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The study of the negative regulation of thyrotropin b by thyroid hormone and its receptor

甲状腺激素及其受体对促甲状腺素b负调节作用的研究

基本信息

批准号：
12671077
负责人：
SASAKI Shigekazu
金额：
$ 1.22万
依托单位：
Hamamatsu University School of Medicine
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

项目摘要

It has been postulated that, in the TSH beta promoter, a DMA sequence immediately down stream to the transcription start site may have critical role to mediate the negative regulation driven by thyroid hormone (T3) and its receptor (TR) (Chin, W. W. et al.(1993) Recent. Prog.Horm. Res. 48 : 393-414). We designated this region as Negative Regulatory Element (NRE) and reported that histone deacetylase (HDAC) 2 may be recruited by TR in a T3 dependent manner (Sasaki S, et al. (1999) EMBO J. 18(19):5389-5398). The inspection of NRE sequence predicted the possibility that high mobility group (HMG) proteins may bind NRE and modulate the function of T3 bound TR Using gel shift assay, we confirmed that HMG protein really bind to NRE in T alpha T1 cell which has been established recently as thyrotroph cell line. In addition, we identified a HMG protein, Sox11, using RT-PCR with degenerated primers. The existence of Sox11 was confirmed by the gel sift assay with specific antibody. Furthermore, we established an experimental system in which we can detect the negative regulation of TSH beta gene in monkey kidney cell line, CV 1. The establishment of this experimental system enabled us to analyze the precise mechanisms of the negative regulation of TSH beta gene much easier than before. Using this system, we found that the overexpression of Sox11 eliminated the negative regulation of TSH beta gene by T3 and TR. Surprisingly, however, this system revealed that the negative regulation was maintained even after the destruction of NRE. Further studies suggested the possibility that the transcription factors, Pit1 and GATA2, may not only be critical to activate TSH beta promoter but also the main target of negative regulation by liganded TR.

据推测，在TSH β启动子中，紧邻转录起始位点下游的DMA序列可能对介导由甲状腺激素（T3）及其受体（TR）驱动的负调节具有关键作用（Chin，W. W.等人03 The Dog of the Woman（1993）Prog.Horm. Res. 48：393-414）。我们将该区域命名为负调控元件（NRE），并报道组蛋白脱乙酰酶（HDAC）2可以以T3依赖性方式被TR募集（Sasaki S，et al.（1999）EMBO J.18（19）：5389-5398）。通过对NRE序列的检测，推测了高迁移率族蛋白（HMG）可能与NRE结合，并调节T3结合TR的功能。此外，我们确定了HMG蛋白，Sox 11，使用RT-PCR简并引物。通过特异性抗体的凝胶筛选试验证实了Sox 11的存在。此外，我们建立了一个实验系统，我们可以检测TSH β基因在猴肾细胞系CV 1中的负调控。该实验系统的建立使我们能够更容易地分析TSH β基因负调控的确切机制。使用该系统，我们发现Sox 11的过表达消除了T3和TR对TSH β基因的负调节。然而，令人惊讶的是，该系统显示，即使在NRE被破坏后，负调节仍得以维持。进一步的研究表明，转录因子Pit 1和GATA 2可能不仅是激活TSH β启动子的关键，而且可能是配体TR负调控的主要靶点。