Loaiza-Pérez A I, Seisdedos M T, Kleiman de Pisarev D L, Sancovich H A, Randi A S, Ferramola de Sancovich A M, Santisteban P
Instituto de Investigaciones Biomédicas Alberto Sols, Consejo Superior de Investigaciones Científicas y Universidad Autónoma de Madrid, Spain.
Endocrinology. 1999 Sep;140(9):4142-51. doi: 10.1210/endo.140.9.6996.
Hexachlorobenzene (HCB) is a dioxin-type chemical that acts mainly through the aryl hydrocarbon receptor. Chronic exposure of rats to HCB increases the activity of malic enzyme (ME). In this report, we show that this increase is correlated with an induction of ME messenger RNA (mRNA) levels, with the maximal HCB effect achieved after 9 days of intoxication. This effect is specific for ME, as other liver enzymes, such as glyceraldehyde-3-phosphate dehydrogenase, phosphoenol pyruvate carboxykinase, and mitochondrial alpha-glycerol-3-phosphate dehydrogenase, are not affected by HCB. The induction of ME mRNA levels is accompanied by an increase in ME promoter activity, as demonstrated by transient transfection experiments performed in rat hepatoma H35 cells. In an attempt to identify the cis-regulatory elements responsible for the HCB effect, different promoter deletions and mutations were used. The results obtained localize the responsive region between positions -315 and -177. This region does not contain either consensus xenobiotic response or activating protein-1 elements, the two main mediators of dioxin compounds described to date. In contrast, a thyroid hormone response element (TRE) is located between -281 to -261. Deletions and mutations of the TRE element do not respond to HCB, demonstrating that this element mediates the response of this dioxin-type compound. As ME gene expression is regulated mainly by thyroid hormones, we next investigated the role of T3 receptor (T3R) in the ME gene transcriptional induction mediated by HCB. Using Scatchard analysis, we show that neither T3R binding features for its ligand nor alpha1 or beta1T3R mRNA levels are changed with the toxic. In gel shift assays, however, we observed that protein/DNA complexes formed on TRE from the ME promoter were induced by HCB. Using an oligonucleotide with a mutation that eliminates the TRE function, we demonstrate a loss of the induced protein/DNA complexes. Together, these data suggest that the dioxin-type compound HCB increases ME gene transcription by modulating the levels of still unidentified nuclear proteins that bind to the TRE element of the ME promoter.
六氯苯（HCB）是一种二噁英类化学物质，主要通过芳烃受体发挥作用。大鼠长期接触HCB会增加苹果酸酶（ME）的活性。在本报告中，我们表明这种增加与ME信使核糖核酸（mRNA）水平的诱导相关，中毒9天后达到最大HCB效应。这种效应是ME特有的，因为其他肝脏酶，如甘油醛-3-磷酸脱氢酶、磷酸烯醇丙酮酸羧激酶和线粒体α-甘油-3-磷酸脱氢酶，不受HCB影响。ME mRNA水平的诱导伴随着ME启动子活性的增加，这在大鼠肝癌H35细胞中进行的瞬时转染实验中得到了证明。为了确定负责HCB效应的顺式调节元件，使用了不同的启动子缺失和突变。获得的结果将反应区域定位在-315至-177位之间。该区域既不包含已知的异生素反应元件，也不包含激活蛋白-1元件，这是迄今为止描述的二噁英化合物的两个主要介质。相反，一个甲状腺激素反应元件（TRE）位于-281至-261之间。TRE元件的缺失和突变对HCB无反应，表明该元件介导了这种二噁英类化合物的反应。由于ME基因表达主要受甲状腺激素调节，我们接下来研究了T3受体（T3R）在HCB介导的ME基因转录诱导中的作用。使用Scatchard分析，我们表明其配体的T3R结合特征以及α-1或β-1 T3R mRNA水平均未因中毒而改变。然而，在凝胶迁移实验中，我们观察到由ME启动子的TRE形成的蛋白质/DNA复合物被HCB诱导。使用具有消除TRE功能的突变的寡核苷酸，我们证明了诱导的蛋白质/DNA复合物消失。总之，这些数据表明二噁英类化合物HCB通过调节与ME启动子的TRE元件结合的仍未鉴定的核蛋白水平来增加ME基因转录。
