Jonckheere Nicolas, Van Der Sluis Maria, Velghe Amélie, Buisine Marie-Pierre, Sutmuller Marjolein, Ducourouble Marie-Paule, Pigny Pascal, Büller Hans A, Aubert Jean-Pierre, Einerhand Alexandra W C, Van Seuningen Isabelle
Unité INSERM U560, Place de Verdun, 59045 Lille Cedex, France.
Biochem J. 2004 Feb 1;377(Pt 3):797-808. doi: 10.1042/BJ20030948.
The nucleotide sequence of the pMS1 clone was submitted to the GenBank Nucleotide Sequence Database under accession number AF288076. 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-beta (transforming growth factor-beta), and Sp1, an important factor in the regulation of MUC5AC. This led us to study Muc5ac regulation by TGF-beta. We show that exogenous addition of TGF-beta 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-beta-mediated up-regulation of Muc5ac and enhance our understanding as to how Muc5ac is regulated in certain pathologies of the gastrointestinal tract.
pMS1克隆的核苷酸序列已提交至GenBank核苷酸序列数据库，登录号为AF288076。胃肠道癌症中粘蛋白基因表达的变化被认为与该疾病的发展有关。在我们实验室，我们之前已表明MUC5AC在直肠乙状结肠绒毛状腺瘤中异常表达。然而，这种表达改变的调控机制尚不清楚。为了在转录水平研究其调控，我们分离并鉴定了小鼠Muc5ac粘蛋白基因5'侧翼区域的5.5 kb片段。启动子两侧有一个TATA盒，转录起始位点位于TATA盒下游22 bp处。序列分析显示，Smad4（转化生长因子-β激活的信号级联反应中的一个关键因子）和Sp1（MUC5AC调控中的一个重要因子）的结合位点密度很高。这促使我们研究TGF-β对Muc5ac的调控。我们发现，向细胞中外源添加TGF-β可诱导Muc5ac内源性表达、启动子活性以及Smad4与启动子的结合。通过共转染研究，我们表明Smad4对Muc5ac启动子激活至关重要，且它不与Smad2或Smad3协同作用。通过凝胶阻滞和共转染试验，我们确定Sp1和Sp3是Muc5ac表达的重要调节因子，并表明Smad4和Sp1以协同方式反式激活Muc5ac启动子活性。总之，这些结果为TGF-β介导的Muc5ac上调的分子机制带来了新的见解，并增强了我们对Muc5ac在胃肠道某些病理状态下如何被调控的理解。
