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J. Biol. Chem., Vol. 276, Issue 18, 15386-15396, May 4, 2001
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From the 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 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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AJ012453.
Aberrant Expression of Human Mucin Gene
MUC5B in Gastric Carcinoma and Cancer Cells
IDENTIFICATION AND REGULATION OF A DISTAL PROMOTER*
§,¶
,**,**,**, and
Unité INSERM 377, Place de Verdun,
59045 Lille Cedex, France and Laboratoires ** de Biochimie et de
Biologie Moléculaire and ¶ d'Endocrinologie de
l'Hôpital C. Huriez, Centre Hospitalier Régional et
Universitaire, 59037 Lille Cedex, France, and Faculté de
Médecine, Université de Lille II,
59045 Lille France
*
This work was supported by l'Association de Recherche
contre le Cancer Grant 5785 and a grant from le Comité du Nord de
la Ligue Nationale contre le Cancer.The costs of publication of this article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.:
33-320-29-88-65; Fax: 33-320-53-85-62; E-mail:
vanseuni@lille.inserm.fr.
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