Transcriptional Regulation of the N-Acetylglucosaminyltransferase V Gene in Human Bile Duct Carcinoma Cells (HuCC-T1) Is Mediated by Ets-1

Abstract

N-Acetylglucosaminyltransferase V (GnT-V) catalyzes the transfer of N-acetylglucosamine from UDP-N-acetylglucosamine to ␣-6-D-mannoside to produce the ␤1-6 linked branching of N-glycan oligosaccharides, which controls the polylactosamine content. The expression of N-acetylglucosaminyltransferase V, which contains 17 exons and spans 155 kilobase pairs, is expressed in a tissue-and cell type-specific manner and is regulated at the level of transcription by multiple promoters (Saito, H., Gu, J., Nishikawa, A., Ihara, Y., Fujii, J., Kohgo, Y., and Taniguchi, N. (1995) Eur. J. Biochem. 233, 18 -26). To elucidate the mechanism by which the GnT-V gene is expressed in a cell-and tissue-specific manner, cell-restricted expression was analyzed using the 5-upstream regions of the human GnT-V gene spanning base pairs ؊2760 to ؉23 in a human bile duct carcinoma cell line, HuCC-T1. We characterized two cis-acting elements that are potentially important in HuCC-T1 cell-specific expression. The two elements each contain an Ets-1 binding site, 5-GGA-3. Specific binding of Ets-1 to the respective elements was demonstrated by competition analysis as well as by antibody supershift experiments. Cotransfection of an Ets-1 expression plasmid along with a GnT-V promoter-luciferase reporter plasmid revealed the participation of Ets-1 in the regulation of the GnT-V gene transcription. These data indicated that the transcriptional regulation of the GnT-V gene was mediated by transcription factor Ets-1.Cell surface oligosaccharides linked to asparagine residues of membrane glycoproteins are thought to participate in a variety of specific biological interactions (1). The high branching of N-glycans seemed to be related to malignancy potential, in particular, increased ␤1-6 branching of N-glycans being directly linked to increased tumor metastasis (2-6). This structure is synthesized by UDP-N-acetylglucosamine:␣-6-D-mannoside ␤1-6-N-acetylglucosaminyltransferase (GnT-V) 1 (EC 2.4.1.155), as shown in Scheme 1. The expression of the GnT-V gene is induced by viral and oncogene transfection, transforming growth factor-␤, and phorbol ester (7-10), suggesting that complicated mechanisms for the induction exist. We and another group succeeded in its purification and cloning of its cDNA (11, 12). Messenger RNA was detected as two bands for HepG2 and MCF7 cells, and LEC rat liver, but the level is not always consistent with its enzymatic activity (13-15). To determine the details of the gene regulation, analysis of the 5Ј-upstream regions of GnT-V was needed. Recently, we isolated the human GnT-V genomic DNA, which contains 17 exons and spans 155 kb. Some putative consensus sequences for the tissue-specific transcription factors have been found in two possible promoter regions of the 5Ј-upstream regions of the GnT-V gene (16).The Ets transcription factor family shares a common DNA binding domain that interacts specifically with sequences containing a common core trinucleotide sequence, GGA (17). Ets binding sites have been identified in the regulator...

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