human trefoil factor, TFF1, by gastrin

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University of Newcastle (2003) J Physiol 549P, C12

Oral Communications: Transcriptional regulation of the human trefoil factor, TFF1, by gastrin

Zara E. Khan *, Timothy C. Wang† and Rod Dimaline*

*Physiological Laboratory, University of Liverpool, Crown Street, Liverpool L69 3BX and †University of Massachusetts Medical Center, Worcester, MA, USAThe trefoil peptide TFF1 is expressed in surface mucous cells of the gastric epithelium. Trefoil factors are important for restitution and repair of the epithelium and are rapidly up-regulated in response to injury. TFF1 may also act as a gastric tumour suppressor (Park et al. 2000). We identified TFF1 as a gastrin-sensitive gene for the first time by mRNA differential display of gastric corpus from gastrin knockout (GAS-KO) mice versus wild-type (WT) C57/BL6 controls. Gastrin-stimulated expression of TFF1 in gastric corpus from humanely killed mice, and in the gastric cancer cell line AGS-GR (Watson et al. 2001), was determined by Northern blot. Regulation of TFF1 transcription in AGS-GR cells was studied using promoter-reporter assays and electrophoretic mobility shift assays (EMSA).

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TFF1 mRNA abundance in GAS-KO mice was reduced to 63.0 ± 7.0 % of that in WT controls (mean ± S.E.M., P < 0.05, Student’s unpaired t test, n = 5) and increased in a hypergastrinaemic transgenic strain (INS-GAS) to 142.0 ± 10.0 % that of WT controls (P < 0.05). TFF1 mRNA abundance was acutely regulated by gastrin (10-8 M) in AGS-GR cells, increasing to 14.3 ± 3.6-fold above vehicle treated controls (P < 0.02, n = 3) after 3 h and 30.1 ± 6.3-fold after 15 h. A fragment of approximately 1.4 kilobases of the human TFF1 promoter was cloned into the luciferase reporter vector pXP2. Luciferase expression in AGS-GR cells was dose-dependently increased by gastrin (779 ± 137 % of that of vehicle control, P < 0.001, n = 9, with 10-9 M gastrin). TFF1-luciferase expression was induced both directly, and by transactivation through neighbouring cells. The response to gastrin mapped to a 16 bp GC-rich region incorporating overlapping consensus binding sites for the transcription factors SP1 and MAZ. Mutation through this region reduced gastrin-stimulated luciferase expression to between 15 and 36 % of that seen with WT constructs (P < 0.05, n = 3, ANOVA). In EMSAs a radiolabelled probe corresponding to this 16bp region of the TFF1 promoter bound to nuclear extracts from gastrin-stimulated but not unstimulated AGS-GR cells. Binding was dependent upon intact SP1 and MAZ consensus sites, and was disrupted by incubation with antibodies to MAZ or SP3.

We conclude that gastrin exerts tonic control of TFF1 expression, but also has the potential for rapid up-regulation of this trefoil factor. TFF1 is a potential candidate to counter the proliferative effects of gastrin that may occur in response to mucosal injury.

This work is supported by the Wellcome Trust and MRC.

Where applicable, experiments conform with Society ethical requirements.

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