Kwon Haw-Young, Dae Hyun-Mi, Song Na-Ri, Kim Kyoung-Sook, Kim Cheorl-Ho, Lee Young-Choon
Department of Biotechnology, Dong-A University, Busan, 604-714, Korea.
Mol Cells. 2009 Jan 31;27(1):113-8. doi: 10.1007/s10059-009-0012-4. Epub 2008 Oct 13.
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-kappaB, 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-kappaB 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.
在本研究中，我们展示了丙戊酸（VPA）在人神经母细胞瘤SK-N-BE(2)-C细胞中对人GD3合酶（hST8Sia I）的转录调控。为阐明VPA刺激的SK-N-BE(2)-C细胞中hST8Sia I基因表达调控的潜在机制，我们对hST8Sia I基因的启动子区域进行了特征分析。通过瞬时表达方法对hST8Sia I基因5'侧翼区域进行功能分析表明，包含转录因子c-Ets-1、CREB、AP-1和NF-κB假定结合位点的-1146至-646区域，在SK-N-BE(2)-C细胞中作为hST8Sia I的VPA诱导型启动子发挥作用。定点诱变和电泳迁移率变动分析表明，-731至-722处的NF-κB结合位点对SK-N-BE(2)-C细胞中VPA诱导的hST8Sia I表达至关重要。此外，如通过逆转录-聚合酶链反应（RT-PCR）和荧光素酶测定所确定的，SK-N-BE(2)-C细胞中VPA诱导的hST8Sia I转录活性受到c-Jun N末端激酶（JNK）抑制剂SP600125和蛋白激酶C（PKC）抑制剂GO6976的强烈抑制。这些结果表明，VPA通过PKC/JNK信号通路在SK-N-BE(2)-C细胞中显著调节hST8Sia I基因表达的转录调控。
