Transcriptional up-regulation of inhibitory PAS domain protein gene expression by hypoxia-inducible factor 1 (HIF-1) -: A negative feedback regulatory circuit in HIF-1-mediated signaling in hypoxic cells
Transcriptional up-regulation of inhibitory PAS domain protein gene expression by hypoxia-inducible factor 1 (HIF-1) -: A negative feedback regulatory circuit in HIF-1-mediated signaling in hypoxic cells
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DOI：
10.1074/jbc.m700732200
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发表时间：
2007-05-11
影响因子：
4.8
通讯作者：
Morimoto, Chikao
中科院分区：
文献类型：
--
作者：
Makino, Yuichi;Uenishi, Rie;Morimoto, Chikao
抑制性PAS(Per/Arnt/Sim)结构域蛋白(IPAS)是低氧诱导转录因子(HIF)的主要负调控因子，可能与眼部无血管角膜等组织的血管生成负调控有关。我们以前已经证明，在低氧组织中，IPAS mRNA的表达上调，这至少部分涉及到低氧依赖的IPAS/HIF-3α基因转录本的选择性剪接。在目前的研究中，我们证明了低氧驱动的转录机制也在IPAS基因表达的增强中发挥作用。分离和分析iPAs基因第一外显子两侧的启动子区域，发现在-834~-799位置有一个功能性的低氧反应元件，而HIF-3α第一外显子上游的序列对低氧刺激几乎没有反应。瞬时转染实验表明，在低氧条件下，HIF-1α通过功能性低氧反应元件介导IPAS启动子的激活，并且HIF-1α的组成活性形式足以在正常缺氧细胞中诱导启动子的激活。此外，染色质免疫沉淀和电泳迁移率改变分析表明，HIF-1复合体以低氧依赖的方式与该元件结合。综上所述，HIF-1通过不同于RNA剪接的机制直接上调IPAS基因的表达，在对缺氧/缺血条件的适应性反应中提供更高水平的负反馈基因调控。
The inhibitory PAS (Per/Arnt/Sim) domain protein (IPAS), a dominant negative regulator of hypoxia-inducible transcription factors (HIFs), is potentially implicated in negative regulation of angiogenesis in such tissues as the avascular cornea of the eye. We have previously shown IPAS mRNA expression is up-regulated in hypoxic tissues, which at least in part involves hypoxia-dependent alternative splicing of the transcripts from the IPAS/HIF-3 alpha locus. In the present study, we demonstrate that a hypoxia-driven transcriptional mechanism also plays a role in augmentation of IPAS gene expression. Isolation and analyses of the promoter region flanking to the first exon of IPAS gene revealed a functional hypoxia response element at position -834 to -799, whereas the sequence upstream of the HIF-3 alpha first exon scarcely responded to hypoxic stimuli. A transient transfection experiment demonstrated that HIF-1 alpha mediates IPAS promoter activation via the functional hypoxia response element under hypoxic conditions and that a constitutively active form of HIF-1 alpha is sufficient for induction of the promoter in normoxic cells. Moreover, chromatin immunoprecipitation and electrophoretic mobility shift assays showed binding of the HIF-1 complex to the element in a hypoxia-dependent manner. Taken together, HIF-1 directly up-regulates IPAS gene expression through a mechanism distinct from RNA splicing, providing a further level of negative feedback gene regulation in adaptive responses to hypoxic/ischemic conditions.