Aberrant expression of human mucin gene MUC5B in gastric carcinoma and cancer cells. Identification and regulation of a distal promoter.
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.
Perrais
M
M
Unité INSERM 377, Place de Verdun, 59045 Lille Cedex, France.
Pigny
P
P
Buisine
M P
MP
Porchet
N
N
Aubert
J P
JP
Van Seuningen-Lempire
I
I
D000230:Adenocarcinoma / Q000235:genetics* / Q000473:pathology; D001483:Base Sequence; D004247:DNA; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D008969:Molecular Sequence Data; D055257:Mucin-5B; D009077:Mucins / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics; D015398:Signal Transduction; D013274:Stomach Neoplasms / Q000235:genetics* / Q000473:pathology; D016385:TATA Box; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
C524578:MUC5B protein, human; D055257:Mucin-5B; D009077:Mucins; D012333:RNA, Messenger; D004247:DNA