Ohmori M, Harii N, Endo T, Onaya T
Third Department of Internal Medicine, Yamanashi Medical University, Tamaho, Japan.
Endocrinology. 1999 Oct;140(10):4651-8. doi: 10.1210/endo.140.10.7021.
Tumor necrosis factor-alpha (TNF-alpha) is known to modulate the expression of thyroid-specific genes, such as thyroglobulin (TG), contributing to the pathogenesis of autoimmune thyroid disease. In the present study, we show that TNF-alpha suppresses DNA-binding activity of thyroid transcription factors, Pax-8 and thyroid transcription factor-1 (TTF-1), which is, in part, involved in TNF-alpha-induced decrease in TG gene expression. Transfected into rat thyroid FRTL-5 cells, the activity of reporter plasmid containing the rat TG promoter ligated to a luciferase gene was significantly suppressed in the presence of TNF-alpha. In gel mobility shift analyses, protein-DNA complexes formed by TTF-1 and Pax-8 were reduced when the nuclear extracts prepared from TNF-alpha-treated FRTL-5 cells were used. The suppressive effect of TNF-alpha on TTF-1-DNA complex formation is, in part, caused by suppression of TTF-1 gene transcription by TNF-alpha. Expressions of TTF-1 messenger RNA and protein, which were assessed by Northern blot and Western blot analyses, respectively, were decreased by TNF-alpha treatment of FRTL-5 cells. In contrast, TNF-alpha did not affect the expression of Pax-8 messenger RNA. Treatment of FRTL-5 cells with TNF-alpha caused a decrease in Pax-8 protein in nuclear extracts and accumulation of the protein in the cytoplasm, as assessed by Western blot analyses. Mutation of the TTF-1/Pax-8-binding site lost the TNF-alpha-induced decrease in TG promoter activity in a transfection experiment. These results indicate that TNF-alpha suppresses the activity of TTF-1 and Pax-8 by different mechanisms, which, in part, seem to be involved in TNF-alpha-induced decrease in TG gene expression.
已知肿瘤坏死因子-α（TNF-α）可调节甲状腺特异性基因的表达，如甲状腺球蛋白（TG），这在自身免疫性甲状腺疾病的发病机制中起作用。在本研究中，我们发现TNF-α抑制甲状腺转录因子Pax-8和甲状腺转录因子-1（TTF-1）的DNA结合活性，这在一定程度上参与了TNF-α诱导的TG基因表达下降。将连接有荧光素酶基因的大鼠TG启动子的报告质粒转染到大鼠甲状腺FRTL-5细胞中，在存在TNF-α的情况下，其活性显著受到抑制。在凝胶迁移率变动分析中，当使用从TNF-α处理的FRTL-5细胞制备的核提取物时，由TTF-1和Pax-8形成的蛋白质-DNA复合物减少。TNF-α对TTF-1-DNA复合物形成的抑制作用部分是由TNF-α对TTF-1基因转录的抑制引起的。分别通过Northern印迹和Western印迹分析评估的TTF-1信使RNA和蛋白质的表达，在TNF-α处理FRTL-5细胞后降低。相反，TNF-α不影响Pax-8信使RNA的表达。通过Western印迹分析评估，用TNF-α处理FRTL-5细胞导致核提取物中Pax-8蛋白减少，而该蛋白在细胞质中积累。在转染实验中，TTF-1/Pax-8结合位点的突变使TNF-α诱导的TG启动子活性下降消失。这些结果表明，TNF-α通过不同机制抑制TTF-1和Pax-8的活性，这在一定程度上似乎参与了TNF-α诱导的TG基因表达下降。
