Romero Laura, Ng Lorraine, Kirby Gordon M
Department of Biomedical Sciences, University of Guelph, Guelph, ON, Canada N1G 2W1.
Mol Pharmacol. 2006 Jul;70(1):277-86. doi: 10.1124/mol.105.018622. Epub 2006 Apr 11.
The regulation of human GSTA1 by chemical inducers of rodent glutathione S-transferases (GSTs) and the regulatory role of hepatic nuclear factor (HNF) 1 was investigated in Caco-2 cells. Treatment of preconfluent and confluent cells with 12-O-tetra-decanoyl phorbol-13-acetate (TPA), 3-methylcholanthrene (3-MC), 2-tert-butyl-4-hydroxy-anisol (BHA), and phenobarbital (PB) reduced GSTA1 mRNA levels in preconfluent and confluent cells. Constitutive levels of GSTA1 and HNF1alpha mRNA were elevated 6.25- and 50-fold, respectively, in postconfluent cells compared with preconfluent cells. Overexpression of HNF1alpha in cells transfected with a GSTA1 promoter-luciferase construct (pGSTA1-1591-luc) resulted in dose-related increases in reporter activity not observed when an HNF1 response element (HRE) in the proximal promoter was mutated (pGSTA1-DeltaHNF1-luc). TPA, 3-MC, BHA, and PB reduced HNF1alpha mRNA levels in preconfluent and confluent cells and caused marked reductions in luciferase activity in pGSTA1-1591-luc transfectants. Transcriptional repression was abrogated with pGSTA1-DeltaHNF1-luc and with truncated constructs that eliminated a functional HRE. Moreover, cotransfection of pHNF1alpha with pGSTA1-1591-luc partially prevented the reduction in luciferase activity by rodent GST inducers. Immunoblot analysis of DNA binding studies indicate that variant (v)HNF1-C binding to HRE is increased in preconfluent cells treated with 3-MC, BHA, and PB. In addition, overexpression of vHNF1-C repressed GSTA1 transcriptional activity in luciferase reporter assays. Finally, treatment with 3-MC, BHA, and PB increased vHNF1-C mRNA levels in preconfluent cells. These data demonstrate that repression of human GSTA1 transcription by chemical inducers of rodent GSTs occurs, in part, through a mechanism involving the repressive action of vHNF1-C.
在Caco-2细胞中研究了啮齿动物谷胱甘肽S-转移酶（GST）的化学诱导剂对人GSTA1的调控以及肝细胞核因子（HNF）1的调节作用。用12-O-十四烷酰佛波醇-13-乙酸酯（TPA）、3-甲基胆蒽（3-MC）、2-叔丁基-4-羟基茴香醚（BHA）和苯巴比妥（PB）处理未汇合和汇合的细胞，可降低未汇合和汇合细胞中GSTA1 mRNA水平。与未汇合细胞相比，汇合后细胞中GSTA1和HNF1α mRNA的组成水平分别升高了6.25倍和50倍。在用GSTA1启动子-荧光素酶构建体（pGSTA1-1591-luc）转染的细胞中过表达HNF1α导致报告基因活性呈剂量相关增加，而当近端启动子中的HNF1反应元件（HRE）发生突变时（pGSTA1-DeltaHNF1-luc）则未观察到这种增加。TPA、3-MC、BHA和PB降低了未汇合和汇合细胞中HNF1α mRNA水平，并导致pGSTA1-1591-luc转染细胞中荧光素酶活性显著降低。用pGSTA1-DeltaHNF1-luc和消除功能性HRE的截短构建体可消除转录抑制。此外，将pHNF1α与pGSTA1-1591-luc共转染可部分防止啮齿动物GST诱导剂降低荧光素酶活性。DNA结合研究的免疫印迹分析表明，在用3-MC、BHA和PB处理的未汇合细胞中，变体（v）HNF1-C与HRE的结合增加。此外，在荧光素酶报告基因测定中，vHNF1-C的过表达抑制了GSTA1转录活性。最后，用3-MC、BHA和PB处理可增加未汇合细胞中vHNF1-C mRNA水平。这些数据表明，啮齿动物GST的化学诱导剂对人GSTA1转录的抑制部分是通过一种涉及vHNF1-C抑制作用的机制发生的。
