Regulation of glucocorticoid-inducible hydroxysteroid sulfotransferase (SULT2A-40/41) gene transcription in primary cultured rat hepatocytes: Role of CCAAT/enhancer-binding protein liverenriched transcription factors
Regulation of glucocorticoid-inducible hydroxysteroid sulfotransferase (SULT2A-40/41) gene transcription in primary cultured rat hepatocytes: Role of CCAAT/enhancer-binding protein liverenriched transcription factors
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DOI：
10.1124/dmd.104.000281
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发表时间：
2005-01-01
影响因子：
3.9
通讯作者：
Runge-Morris, M
中科院分区：
文献类型：
--
作者：
Fang, HL;Abdolalipour, M;Runge-Morris, M
探讨糖皮质激素受体（GR）诱导大鼠肝羟类固醇磺基转移酶（SULT 2A-40/41）基因转录的机制。我们先前报道了相对于转录起始位点由-158至-77个核苷酸限定的SULT 2A-40/41 5 '侧翼区的区域足以支持GR诱导的表达。SULT 2A-40/41基因的该区域不包含共有糖皮质激素受体反应元件，但包含两个肝脏富集CCAAT/增强子结合蛋白（C/EBP）转录因子的共有位点。在本研究中，将原代培养的大鼠肝细胞与GR激活浓度（10（-7）M）的强效糖皮质激素、地塞米松或曲安奈德（TA）孵育，通过Western印迹或体外DNA结合活性分析测定，在SULT 2A-40/41 mRNA和蛋白水平增加之前，C/EBP α和C/EBP β核蛋白含量迅速增加。SULT 2A-40/41报告基因质粒与显性阴性C/EBP表达质粒的瞬时共转染完全阻断TA诱导的SULT 2A-40/41报告基因表达。近端SULT 2A 40/41 5 '侧翼区的接头扫描和定点突变，辅以体外DNA结合分析，表明更远端的C/EBP位点对控制SULT 2A-40/41启动子活性很重要。这些数据支持GR诱导的C/EBPalpha和C/EBPbeta表达在肝SULT 2A-40/41表达的反式激活中的作用。
The mechanism responsible for glucocorticoid receptor (GR)-mediated induction of rat hepatic hydroxysteroid sulfotransferase (SULT2A-40/41) gene transcription was investigated. We previously reported that the region of the SULT2A-40/41 5'-flanking region delimited by -158 to -77 nucleotides relative to the transcription start site was sufficient to support GR-inducible expression. This region of the SULT2A-40/41 gene does not contain a consensus glucocorticoid receptor-responsive element, but does contain two consensus sites for liver-enriched CCAAT/enhancer-binding protein (C/EBP) transcription factors. In the present study, incubation of primary cultured rat hepatocytes with a GR-activating concentration (10(-7) M) of a potent glucocorticoid, dexamethasone or triamcinolone acetonide ( TA), rapidly produced increases in C/EBPalpha and C/EBPbeta nuclear protein contents, as measured by Western blot or in vitro DNA-binding activity analysis, that preceded increases in SULT2A-40/41 mRNA and protein levels. Transient cotransfection of SULT2A-40/41 reporter plasmids with a dominant negative C/EBP expression plasmid completely blocked TA-inducible SULT2A-40/41 reporter gene expression. Linker scanning and site-directed mutagenesis of the proximal SULT2A40/41 5'-flanking region, complemented by in vitro DNA-binding analyses, indicated that the more distal C/EBP site was important for controlling SULT2A-40/41 promoter activity. These data support a role for GR-inducible C/EBPalpha and C/EBPbeta expression in the transactivation of hepatic SULT2A-40/41 expression.