Guo Feifan, Bakal Keren, Minokoshi Yasuhiko, Hollenberg Anthony N
Thyroid Unit, Division of Endocrinology, Beth Israel Deaconess Medical Center, 330 Brookline Avenue, Boston Massachusetts 02215, USA.
Endocrinology. 2004 May;145(5):2221-7. doi: 10.1210/en.2003-1312. Epub 2004 Feb 5.
The regulation of TRH gene expression in the paraventricular nucleus of the hypothalamus (PVH) by leptin is critical for normal function of the thyroid axis in rodents and humans. The TRH neuron in the PVH expresses both leptin and melanocortin-4 receptors, suggesting that both signaling systems may regulate TRH gene expression in vivo. Indeed, the TRH promoter responds to both of these signaling pathways in cell culture through identified cis-acting elements, which include signal transducer and activator of transcription (STAT) 3 and cAMP-response element binding protein binding sites that mediate leptin and melanocortin responses, respectively. To determine whether leptin signaling can directly target the TRH promoter in vivo, we developed a chromatin immunoprecipitation assay to use on leptin-treated animals. After a single injection of leptin in fasting animals, we could detect a significant increase in TRH gene expression in the PVH that correlated well with the induction of phosphorylated-STAT3 in the hypothalamus. Furthermore, using a STAT3 antibody, we could immunoprecipitate the STAT-binding site containing regions of both the TRH promoter and the promoter of the suppressor of cytokine signaling-3 gene, another well-defined target of leptin action. In contrast, upstream regions of these promoters that lack STAT sites were not precipitated. Taken together these experiments demonstrate that STAT3 mediates transcriptional effects of leptin in vivo and that the TRH promoter is a likely direct site of leptin action. In addition, these experiments demonstrate that chromatin immunoprecipitation can be used to characterize leptin-signaling in vivo.
瘦素对下丘脑室旁核（PVH）中促甲状腺激素释放激素（TRH）基因表达的调节，对啮齿动物和人类甲状腺轴的正常功能至关重要。PVH中的TRH神经元同时表达瘦素和黑皮质素-4受体，这表明这两种信号系统可能在体内调节TRH基因表达。实际上，在细胞培养中，TRH启动子通过已确定的顺式作用元件对这两种信号通路作出反应，这些元件包括分别介导瘦素和黑皮质素反应的信号转导和转录激活因子（STAT）3以及环磷酸腺苷反应元件结合蛋白结合位点。为了确定瘦素信号在体内是否能直接作用于TRH启动子，我们开发了一种染色质免疫沉淀测定法用于瘦素处理的动物。在禁食动物单次注射瘦素后，我们能检测到PVH中TRH基因表达显著增加，这与下丘脑中磷酸化STAT3的诱导密切相关。此外，使用STAT3抗体，我们能够免疫沉淀TRH启动子以及细胞因子信号抑制因子-3基因启动子中包含STAT结合位点的区域，细胞因子信号抑制因子-3基因是瘦素作用的另一个明确靶点。相比之下，这些启动子中缺乏STAT位点的上游区域未被沉淀。这些实验共同表明，STAT3在体内介导瘦素的转录作用，并且TRH启动子可能是瘦素作用的直接位点。此外，这些实验表明染色质免疫沉淀可用于在体内表征瘦素信号。
