Leptin signaling targets the thyrotropin-releasing hormone gene promoter in vivo
Leptin signaling targets the thyrotropin-releasing hormone gene promoter in vivo
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DOI：
10.1210/en.2003-1312
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发表时间：
2004-05-01
期刊：
影响因子：
4.8
通讯作者：
Hollenberg, AN
中科院分区：
文献类型：
--
作者：
Guo, FF;Bakal, K;Hollenberg, AN
瘦素对下丘脑室旁核（PVH）TRH基因表达的调控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都可以调节体内TRH基因的表达。事实上，TRH启动子通过鉴定的顺式作用元件对细胞培养物中的这两种信号传导途径做出响应，所述顺式作用元件包括分别介导瘦素和黑皮质素反应的信号转导子和转录激活子（STAT）3和cAMP反应元件结合蛋白结合位点。为了确定瘦素信号传导是否可以直接靶向体内TRH启动子，我们开发了一种染色质免疫沉淀试验用于瘦素治疗的动物。在禁食动物中单次注射瘦素后，我们可以检测到PVH中TRH基因表达的显着增加，这与下丘脑中磷酸化STAT 3的诱导密切相关。此外，使用STAT 3抗体，我们可以免疫沉淀的STAT结合位点包含TRH启动子和细胞因子信号转导抑制因子-3基因的启动子，另一个明确的目标瘦素的行动。相比之下，这些启动子的上游区域缺乏STAT位点没有沉淀。总之，这些实验表明，STAT 3介导的瘦素在体内的转录作用，TRH启动子是一个可能的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表征瘦素信号在体内。
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 immuno-precipitation 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.