Transcriptional activation of the murine Muc5ac mucin gene in epithelial cancer cells by TGF-beta/Smad4 signalling pathway is potentiated by Sp1

Changes in the expression of mucin genes in gastrointestinal cancers is thought to contribute to the development of the disease. In our laboratory we have shown previously that MUC5AC is aberrantly expressed in rectosigmoid villous adenomas. However, the regulatory mechanisms underlying that altered profile of expression is unknown. In order to study its regulation at the transcriptional level, we have isolated and characterized 5.5 kb of the 5´-flanking region of the mouse Muc5ac mucin gene. The promoter is flanked by a TATA box and a transcriptional start site is located 22 bp downstream of the TATA box. Analysis of the sequence showed a high density of binding sites for Smad4, an essential factor in the signalling cascade activated by TGF-β (transforming growth factor-β), and Sp1, an important factor in the regulation of MUC5AC. This led us to study Muc5ac regulation by TGF-β. We show that exogenous addition of TGF-β to the cells induces Muc5ac endogenous expression, promoter activity and Smad4 binding to the promoter. By co-transfection studies we show that Smad4 is essential for Muc5ac promoter activation and that it does not synergize with Smad2 or Smad3. By gel-retardation and co-transfection assays, we identified Sp1 and Sp3 as important regulators of Muc5ac expression and showed that Smad4 and Sp1 act in a co-operative manner to transactivate Muc5ac promoter activity. Altogether these results bring new insights into the molecular mechanisms of TGF-β-mediated up-regulation of Muc5ac and enhance our understanding as to how Muc5ac is regulated in certain pathologies of the gastrointestinal tract.