Study of gene expression on toxicity of dioxin in cultured cells

2，3，7，8-四氯二苯并对二恶英(TCDD)是近年来备受关注的一种剧毒化合物，可引起多种毒性反应。TCDD的大部分毒性效应被认为是由于基因表达的改变造成的。在这项研究中，我们研究了单次给予TCDD后，大鼠肝脏细胞质中参与TCDD依赖的mRNA稳定的反式作用因素。紫外线交联研究表明，50 kDa(P50)的细胞质蛋白选择性地识别尿激酶型纤溶酶原激活物(UPA)mRNA3‘非翻译区。我们还表明，TCDD激活p50是通过蛋白磷酸化级联反应而不是通过从头合成蛋白来介导的。我们先前报道，在3T3-L1细胞中，随着脂肪分化的进行，芳烃受体(AhR)蛋白水平下降。AhR是TCDD及其相关化合物的受体。利用烟草花叶病抗性克隆…的研究更多的3T3-L1细胞提示AhR可能参与了脂肪分化的负调控。为了证实这一假设，将高表达全长正义AhR mRNA、反义AhR mRNA的载体或对照载体稳定地导入3T3-L1成纤维细胞。对这些克隆与对照细胞分化潜能的比较表明，AhR的过表达抑制了脂肪细胞的形态分化和脂肪细胞相关基因的诱导，而AhR的表达降低导致了更大的形态分化和脂肪细胞相关基因的表达。C/EBPα和PPARγ2的激活恢复了AhR过表达细胞的分化能力。与对照细胞相比，过表达AhR的细胞表现出更高的p42/p44 MAPK活性。我们还表明，AhR的激活减缓了克隆性扩张。这些结果有力地表明AhR是3T3-L1细胞脂肪分化的负调控因子。较少

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), a highly toxic compound that has recently attracted much attention as an environmental contaminant, elicits a variety of toxic responses. Most of the toxic effects of TCDD are thought to result from alteration of gene expression. In this study, we investigated the trans-acting factors involved in TCDD-dependent mRNA stabilization in a rat liver cytoplasm after a single dose administration of TCDD.UV-crosslinking study showed that the cytoplasmic protein of 50 kDa (p50) selectively recognized the 3' untranslated region of the urokinase-type plasminogen activator (uPA) mRNA.We also showed that the activation of p50 by TCDD is mediated through a protein phosphorylation cascade but not via de novo protein synthesis.We previously reported that a level of arylhydrocarbon receptor (AhR) protein decreased with ongoing adipose differentiation in 3T3-L1 cells. The AhR is the receptor for TCDD and related compounds. Studies using a TCDD-resistant clone … More of 3T3-L1 cells suggested that the AhR may be involved in the negative regulation of adipose differentiation. To confirm this hypothesis, 3T3-L1 fibroblast cells were stably transfected with a vector expressing high levels of full length sense AhR mRNA, antisense AhR mRNA, or a control vector. Comparison of the differentiation potency of these clones with that of control cells showed that overexpression of the AhR suppressed morphological differentiation as well as inductionof adipocyte-related genes, whereas decreased expression of the AhR induced much greater morphological differentiation and expression of adipocyte-related genes. Activation of C/EBPα and PPARγ2 restored the ability of the AhR-overexpressing cells to differentiate. The cells overexpressing the AhR exhibited the higher p42/p44 MAPkinase activity compared with the control cells. We also showed that activation of the AhR slowed clonal expansion. These results strongly suggest that AhR is a negative regulator of adipose differentiation in 3T3-L1 cells. Less