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Article Dans Une Revue Journal of Biological Chemistry Année : 2001

Aberrant Expression of Human Mucin Gene MUC5B in Gastric Carcinoma and Cancer Cells

Résumé

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 A23187 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 methy-lated 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. Mucins are high molecular weight O-glycoproteins synthesized by epithelial cells as large secreted or membrane-bound glycoproteins (1). So far, eight mucin genes have been well characterized (MUC1, MUC2, MUC3, MUC4, MUC5AC, MUC5B, MUC6, and MUC7) (1), and cDNAs have been proposed for MUC8, MUC9, MUC11, and MUC12 (1, 2). Numerous studies have now demonstrated that the expression of mucin genes is tissue-and cell-specific and that their expression is altered during the pathogenesis of several diseases, which suggests that human mucin gene expression is tightly regulated and that they may play important roles during cell differentiation and carcinogenesis (3-8). In normal stomach, MUC5AC is expressed at the surface/ foveolar epithelium and MUC6 in the mucous neck cells and in the antral glands (9-11). Other mucin genes expressed in normal gastric mucosae are MUC1 and to a lesser extent MUC2, MUC3, and MUC4. In gastric carcinomas, a decrease of MUC1, MUC5AC and MUC6 expression and an increase of MUC2, MUC3, and MUC4 expression has already been demonstrated (11-15). Human mucin gene MUC5B has been extensively studied in our laboratory. Studies of MUC5B genomic sequence (39.1 kb) 1 showed that it encodes a high molecular weight polypeptide (627,000) (16-20). MUC5B mucin gene is localized on chromosome 11 (band p15) and is clustered with three other mucin genes: MUC2, MUC6, and MUC5AC (21). In normal adult, MUC5B is essentially expressed in trachea, bronchi, submax-illary glands, pancreas, gallbladder, and endocervix (4, 22-24). In cancer, MUC5B has been shown to be highly expressed in colon carcinoma (5), in HT-29 treated with methotrexate (19, 20, 25), and LS174T (20, 24, 26) mucus-secreting colon cancer cell lines. We recently characterized the first 956 nucleotides located upstream of MUC5B transcription start site and studied the promoter functional activity in colon cancer cells (20). The region is characterized by the presence of a TATA box and numerous putative binding sites for ubiquitous (Sp1) and specific transcription factors (NF-B, c-Myc). The high expression of MUC5B was correlated with the mucus-secreting phenotype of the LS174T colon cancer cell line. Introns 1 and 37 of MUC5B have also been studied in our laboratory because they contain tandemly repeated GA-and GC-rich sequences that
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hal-02905839 , version 1 (23-07-2020)

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Michael Perrais, Pascal Pigny, Marie-Pierre Buisine, Nicole Porchet, Jean-Pierre Aubert, et al.. Aberrant Expression of Human Mucin Gene MUC5B in Gastric Carcinoma and Cancer Cells: IDENTIFICATION AND REGULATION OF A DISTAL PROMOTER. Journal of Biological Chemistry, 2001, ⟨10.1074/jbc.M010534200⟩. ⟨hal-02905839⟩

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