ROLE OF ROS IN HYPOXIA-MEDIATED REGULATION OF THE PHENYLETHANOLAMINE N-METHYLTRANSFERASE GENE

氧是哺乳动物细胞正常细胞功能和体内平衡的重要组成部分。哺乳动物能够引发对缺氧的应激反应，以促进清除或适应缺氧。哺乳动物的应激反应是通过激活下丘脑-垂体-肾上腺（HPA）轴和交感神经-肾上腺（SA）轴介导的，导致应激激素、皮质酮/皮质醇和肾上腺素的释放。肾上腺素主要由肾上腺的嗜铬细胞合成和释放，是负责“战斗或逃跑反应”的神经递质/神经激素。虽然肾上腺素的合成可能在缺氧胁迫下通过HPA和SA轴进行调节，但研究表明，肾上腺染色质细胞对氧敏感，因此可能调节稳态机制，包括儿茶酚胺的生物合成，独立于激素和神经激活。这在染色质细胞交感神经支配之前的胎儿发育过程中是至关重要的。迄今为止的研究支持活性氧（ROS）参与细胞对缺氧的反应。此外，最近报道的研究表明，氧化应激和ROS改变了表观遗传基因的调控机制。然而，ROS在参与肾上腺素合成调控的细胞机制中的作用，特别是PNMT基因的表观遗传调控尚不清楚。本提案将利用细胞到系统生物学的方法来研究缺氧在PNMT基因调控中的作用。利用体外PC12细胞培养模型和先进的体外细胞和分子技术，将描述PNMT基因在缺氧和ROS反应中调控的细胞和分子机制。此外，表观遗传调控因子在介导细胞缺氧反应中的作用也将被研究。此外，将利用啮齿动物体内缺氧暴露模型来检验体外鉴定的分子调节机制是否参与整个生物体对缺氧应激的反应。这项资助的结果将提供对缺氧反应的基本细胞过程的新见解。此外，它还将深入了解应激激素肾上腺素在哺乳动物对缺氧的应激反应和适应中的作用。

Oxygen is a critical component of normal cellular function and homeostasis in mammalian cells. Mammals are capable of eliciting a stress-response to reduced oxygen to facilitate either removal or adaptation to hypoxia. The mammalian stress-response is mediated via activation of the hypothalamic-pituitary-adrenal (HPA) axis and the sympatho-adrenal (SA) axis, resulting in the release of the stress hormones, cortisosterone/cortisol and adrenaline. Adrenaline is synthesized and released predominantly by the chromaffin cells of the adrenal glands and is the neurotransmitter/neurohormone responsible for the “fight or flight response”. Although adrenaline synthesis may be regulated via the HPA and SA axis in response to hypoxic stress, studies have shown that adrenal chromaffin cells are oxygen sensitive and may therefore regulate homeostatic mechanism, including catecholamine biosynthesis, independent of hormonal and neural activation. This is critically important during fetal development prior to sympathetic innervation of chromaffin cells. Studies to date support a role of reactive oxygen species (ROS) is involved in the cellular responses to hypoxia. Furthermore, recently reported studies suggest that oxidative stress and ROS alters epigenetic gene regulation mechanism. However the role of ROS in cellular mechanisms involved in the regulation of the adrenaline synthesis and in particular epigenetic regulation of the PNMT gene is not known. This proposal will utilize a cell to systems biology approach to examine the role of hypoxia in the regulation of the PNMT gene. Utilizing an in-vitro PC12 cell culture model and advanced in vitro cellular and molecular techniques, the cellular and molecular mechanism involved in the regulation of the PNMT gene in response to hypoxia and ROS will be delineated. In addition, the role of epigenetic regulators in mediating the hypoxic cellular response will also be investigated. Furthermore, in vivo rodent models of hypoxia exposure will be utilized to examine the whether the molecular regulatory mechanisms identified in-vitro are involved in the whole organism's response to hypoxic stress. The results from this grant will provide new insight into the fundamental cellular processes involved in response to hypoxia. In addition, it will also provide insight into the role of stress the hormone, adrenaline, in the mammalian stress response to and in the adaptation to hypoxia.