Murakami Itsuo, Takeuchi Sakae, Kudo Toshiyuki, Sutou Shizuyo, Takahashi Sumio
Department of Biology, Faculty of Science, Okayama University, 3-1-1, Tsusima-naka, Okayama, Japan.
J Endocrinol. 2007 May;193(2):279-90. doi: 10.1677/JOE-06-0143.
Tpit/Pitx-responsive element (Tpit/PitxRE), which binds transcription factors Tpit and Pitx1, confers cell-type specific expression of proopiomelanocortin (POMC) gene in pituitary corticotrops where the gene expression is mainly regulated by corticotropin-releasing hormone (CRH) and glucocorticoids (Gcs). CRH stimulates POMC gene expression, which is mediated by the accumulation of intracellular cAMP and requires binding of Nur factors to Nur-responsive element (NurRE). Gcs antagonize NurRE-dependent POMC gene expression through direct interaction between glucocorticoid receptors and Nur factors. We examined whether Tpit/PitxRE and NurRE are involved in CRH/cAMP-induced activation and Gc-induced repression of POMC gene expression by reporter assay in AtT-20 corticotropic cells. Deletion and mutation of Tpit/PitxRE markedly reduced basal activity of the promoter, and those of NurRE decreased the levels of the CRH/cAMP-induced activation. Nifedipine, KN-62, and W-7, specific inhibitors of the L-type calcium channel, calmodulin-dependent protein kinase II, and calmodulin respectively, attenuated CRH/cAMP-induced activation of promoters with three copies of either Tpit/PitxRE or NurRE, indicating that both Tpit/PitxRE and NurRE mediate CRH-induced activation of POMC gene expression in a calcium-dependent manner. Deletion and mutation of Tpit/PitxRE abolished dexamethasone (DEX)-induced repression of POMC gene expression, while those of NurRE did not, indicating that Tpit/PitxRE predominantly mediates Gc-induced repression of POMC transcription. However, DEX treatment attenuated activities of promoters with three copies of either Tpit/PitxRE or NurRE, suggesting that Gcs act at NurRE as well as Tpit/PitxRE to repress POMC gene expression. We conclude that Tpit/PitxRE is an important element by which CRH and Gcs regulate the POMC gene expression.
Tpit/Pitx反应元件（Tpit/PitxRE）可结合转录因子Tpit和Pitx1，赋予阿黑皮素原（POMC）基因在垂体促肾上腺皮质激素细胞中的细胞类型特异性表达，该基因的表达主要受促肾上腺皮质激素释放激素（CRH）和糖皮质激素（Gcs）调控。CRH刺激POMC基因表达，这是由细胞内cAMP的积累介导的，并且需要Nur因子与Nur反应元件（NurRE）结合。Gcs通过糖皮质激素受体与Nur因子之间的直接相互作用拮抗NurRE依赖性POMC基因表达。我们通过在AtT-20促肾上腺皮质激素细胞中进行报告基因检测，研究了Tpit/PitxRE和NurRE是否参与CRH/cAMP诱导的激活以及Gc诱导的POMC基因表达抑制。Tpit/PitxRE的缺失和突变显著降低了启动子的基础活性，而NurRE的缺失和突变则降低了CRH/cAMP诱导的激活水平。硝苯地平、KN-62和W-7分别是L型钙通道、钙调蛋白依赖性蛋白激酶II和钙调蛋白的特异性抑制剂，它们减弱了具有三个拷贝Tpit/PitxRE或NurRE的启动子的CRH/cAMP诱导的激活，表明Tpit/PitxRE和NurRE均以钙依赖性方式介导CRH诱导的POMC基因表达激活。Tpit/PitxRE的缺失和突变消除了地塞米松（DEX）诱导的POMC基因表达抑制，而NurRE的缺失和突变则没有，表明Tpit/PitxRE主要介导Gc诱导的POMC转录抑制。然而，DEX处理减弱了具有三个拷贝Tpit/PitxRE或NurRE的启动子的活性，表明Gcs在NurRE以及Tpit/PitxRE处起作用以抑制POMC基因表达。我们得出结论，Tpit/PitxRE是CRH和Gcs调节POMC基因表达的重要元件。
