Liu P C, Phillips M A, Matsumura F
Department of Environmental Toxicology, University of California, Davis, 95616, USA.
Mol Pharmacol. 1996 Jun;49(6):989-97.
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and related compounds elicit multiple effects on the function of adipose tissue and adipogenic cell lines, including the suppression of adipocyte differentiation. We began to examine the mechanism by which TCDD inhibits differentiation of the established preadipocyte cell line 3T3-L1. Examination of the expression of several early marker genes of preadipocyte differentiation through Northern blot analysis and of differentiation-dependent mitosis showed that TCDD did not interfere with the earliest known responses of preadipocytes to inducers of differentiation. Analysis of mRNA for three isoforms of the CCAAT/enhancer binding protein (C/EBP) revealed that TCDD (5 nM) selectively inhibited the induction of C/EBP alpha mRNA but did not block the induction of C/EBP beta and C/EBP delta in response to differentiation inducers. The differentiation-dependent induction of PPAR gamma mRNA was also blocked by TCDD. Immunoblot analysis with specific antibodies to each C/EBP isoform demonstrated that the levels of C/EBP delta and C/EBP beta protein were rapidly induced (by day 1) and then abrogated by day 4 and 8, respectively, in solvent-treated (control) cells. In TCDD-treated cells, however, the levels of C/EBP beta and C/EBP delta protein persisted at these time points. In contrast, C/EBP alpha protein was markedly suppressed by TCDD in concordance with its level of RNA. Both translational products of C/EBP alpha, p30 and p42, were dose-dependently decreased by TCDD. Gel shift analysis of nuclear extract binding to an oligonucleotide containing a C/EBP DNA recognition sequence revealed no difference between extracts from control and TCDD-treated cells in the binding pattern at day 2 of differentiation. At days 4 and 8, the band corresponding to the C/EBP alpha/DNA complex (as determined with supershift assays) was dramatically decreased in the treated extracts in comparison to control extracts. In contrast, a band corresponding to a C/EBP beta/DNA complex was found to be enriched in the treated samples. These data indicate that suppression of differentiation in the 3T3-L1 preadipocyte cell line by TCDD occurs at a short but defined period, during the differentiation program, and involves altered regulation of C/EBP, including the inhibition of C/EBP alpha.
2,3,7,8-四氯二苯并-对-二恶英（TCDD）及相关化合物对脂肪组织和脂肪生成细胞系的功能具有多种影响，包括抑制脂肪细胞分化。我们开始研究TCDD抑制已建立的前脂肪细胞系3T3-L1分化的机制。通过Northern印迹分析检测几种前脂肪细胞分化早期标记基因的表达以及分化依赖性有丝分裂，结果表明TCDD不会干扰前脂肪细胞对分化诱导剂的已知最早反应。对CCAAT/增强子结合蛋白（C/EBP）三种同工型的mRNA分析显示，TCDD（5 nM）选择性抑制C/EBPα mRNA的诱导，但不阻断C/EBPβ和C/EBPδ对分化诱导剂反应的诱导。PPARγ mRNA的分化依赖性诱导也被TCDD阻断。用针对每种C/EBP同工型的特异性抗体进行免疫印迹分析表明，在溶剂处理（对照）细胞中，C/EBPδ和C/EBPβ蛋白水平在第1天迅速诱导，然后分别在第4天和第8天消失。然而，在TCDD处理的细胞中，C/EBPβ和C/EBPδ蛋白水平在这些时间点持续存在。相比之下，C/EBPα蛋白在TCDD处理下与其RNA水平一致地被显著抑制。C/EBPα的两种翻译产物p30和p42均被TCDD剂量依赖性降低。对与含有C/EBP DNA识别序列的寡核苷酸结合的核提取物进行凝胶迁移分析显示，在分化第2天，对照细胞和TCDD处理细胞的提取物在结合模式上没有差异。在第4天和第8天，与C/EBPα/DNA复合物对应的条带（通过超迁移分析确定）在处理后的提取物中与对照提取物相比显著减少。相反，在处理后的样品中发现与C/EBPβ/DNA复合物对应的条带富集。这些数据表明，TCDD对3T3-L1前脂肪细胞系分化的抑制发生在分化程序中的一个短暂但确定的时期，并且涉及C/EBP调节的改变，包括对C/EBPα的抑制。
