Expression of the human CMP-NeuAc:GM3 α2,8-sialyltransferase (GD3 synthase) gene through the NF-κB activation in human melanoma SK-MEL-2 cells

Abstract

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-κB. Electrophoretic mobility shift assays using specific competitors, chromatin immunoprecipitation assay and site-directed mutagenesis demonstrated that only NF-κB element in this region is required for the promoter activity in SK-MEL-2 cells. These results indicate that NF-κB plays an essential role in the transcriptional activity of human GD3 synthase gene essential for GD3 synthesis in SK-MEL-2 cells.