Chen Xinrong, Zhang Jimei, Baker Sharon M, Chen Guangping
Department of Physiological Sciences, Center for Veterinary Health Sciences, Oklahoma State University, Stillwater, OK 74078, USA.
Toxicology. 2007 Mar 7;231(2-3):224-33. doi: 10.1016/j.tox.2006.12.019. Epub 2006 Dec 22.
Sulfotransferases (SULTs) catalyzed sulfation is important in the regulation of biological activities of hormones and neurotransmitters, the metabolism of drugs, and the detoxification of xenobiotic toxicants. Sulfation also leads to the bioactivation of procarcinogens. Human dehydroepiandrosterone sulfotransferase (hSULT2A1) is a major SULT catalyzing the sulfation of hydroxysteroids and xenobiotic alcohols. Our previous studies had shown that the anti-folate drug methotrexate (MTX) can up-regulate several major isoforms of human SULTs. To determine the mechanisms controlling the regulation of hSULT2A1, the 5'-flanking region of hSULT2A1 was constructed into the pGL3-Basic luciferase reporter vector. The transcriptional regulation mechanism of hSULT2A1 promoter was studied using Caco-2 cell line based on the reporter gene assay. Nuclear receptor co-transfection results indicated that human constitutive androstane receptor (hCAR) and human retinoid X receptor alpha (hRXRalpha) were involved in the transcriptional regulation of hSULT2A1. RNA interference experiments further proved the role of hCAR in hSULT2A1 regulation. Progressive promoter deletion, DNA sequence alignment, and site directed promoter mutation results suggested that an imperfect inverted repeat DNA motif, IR2 (-186AGCTCAGATGACCC-173), within the hSULT2A1 promoter region mediated the hSULT2A1 induction by MTX. Furthermore, electrophoretic mobility shift assay and super shift assay were employed to characterize the interactions of hCAR and hRXRalpha with the IR2 element. In summary, we identified an IR2 DNA cis-element located at -186/-173 of hSULT2A1 promoter region; the IR2 element mediates the MTX induction of hSULT2A1 through interacting with hCAR and hRXRalpha.
磺基转移酶（SULTs）催化的硫酸化作用在激素和神经递质生物活性的调节、药物代谢以及外源性毒物的解毒过程中起着重要作用。硫酸化作用还会导致前致癌物的生物活化。人脱氢表雄酮磺基转移酶（hSULT2A1）是催化羟类固醇和外源性醇类硫酸化的主要SULT。我们之前的研究表明，抗叶酸药物甲氨蝶呤（MTX）可以上调人SULTs的几种主要同工型。为了确定控制hSULT2A1调节的机制，将hSULT2A1的5'-侧翼区域构建到pGL3-Basic荧光素酶报告载体中。基于报告基因测定，使用Caco-2细胞系研究了hSULT2A1启动子的转录调节机制。核受体共转染结果表明，人组成型雄烷受体（hCAR）和人视黄酸X受体α（hRXRα）参与了hSULT2A1的转录调节。RNA干扰实验进一步证明了hCAR在hSULT2A1调节中的作用。启动子逐步缺失、DNA序列比对和定点启动子突变结果表明，hSULT2A1启动子区域内一个不完美的反向重复DNA基序IR2（-186AGCTCAGATGACCC-173）介导了MTX对hSULT2A1的诱导作用。此外，采用电泳迁移率变动分析和超迁移分析来表征hCAR和hRXRα与IR2元件的相互作用。总之，我们在hSULT2A1启动子区域-186/-173处鉴定出一个IR2 DNA顺式元件；IR2元件通过与hCAR和hRXRα相互作用介导MTX对hSULT2A1的诱导作用。
