Regulation of Proline Oxidase for Bioenergetics during Nutrient Stress

我们之前已经证明，缺糖可以激活POX，以维持自噬和存活。由于营养缺乏通常伴随着低氧，我们测试了低氧对POX表达的影响。通过实时定量聚合酶链式反应或荧光素酶活性检测，在实时定量聚合酶链式反应或荧光素酶活性检测中，各种低氧培养细胞均表现出POX基因表达的增加。Western印迹显示POX mRNA和POX蛋白随低氧(5%、0.5%、0.05%氧气)和低氧时间的延长而增加。有趣的是，痘的增加并不是由HIF-1α介导的。相反，痘反应是由AMPK介导的。与对AMPK的依赖一致，我们发现ATP水平随着低氧的降低而降低，随着POX的下调而进一步降低。重要的是，用siRNA敲除POX后，缺氧导致的细胞增殖下降的情况更加明显。虽然POX在很大程度上是缺氧时ROS增加的原因，但通过PARP裂解检测，POX并没有诱导细胞凋亡。相反，低氧诱导了自噬，而这种自噬通过siRNA击倒POX而减少。虽然在组织培养中发现了POX的诱导机制，但我们希望在体内证实低氧对异种移植瘤的影响。在约翰霍普金斯大学克里斯汀·格伦德斯实验室的合作下，我们测试了体内肿瘤中POX的表达。该模型使用在低氧反应元件(HRE)控制的启动子控制下表达绿色荧光蛋白(GFP)的细胞。我们首先发现，这些暴露在低氧或化学低氧(CoCl2)中的细胞表达POX，正如qPCR所监测的那样。然后将这些细胞注射到裸鼠体内，当肿瘤达到血管形成不足的大小时，将其取出，切片，并对GFP和POX进行染色。在组织技术实验室的米里亚姆·安弗博士和唐娜·布彻的帮助下，建立了一种鸡抗GFP抗体和兔抗痘抗体的方法。二次抗体的差异荧光分别区分了GFP和POX的表达。利用一个数学模型，建立了共焦模型。因此，在体内证实了低氧和POX表达之间的偶联。

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 Glundes 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.