Olnes M J, Verma M, Kurl R N
Graduate Program in Pathobiology, Brown University School of Medicine, Providence, Rhode Island, USA.
J Pharmacol Exp Ther. 1996 Dec;279(3):1566-73.
As an approach to understanding the molecular mechanism(s) of thymic gene expression mediated by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), we investigated the effect of TCDD on expression of prostaglandin G/H synthase-2 (PGHS-2) in rat thymocytes by reverse transcription-polymerase chain reaction. Incubation of thymocytes with increasing doses of TCDD resulted in inhibition of PGHS-2 gene expression in a concentration-dependent manner, with an IC50 of 10 nM. In contrast, TCDD had no appreciable effect on expression of glyceraldehyde phosphate dehydrogenase. Because the xenobiotic-responsive element is conserved in the PGHS-2 promoter from several animal species, it seems likely that inhibition of PGHS-2 expression by TCDD may occur at the level of transcription. To test this hypothesis in cultured thymocytes, we characterized the Ah receptor in the thymoma cell line WEHI 7.1. Reverse transcription-polymerase chain reaction experiments indicated that TCDD inhibited PGHS-2 expression in this cell line. Sucrose density gradient centrifugation experiments indicated that WEHI 7.1 cytosol exhibited 9 to 10S ligand-binding activity characteristic of the Ah receptor. The viability of WEHI 7.1 cells incubated with TCDD was comparable to that of control cells, whereas dexamethasone induced toxicity in a concentration-dependent manner. Transient transfection experiments using PGHS-2 promoter fragments ligated into a chloramphenicol acetyltransferase reporter plasmid suggested that TCDD inhibits PGHS-2 transcription, and deletion of the xenobiotic-responsive element failed to exhibit this repression. These results demonstrate that TCDD is a potent inhibitor of PGHS-2 gene expression, and they represent the first mechanistic evidence for TCDD-dependent inhibition of transcription in thymocytes.
作为一种理解由2,3,7,8-四氯二苯并-对-二恶英（TCDD）介导的胸腺基因表达分子机制的方法，我们通过逆转录-聚合酶链反应研究了TCDD对大鼠胸腺细胞中前列腺素G/H合酶-2（PGHS-2）表达的影响。用递增剂量的TCDD孵育胸腺细胞导致PGHS-2基因表达呈浓度依赖性抑制，半数抑制浓度（IC50）为10 nM。相比之下，TCDD对甘油醛-3-磷酸脱氢酶的表达没有明显影响。由于几种动物物种的PGHS-2启动子中的外源性反应元件是保守的，因此TCDD对PGHS-2表达的抑制可能发生在转录水平。为了在培养的胸腺细胞中验证这一假设，我们对胸腺瘤细胞系WEHI 7.1中的芳烃受体进行了表征。逆转录-聚合酶链反应实验表明TCDD抑制了该细胞系中PGHS-2的表达。蔗糖密度梯度离心实验表明，WEHI 7.1细胞溶质表现出芳烃受体特有的9至10S配体结合活性。用TCDD孵育的WEHI 7.1细胞的活力与对照细胞相当，而地塞米松以浓度依赖性方式诱导毒性。使用连接到氯霉素乙酰转移酶报告质粒中的PGHS-2启动子片段进行的瞬时转染实验表明，TCDD抑制PGHS-2转录，并且外源性反应元件的缺失未能表现出这种抑制作用。这些结果表明，TCDD是PGHS-2基因表达的有效抑制剂，它们代表了TCDD依赖性抑制胸腺细胞转录的首个机制证据。
