Mechanisms of Oxygen Sensing

描述(由申请人提供)：适当调节细胞对低氧的反应对于维持细胞和生物体的活力和功能至关重要。尽管在过去的二十年里，关于细胞的缺氧反应已经有了很多的了解，但我们对氧气感知的理解仍然存在一些根本的差距。我们以前的工作表明，低氧反应过程中线粒体活性氧物种(ROS)的产生是诱导低氧诱导转录因子(HIF)的关键。然而，线粒体如何在低氧条件下产生更高水平的ROS尚不清楚。我们将测试分离的线粒体在低氧条件下产生ROS的能力。由于我们的工作已经证明线粒体复合体III的QO位点对缺氧时ROS的产生至关重要，因此我们还将测定分离的线粒体复合体III在低氧条件下产生ROS的能力。我们的初步数据表明，ROS通过抑制针对HIF降解的Pro羟基酶结构域蛋白2(PHD2)来调节HIF。ROS如何调控PHD2尚不清楚。因此，我们将研究PHD2的氨基末端(非催化)结构域在低氧时调节PHD2功能中的作用。以前的报道表明，这是一个抑制调节域，尽管抑制的机制尚不清楚。最后，我们将建立第一个线粒体氧感应的小鼠模型，以展示线粒体ROS在体内HIF调节中的作用。表皮通过调节流向肾脏的血液来发挥氧气传感器的作用，肾脏在缺氧时会产生促红细胞生成素。因此，我们将有条件地敲除角质形成细胞中的线粒体转录因子组，从而耗尽这些表皮细胞中线粒体和ROS的产生。然后，我们将让这些小鼠处于缺氧状态，以证明线粒体在体内作为氧气感受器的作用。与公共卫生相关：虽然健康的人在高海拔地区遇到低氧，但在缺血再灌注损伤、心肺疾病和癌症期间会出现低氧的病理条件。由于这些情况是导致死亡的主要原因，因此需要进一步研究细胞对缺氧的反应机制。因此，我们将进一步探讨线粒体ROS在调节细胞缺氧反应中的作用。

DESCRIPTION (provided by applicant): Proper regulation of the cellular response to hypoxia is of critical importance for the maintenance of cellular and organismal viability and function. Although much has been learned about the cellular hypoxic response in the last twenty years, several fundamental gaps in our understanding of oxygen sensing still exist. Our previous work has shown that production of mitochondrial Reactive Oxygen Species (ROS) during the hypoxic response is critical for the induction of the Hypoxia Inducible transcription Factor (HIF). It is unknown however, how mitochondria produce increased levels of ROS under conditions of low oxygen. We will test the ability of isolated mitochondria to produce ROS in response to low oxygen. Because our work has demonstrated that the QO site of mitochondrial complex III is critical for ROS production during hypoxia, we will also determine the ability of isolated mitochondrial complex III to produce ROS under conditions of low oxygen. Our preliminary data demonstrates that ROS regulate HIF through the inhibition of the Prolyl Hydroxylase Domain protein 2 (PHD2), which targets HIF for degradation. How ROS regulate PHD2 remains unknown. We will therefore examine the role of the amino-terminal (non-catalytic) domain of PHD2 In the regulation of PHD2 function during hypoxia. Previous reports suggest that this is an inhibitory regulatory domain, although the mechanism of inhibition is unexplored. Finally, we will create the first mouse model of mitochondrial oxygen sensing to demonstrate the role of mitochondrial ROS in HIF regulation in vivo. Epidermis acts as an oxygen sensor by regulating blood flow to the kidneys, which produce erythropoietin during hypoxia. We will therefore conditionally knock out the mitochondrial transcription factor TEAM in keratinocytes, thus depleting mitochondria and ROS production in these epidermal cells. We will then subject these mice to hypoxia to demonstrate the role of mitochondria as oxygen sensors in vivo. Relevance to Public Heath: While healthy individuals encounter hypoxia at high altitudes, pathological conditions of hypoxia arise during Ischemia-reperfusion injury, heart and lung disease, and cancer. Because these conditions represent major causes of mortality, further study of the mechanisms by which cells respond to hypoxia is needed. We will thus further explore the role of mitochondrial ROS in regulating the cellular hypoxic response.