Perrais M, Pigny P, Buisine M P, Porchet N, Aubert J P, Van Seuningen-Lempire I
Unité INSERM 377, Place de Verdun, 59045 Lille Cedex, France.
J Biol Chem. 2001 May 4;276(18):15386-96. doi: 10.1074/jbc.M010534200. Epub 2001 Jan 30.
In gastric cancer, altered expression of MUC1, MUC2, MUC5AC, and MUC6 mucin genes has already been described. We show in this report by the means of in situ hybridization, reverse transcriptase-polymerase chain reaction, and transfection assays that MUC5B is also abnormally expressed in gastric carcinomatous tissues and cell lines. We thus undertook to elucidate the molecular mechanisms that regulate the transcription of MUC5B in gastric cancer cells. To this end, high expressing (KATO-III) and low expressing (AGS) gastric cancer cell lines were chosen to study human mucin gene MUC5B expression and promoter activity. Sequencing of the promoter region revealed a distal TATA box located 1 kilobase upstream of the proximal TATA box. Functional activity of the promoter was addressed by using deletion mutants covering 2044 nucleotides upstream of the MUC5B transcription start site. We identified a distal promoter 10 times more active than the proximal promoter in KATO-III cells. In AGS cells, both promoters, much less active, showed the same range of activity. Binding assays allowed us to show that the transcription factor ATF-1 binds to a cis-element present in the distal promoter. Sp1, which binds to both promoters specifically transactivates the proximal promoter. Treatment of transfected cells with PMA, cholera toxin A subunit, and calcium ionophore showed that only PMA led to a substantial activation of the distal promoter. MUC5B 5'-flanking region having a high GC content, influence of methylation on the MUC5B expression was assessed. Our results indicate that repression of MUC5B expression visualized in AGS cells is due in part to the presence of numerous methylated cytosine residues throughout the 5'-flanking region. Altogether these results demonstrate that MUC5B expression in gastric cancer cells is governed by a highly active distal promoter that is up-regulated by protein kinase C and that repression is under the influence of methylation.
在胃癌中，已有人描述过MUC1、MUC2、MUC5AC和MUC6粘蛋白基因的表达改变。我们在本报告中通过原位杂交、逆转录聚合酶链反应和转染试验表明，MUC5B在胃癌组织和细胞系中也异常表达。因此，我们着手阐明调节胃癌细胞中MUC5B转录的分子机制。为此，选择高表达（KATO-III）和低表达（AGS）的胃癌细胞系来研究人粘蛋白基因MUC5B的表达和启动子活性。启动子区域测序显示，在近端TATA盒上游1千碱基处有一个远端TATA盒。通过使用覆盖MUC5B转录起始位点上游2044个核苷酸的缺失突变体来研究启动子的功能活性。我们发现，在KATO-III细胞中，远端启动子的活性比近端启动子高10倍。在AGS细胞中，两个启动子的活性都低得多，但活性范围相同。结合试验表明，转录因子ATF-1与远端启动子中存在的一个顺式元件结合。与两个启动子都结合的Sp1特异性地反式激活近端启动子。用佛波酯、霍乱毒素A亚基和钙离子载体处理转染细胞表明，只有佛波酯能导致远端启动子的显著激活。由于MUC5B 5'-侧翼区具有高GC含量，因此评估了甲基化对MUC5B表达的影响。我们的结果表明，AGS细胞中可见的MUC5B表达抑制部分归因于整个5'-侧翼区存在大量甲基化胞嘧啶残基。总之，这些结果表明，胃癌细胞中MUC5B的表达受一个高活性远端启动子的调控，该启动子受蛋白激酶C上调，而抑制作用受甲基化影响。
