Mori K, Stone S, Braverman L E, Devito W J
Division of Endocrinology, University of Massachusetts Medical Center, Worcester, 01655, USA.
Endocrinology. 1996 Apr;137(4):1313-8. doi: 10.1210/endo.137.4.8625905.
Recent studies suggest that protein tyrosine phosphorylation may play a role in the regulation of thyroid growth and function. In the present study, we used genistein, a specific inhibitor of tyrosine phosphorylation, to determine if tyrosine phosphorylation is involved in the regulation of type I 5'-deiodinase (5'D-I) expression in FRTL-5 cells and type II 5'-deiodinase (5'D-II) in rat astrocytes. Incubation of FRTL-5 cells with genistein (100 microM) for 3 days had no effect on cell viability as assessed by trypan blue exclusion. In TSH-deprived cells, incubation of FRTL-5 cells with genistein (100 microM) resulted in a modest, but not significant, decrease in 5'D-I activity. Incubation of FRTL-5 cells with TSH (100 microU/ml), Bu2cAMP (0.5 mM) or forskolin (1 microM) resulted in marked increases in 5'D-I activity. In the presence of genistein (100 microns), however, the TSH, Bu2cAMP and forskolin-induced increases in 5'D-I activity were completely inhibited. In Bu2cAMP-stimulated FRTL-5 cells, incubation with genistein (1, 10, and 100 microM) resulted in a dose-dependent decrease in 5'D-I activity, with 100 microns genistein completely blocking the Bu2cAMP-induced increase in 5'D-I activity. Similarly, we found that in FRTL-5 cells, genistein (100 microns) completely blocked the Bu2cAMP-induced increase in 5'D-I messenger RNA (mRNA) levels, DNA synthesis as assessed by [3H]thymidine incorporation, and the T3-induced increase in 5'D-I activity. To determine if addition of genistein to FRTL-5 cells resulted in a general inhibition of Bu2cAMP-induced responses, we examined its effect on the Bu2cAMP-induced increase in c-fos mRNA levels. Bu2cAMP-induced c-fos mRNA levels were not affected by the treatment of cells with genistein (100 microM). We then examined the effect of genistein on the Bu2cAMP and hydrocortisone-induced 5'D-II activity in cultured rat astrocytes. Genistein (100 microM) had no effect on cell viability as assessed by trypan blue exclusion. In serum deprived astrocytes, addition of Bu2cAMP (1 mM) and hydrocortisone (100 nM) resulted in a 110-fold increase in 5'D-II activity. Addition of genistein (100 microM) to stimulated astrocytes completely blocked the Bu2cAMP and hydrocortisone-induced increase in 5'D-II activity. The present data suggest that tyrosine phosphorylation-dephosphorylation may play an important role in the regulation of thyroid hormone deiodination and action in the thyroid and brain.
最近的研究表明，蛋白质酪氨酸磷酸化可能在甲状腺生长和功能的调节中起作用。在本研究中，我们使用染料木黄酮（一种酪氨酸磷酸化的特异性抑制剂）来确定酪氨酸磷酸化是否参与FRTL-5细胞中I型5'-脱碘酶（5'D-I）表达的调节以及大鼠星形胶质细胞中II型5'-脱碘酶（5'D-II）的调节。用染料木黄酮（100 microM）孵育FRTL-5细胞3天，通过台盼蓝排斥法评估，对细胞活力没有影响。在缺乏促甲状腺激素（TSH）的细胞中，用染料木黄酮（100 microM）孵育FRTL-5细胞导致5'D-I活性适度但不显著降低。用TSH（100 microU/ml）、双丁酰环磷腺苷（Bu2cAMP，0.5 mM）或福斯高林（1 microM）孵育FRTL-5细胞导致5'D-I活性显著增加。然而，在存在染料木黄酮（100 microns）的情况下，TSH、Bu2cAMP和福斯高林诱导的5'D-I活性增加被完全抑制。在Bu2cAMP刺激的FRTL-5细胞中，用染料木黄酮（1、10和100 microM）孵育导致5'D-I活性呈剂量依赖性降低，100 microns染料木黄酮完全阻断了Bu2cAMP诱导的5'D-I活性增加。同样，我们发现，在FRTL-5细胞中，染料木黄酮（100 microns）完全阻断了Bu2cAMP诱导的5'D-I信使核糖核酸（mRNA）水平增加、通过[3H]胸苷掺入评估的DNA合成以及T3诱导的5'D-I活性增加。为了确定向FRTL-5细胞中添加染料木黄酮是否导致对Bu2cAMP诱导反应的普遍抑制，我们检查了其对Bu2cAMP诱导的c-fos mRNA水平增加的影响。用染料木黄酮（100 microM）处理细胞对Bu2cAMP诱导的c-fos mRNA水平没有影响。然后我们检查了染料木黄酮对培养的大鼠星形胶质细胞中Bu2cAMP和氢化可的松诱导的5'D-II活性的影响。通过台盼蓝排斥法评估，染料木黄酮（100 microM）对细胞活力没有影响。在血清缺乏的星形胶质细胞中，添加Bu2cAMP（1 mM）和氢化可的松（100 nM）导致5'D-II活性增加110倍。向受刺激的星形胶质细胞中添加染料木黄酮（100 microM）完全阻断了Bu2cAMP和氢化可的松诱导的5'D-II活性增加。目前的数据表明，酪氨酸磷酸化-去磷酸化可能在甲状腺激素脱碘以及在甲状腺和大脑中的作用调节中起重要作用。
