Transcriptional regulation of the human GD3 synthase gene expression in Fas-induced Jurkat T cells: a critical role of transcription factor NF-kappaB in regulated expression.
ABSTRACT The transcriptional regulation mechanisms involved in the up-regulation of Fas-induced GD3 synthase gene have not yet been elucidated. 5'-Rapid amplification of cDNA end (5'-RACE) using mRNA prepared from Fas-induced Jurkat T cells revealed the presence of multiple transcription start sites of human GD3 synthase gene, and the 5'-end analysis of the longest of its product showed that transcription started from 650 nucleotides upstream of the translational initiation site. Promoter analyses of the 5'-flanking region of the human GD3 synthase gene using luciferase gene reporter system showed strong promoter activity in Fas-induced Jurkat T cells. Deletion study revealed that the region from -1146 to -646 (A of the translational start ATG as position +1) was indispensable for the Fas response. This region lacks apparent TATA and CAAT boxes but contains putative binding sites for transcription factors c-Ets-1, cAMP-responsive element-binding (CREB) protein, activating protein 1 (AP-1), and NF-kappaB. Base-substitution experiment showed that only the NF-kappaB-binding site of putative binding sites is required for the maximal expression induced by Fas. Both DNase I footprint and electrophoretic mobility shift assays with the nuclear extract of Fas-induced Jurkat T cells revealed that NF-kappaB was bound specifically to the probe being mediated by its binding site in the promoter sequence. Taken together, these results indicate that NF-kappaB plays an essential role in the transcriptional activity of human GD3 synthase gene in Fas-induced Jurkat T cells. In addition, the translocation of NF-kappaB-binding protein to nucleus by Fas activation is also crucial for the increased expression of the GD3 synthase gene in Fas-activated Jurkat T cells.
Full-textDOI: · Available from: Soo-Young Cho, Feb 03, 2014
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ABSTRACT: The transporter protein genes and lipids in human ovarian carcinoma-derived KF28 cells with anticancer-drug-sensitive properties were compared with those in resistant cells, taxol-resistant KF28TX, cisplatin-resistant KFr13, and taxol- and cisplatin-resistant KFr13TX, to identify the molecules required for anticancer-drug resistance. In accordance with previous reports, taxol and cisplatin resistance was closely correlated with expression of the multidrug resistance 1 and bile acid export pump, and multidrug resistance-associated protein 2 genes, respectively. In addition, we found a distinct difference in glycosphingolipids between the sensitive and resistant cells. Although GlcCer was the major glycolipid (83.0%) in sensitive cells, GalCer, LacCer and, particularly, Gb(3)Cer were characteristically increased in all resistant cells, irrespective of whether the resistance was to taxol or cisplatin, and comprised 65-84% of total glycosphingolipids. GM3, which was present at 0.04 microg/mg dry weight in the sensitive cells, showed a twofold increase in the taxol-resistant cells, but was absent in the cisplatin-resistant cells. The altered glycolipid composition was proven to be due to enhanced or suppressed expression of the respective sugar transferase genes. In addition, the ceramide moiety of ceramide monohexoside in the sensitive cells constituted 83% of non-hydroxy fatty acids, but that in the resistant cells comprised 67-74% of alpha-hydroxy fatty acids. Thus, cells containing Gb(3)Cer with alpha-hydroxy fatty acids were found to survive selectively in the presence of taxol and cisplatin, and modification of the glycolipid structure was revealed to occur in association with anticancer-drug resistance.Cancer Science 01/2007; 97(12):1321-6. DOI:10.1111/j.1349-7006.2006.00326.x · 3.48 Impact Factor
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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-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.Biochimica et Biophysica Acta 11/2007; 1769(11-12):622-30. DOI:10.1016/j.bbaexp.2007.08.001 · 4.66 Impact Factor
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ABSTRACT: In this study, we have shown the transcriptional regulation of the human GD3 synthase (hST8Sia I) induced by valproic acid (VPA) in human neuroblastoma SK-N-BE(2)-C cells. To elucidate the mechanism underlying the regulation of hST8Sia I gene expression in VPA-stimulated SK-N-BE(2)-C cells, we characterized the promoter region of the hST8Sia I gene. Functional analysis of the 5’-flanking region of the hST8Sia I gene by the transient expression method showed that the -1146 to -646 region, which contains putative binding sites for transcription factors c-Ets-1, CREB, AP-1 and NF-kappa B, functions as the VPA-inducible promoter of hST8Sia I in SK-N-BE(2)-C cells. Site-directed mutagenesis and electrophoretic mobility shift assay indicated that the NF-kappa B binding site at -731 to -722 was crucial for the VPA-induced expression of hST8Sia I in SK-N-BE(2)-C cells. In addition, the transcriptional activity of hST8Sia I induced by VPA in SK-N-BE(2)-C cells was strongly inhibited by SP600125, which is a c-Jun N-terminal kinase (JNK) inhibitor, and GO6976, which is a protein kinase C (PKC) inhibitor, as determined by RT-PCR (reverse transcription-polymerase chain reaction) and luciferase assays. These results suggest that VPA markedly modulated transcriptional regulation of hST8Sia I gene expression through PKC/JNK signal pathways in SK-N-BE(2)-C cells.Molecules and Cells 10/2008; 27(1). DOI:10.1007/s10059-009-0012-4 · 2.24 Impact Factor