Transcriptional regulation of the human beta-1,4-galactosyltransferase V gene in cancer cells: essential role of transcription factor Sp1.
Transcriptional regulation of the human beta-1,4-galactosyltransferase V gene in cancer cells: essential role of transcription factor Sp1.
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发表时间：
2004
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通讯作者：
Takeshi Sato;K. Furukawa
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作者：
Takeshi Sato;K. Furukawa
β-1，4-半乳糖基转移酶(β-1，4-GALT)V是一种结构性表达的酶，它能有效地半乳糖化肿瘤细胞特有的高分支N-糖链中的GlcNAcbeta1-->；6Man基团。在细胞恶性转化时，β-1，4-GalT V基因的表达随着高分支N-糖链数量的增加而增加。凝集素印迹分析表明，反义β-1，4-GalT V基因可显著抑制SH-SY5Y人神经母细胞瘤细胞中高支化N-糖链的半乳糖化，提示β-1，4-GalT V对高支化N-糖链功能具有重要的生物学意义。我们克隆了人β-1，4-GalT V基因的2.3kb的5‘侧翼区，并鉴定了相对于转录起始点的-116/-18区域具有启动子活性。该区域含有几个可能的转录因子结合位点，包括AP2、AP4、N-Myc、Sp1和上游刺激因子。凝胶迁移率分析表明，Sp1与启动子区域的-81/-69位核苷酸结合。Sp1结合位点的突变表明，在癌细胞中，β-1，4-GalT V基因的启动子活性完全受损。反之，将Sp1基因导入A549人肺癌细胞，启动子活性显著增强。米曲霉素A抑制Sp1与其结合部位的结合，降低A549细胞中β-1，4-GalT V基因启动子的激活和表达。这些结果表明Sp1在癌细胞中β-1，4-GalT V基因的转录活性中起重要作用。
Beta-1,4-galactosyltransferase (beta-1,4-GalT) V is a constitutively expressed enzyme that can effectively galactosylate the GlcNAcbeta1-->6Man group of the highly branched N-glycans that are characteristic of tumor cells. Upon malignant transformation of cells, the expression of the beta-1,4-GalT V gene increases in accordance with the increase in the amounts of highly branched N-glycans. Lectin blot analysis showed that the galactosylation of highly branched N-glycans is inhibited significantly in SH-SY5Y human neuroblastoma cells by the transfection of the antisense beta-1,4-GalT V cDNA, indicating the biological importance of the beta-1,4-GalT V for the functions of highly branched N-glycans. We cloned the 2.3-kb 5'-flanking region of the human beta-1,4-GalT V gene, and we identified the region -116/-18 relative to the transcription start site as that having promoter activity. The region was found to contain several putative binding sites for transcription factors, including AP2, AP4, N-Myc, Sp1, and upstream stimulatory factor. Electrophoretic mobility shift assay showed that Sp1 binds to nucleotide positions -81/-69 of the promoter region. Mutations induced in the Sp1-binding site showed that the promoter activity of the beta-1,4-GalT V gene is impaired completely in cancer cells. In contrast, the promoter activity increased significantly by the transfection of the Sp1 cDNA into A549 human lung carcinoma cells. Mithramycin A, which inhibits the binding of Sp1 to its binding site, reduced the promoter activation and expression of the beta-1,4-GalT V gene in A549 cells. These results indicate that Sp1 plays an essential role in the transcriptional activity of the beta-1,4-GalT V gene in cancer cells.