Regulation of Proline Oxidase for Bioenergetics during Nutrient Stress

营养胁迫期间脯氨酸氧化酶对生物能的调节

• 批准号: 8937822
• 负责人: JAMES M PHANG
• 金额: $ 36.8万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8937822
• 关键词: Antibodies; Apoptosis; Attention; Autophagocytosis; Bioenergetics; Biological Assay; Blood Circulation; Cell Proliferation; Cell Survival; Cells; Chemicals; Chickens; Cleaved cell; Clinical; Collaborations; Coupling; Cultured Cells; Dependence; Down-Regulation; Eating; Extracellular Matrix Degradation; Fluorescence; Glucose; Green Fluorescent Proteins; HIF1A gene; Home environment; Hydroxyproline; Hypoxia; In Vitro; Laboratories; Lead; Luciferases; Matrix Metalloproteinases; Measures; Mediating; Messenger RNA; Metabolism; Methods; Modeling; Monitor; Neoplasm Metastasis; Nude Mice; Nutrient; Oryctolagus cuniculus; Oxygen; Process; Proliferating; Proline; Proline Dehydrogenase; Proteins; Regulation; Research Personnel; Response Elements; Signal Transduction; Small Interfering RNA; Staining method; Stains; Stress; Testing; Time; Tumor Cell Invasion; Vascular blood supply; Vascularization; Western Blotting; cancer therapy; defined contribution; deprivation; genetic regulatory protein; in vivo; interest; mathematical model; neoplastic cell; neovascularization; novel strategies; promoter; response; success; tissue culture; tumor; tumor metabolism; tumor xenograft

We previously showed that glucose deprivation can activate POX to maintain autophagy and survival. Since nutrient deprivation is usually accompanied by hypoxia, we tested the effects of hypoxia on POX expression. A variety of cultured cells subjected to hypoxia showed an increase in POX expression either monitored at the level of mRNA by real time PCR or by a luciferase assay for POX promoter activity. POX mRNA and POX protein by Western blot increased as a function of hypoxia (5%, 0.5%, 0.05% oxygen) and duration of hypoxia. Interestingly, the increase in POX is not mediated by HIF-1alpha. Instead, the POX response was mediated by AMPK. Consistent with the dependence on AMPK, we found that ATP levels which were decreased with hypoxia, decreased further with POX knockdown. Importantly, the decrease in cell proliferation due to hypoxia was accentuated by knockdown of POX with siRNA. Although POX was responsible, in large part, for the increase in ROS with hypoxia, it did not induce apoptosis as measured by PARP cleave. Instead, hypoxia induced autophagy and this autophagy was decreased by the knockdown of POX by siRNA. Although the mechanism for POX induction was identified in tissue culture, we desired confirmation of the effects of hypoxia in xenograft tumors in vivo. In collaboration with Kristine Glunde?s laboratory at Johns Hopkins, we tested POX expression in tumors in vivo. This model uses cells expressing green fluorescent protein (GFP) under control of a hypoxia response element (HRE) controlled promoter. We first showed that these cells exposed to hypoxia or chemical hypoxia (CoCl2) expressed POX as monitored by qPCR. These cells were then injected into nude mice and when the tumors had attained a size with inadequate vascularization, they were removed, sectioned and stained for GFP and for POX. With the help of Dr. Miriam Anver and Donna Butcher of the histotechnology lab, a method was developed with a chicken anti-GFP antibody and a rabbit anti-POX antibody. Differential fluorescence of the secondary antibodies distinguished the expression of GFP and POX, respectively. Using a mathematical model, colocalization was established. Thus, the coupling between hypoxia and POX expression was confirmed in vivo. These in vitro and in vivo studies led to the conclusion that with hypoxia, AMPK activates PRODH/POX to produce ROS which initiates autophagy. On the other hand, with low glucose in the presence or absence of hypoxia, AMPK activates PRODH/POX to generate ATP for cell survival. After neovascularization with an adequate blood supply when tumor cells are proliferating, this state is maintained by the downregulation of PRODH/POX by the increased levels of miR-23b* which is increased by c-MYC. We are currently identifying modulatory proteins for this regulatory axis. An interesting finding is that FOXO proteins are involved in the POX-mediated, ROS generated signaling. This mechanism is being actively explored.

我们之前的研究表明，葡萄糖剥夺可以激活POX以维持自噬和存活。由于营养剥夺通常伴随着缺氧，我们测试了缺氧对POX表达的影响。通过实时PCR或荧光素酶检测痘启动子活性，在mRNA水平上监测多种缺氧培养细胞的痘表达增加。Western blot结果显示，低氧（5%、0.5%、0.05%）和缺氧时间对痘mRNA和蛋白表达均有影响。有趣的是，痘的增加不是由hif -1 α介导的。相反，痘反应是由AMPK介导的。与对AMPK的依赖性一致，我们发现ATP水平在缺氧时下降，在POX敲低时进一步下降。重要的是，由于缺氧导致的细胞增殖的减少可以通过siRNA敲低POX来加强。虽然POX在很大程度上导致了缺氧时ROS的增加，但通过PARP切割测量，它并没有诱导细胞凋亡。相反，缺氧诱导自噬，这种自噬通过siRNA敲低POX而减少。虽然在组织培养中确定了POX诱导的机制，但我们希望在体内确认缺氧对异种移植物肿瘤的影响。和克里斯汀·格伦德合作？在约翰霍普金斯大学的实验室里，我们测试了POX在肿瘤体内的表达。该模型使用在缺氧反应元件（HRE）控制启动子控制下表达绿色荧光蛋白（GFP）的细胞。我们首先发现这些暴露于缺氧或化学缺氧（CoCl2）的细胞通过qPCR监测表达POX。然后将这些细胞注射到裸鼠体内，当肿瘤达到一定大小且血管化不足时，将其取出，切片并进行GFP和POX染色。在组织技术实验室的Miriam Anver博士和Donna Butcher的帮助下，我们开发了一种使用鸡抗gfp抗体和兔抗痘抗体的方法。二抗的差异荧光分别区分GFP和POX的表达。利用数学模型，建立了共定位。因此，在体内证实了缺氧与POX表达之间的耦合。这些体外和体内研究得出的结论是，在缺氧情况下，AMPK激活PRODH/POX产生ROS，引发自噬。另一方面，在低葡萄糖存在或不存在缺氧的情况下，AMPK激活PRODH/POX产生ATP以维持细胞存活。肿瘤细胞增殖时，新生血管形成后血供充足，通过c-MYC升高miR-23b*水平下调PRODH/POX来维持这种状态。我们目前正在鉴定这个调节轴的调节蛋白。一个有趣的发现是FOXO蛋白参与了pox介导的ROS产生的信号。这一机制正在积极探索中。