Sequential action of Ets-1 and Sp1 in the activation of the human beta-1,4-galactosyltransferase V gene involved in abnormal glycosylation characteristic of cancer cells.
Malignant transformation is associated with increased gene expression of beta-1,4-galactosyltransferase (beta-1,4-GalT) V, which contributes to the biosynthesis of highly branched N-linked oligosaccharides characteristic of cancer cells. Our previous study showed that expression of the human beta-1,4-GalT V gene is regulated by Sp1 (Sato, T., and Furukawa, K. (2004) J. Biol. Chem. 279, 39574-39583), and a subsequent study showed that the gene expression is also activated by Ets-1, a product of the oncogene (Sato, T., and Furukawa, K. (2005) Glycoconj. J. 22, 365). Herein we report the mechanism of beta-1,4-GalT V gene activation by these transcription factors. The gene expression and promoter activity of beta-1,4-GalT V increased when the ets-1 cDNA was transfected into A549 cells, which contain a small amount of Ets-1, but decreased dramatically when the dominant-negative ets-1 cDNA was transfected into HepG2 cells, which contain a large amount of Ets-1. Luciferase assays using deletion constructs of the beta-1,4-GalT V gene promoter showed that promoter region -116 to +22 is critical for the transcriptional activation of the gene by Ets-1. Despite the presence of one Ets-1-binding site, which overlapped the Sp1-binding site, electrophoretic mobility shift assays showed that the region bound preferentially to Sp1 rather than to Ets-1. To solve this problem, we examined the transcriptional regulation of the human Sp1 gene by Ets-1 and found that the gene expression and promoter activity of Sp1 are regulated by Ets-1 in cancer cells. Functional analyses of two Ets-1-binding sites in the Sp1 gene promoter showed that only Ets-1-binding site -413 to -404 is involved in the activation of the gene by Ets-1. These results indicate that Ets-1 enhances expression of the beta-1,4-GalT V gene through activation of the Sp1 gene in cancer cells.
Sato
Takeshi
T
Laboratory of Glycobiology, Department of Bioengineering, Nagaoka University of Technology, Kamitomioka 1603-1, Nagaoka 940-2188, Japan. taksato@vos.nagaokaut.ac.jp
Furukawa
Kiyoshi
K
D001483:Base Sequence; D001665:Binding Sites; D015152:Blotting, Northern; D045744:Cell Line, Tumor; D005700:Galactosyltransferases / Q000096:biosynthesis* / Q000235:genetics*; D017353:Gene Deletion; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D008954:Models, Biological; D008957:Models, Genetic; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D017434:Protein Structure, Tertiary; D050784:Proto-Oncogene Protein c-ets-1 / Q000378:metabolism / Q000502:physiology*; D016329:Sp1 Transcription Factor / Q000378:metabolism*; D015533:Transcriptional Activation
C494041:ETS1 protein, human; D050784:Proto-Oncogene Protein c-ets-1; D016329:Sp1 Transcription Factor; D005700:Galactosyltransferases; C419408:beta-1,4-galactosyltransferase V