Expression of the human CMP-NeuAc:GM3 alpha2,8-sialyltransferase (GD3 synthase) gene through the NF-kappaB activation in human melanoma SK-MEL-2 cells.
To elucidate the mechanism underlying the regulation of human GD3 synthase gene expression in human melanoma SK-MEL-2 cells, we identified the promoter region of the human GD3 synthase gene. The 5'-rapid amplification of cDNA end (5'-RACE) using mRNA prepared from SK-MEL-2 cells revealed the presence of multiple transcription start sites of human GD3 synthase gene. Promoter analyses of the 5'-flanking region of the human GD3 synthase gene using luciferase gene reporter system showed the strong promoter activity in SK-MEL-2 cells. Deletion study revealed that the region as the core promoter from -1146 to -646 (A of the translational start ATG as position +1) was indispensable for endogenous expression of human GD3 synthase gene. This region lacks apparent TATA and CAAT boxes but contains putative binding sites for transcription factors c-Ets-1, CREB, AP-1 and NF-kappaB. Electrophoretic mobility shift assays using specific competitors, chromatin immunoprecipitation assay and site-directed mutagenesis demonstrated that only NF-kappaB element in this region is required for the promoter activity in SK-MEL-2 cells. These results indicate that NF-kappaB plays an essential role in the transcriptional activity of human GD3 synthase gene essential for GD3 synthesis in SK-MEL-2 cells.
Kang
Nam-Young
NY
Brain Korea 21 Center for Silver-Bio Industrialization, Dong-A University, Busan 604-714, South Korea.
Kim
Cheorl-Ho
CH
Kim
Kyoung-Sook
KS
Ko
Jeong-Heon
JH
Lee
Jai-Heon
JH
Jeong
Yong-Kee
YK
Lee
Young-Choon
YC
D024506:5' Flanking Region; D001483:Base Sequence; D004789:Enzyme Activation; D005732:Gangliosides / Q000235:genetics*; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D008545:Melanoma / Q000235:genetics*; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000096:biosynthesis*; D011401:Promoter Regions, Genetic; D012799:Sialyltransferases / Q000235:genetics*; D024363:Transcription Initiation Site; D014407:Tumor Cells, Cultured
D005732:Gangliosides; D016328:NF-kappa B; C026226:ganglioside, GD3; D012799:Sialyltransferases; C043662:alpha-N-acetylneuraminate alpha-2,8-sialyltransferase