Regulation of Proline Oxidase for Bioenergetics during Nutrient Stress

我们先前显示噻唑烷二酮类，通常用于2型糖尿病的药物，是通过PPARgamma的POX启动子的有效激活剂（Pandhare J等人，J. Biol. Chem.，281：2044，2006; Phang J.等人，，2008;2008：542694）。POX与PPARgamma的偶联强烈表明POX参与生物能量学的调节和对营养胁迫的响应，这一发现使我们考虑mTOR-AMPK信号通路。该途径整合来自生长因子、营养素、能量水平和细胞应激的信号，以调节蛋白质翻译和细胞生长。该途径中的突变与许多肿瘤表型相关。我们测试了雷帕霉素（mTOR的抑制剂）、LY 294002（PI 3-K/Akt的抑制剂）和5-氨基-4-甲酰胺呋喃核糖苷（AICAR）（一种激活AMP导向蛋白激酶（AMPK）的嘌呤类似物）的作用。我们发现，这些在3个不同位点阻断mTOR信号传导的药物均显著激活POX活性。此外，雷帕霉素通过阻断mTOR，抑制蛋白质翻译和细胞生长，并伴随着增加细胞ATP水平，可能是为了维持植物生存状态。有趣的是，通过POX siRNA阻断POX表达或用脱氢脯氨酸抑制POX催化活性显著抑制了雷帕霉素诱导的细胞ATP增加。这些研究表明，脯氨酸可以在PPARgamma和mTOR/AMPK信号传导途径的调节下作为应激底物发挥作用（Pandhare J，et al.，J.细胞。生化、107：759，2009; Phang JM小于I.et等人，138：2008S，2008）。 虽然我们表明，POX表达产生ATP的营养胁迫条件下，ATP的生化来源需要阐明。葡萄糖是培养细胞中ATP的主要来源，因此我们测试了POX过表达是否会增加糖酵解。令人惊讶的是，通过（5）-3H-葡萄糖转化为3 H2O测量的糖酵解不随POX过表达而改变。与此相反，戊糖磷酸分流（PPS）增加超过5倍时，POX诱导。此外，限制葡萄糖（0.05 mM），ATP水平逐渐下降。然而，当POX被诱导时，在存在或不存在添加的脯氨酸的情况下维持ATP水平。推测内源脯氨酸的循环可能介导了这种效应。这些发现表明，当葡萄糖受到限制时，POX通过PPS促进葡萄糖的代谢，并且脯氨酸的循环将从分流产生的NADPH穿梭到ATP产生的还原潜力的来源（Pandhare J，et al.，J.细胞。生化、107：759，2009）。 另一个开创性的发现是，POX介导的信号参与脂质代谢和氧化低密度脂蛋白（oxLDL）启动的信号。用oxLDL处理结肠直肠癌细胞显著诱导POX，并且这种诱导依赖于PPARgamma，因为oxLDL颗粒中所含的氧化代谢物7-酮基胆固醇是PPARgamma的有效激活剂。OxLDL可刺激自噬和凋亡。有趣的是，自噬被siRNA敲低POX部分阻断，而oxLDL激活的细胞凋亡则没有。因此，POX似乎参与oxLDL的自噬激活。为了直接评估这种联系，我们使用DLD-tet-off-POX细胞，当从培养基中去除多西环素时，稳定的转染子中诱导POX。我们发现LC 3-I在POX表达下被切割成LC 3-II，并且LC 3-II被并入自噬体中。重要的是，自噬的关键基因beclin-1的表达由POX的表达诱导。因此，由oxLDL通过PPARgamma诱导的POX的表达是自噬激活的信号传导机制。这是令人感兴趣的，因为我们已经提出POX启动生态吞噬，即微环境中底物（例如胶原蛋白）的消耗。我们的研究表明，生态吞噬不仅先于自噬，而且可能激活自噬。有了上述POX在营养胁迫期间提供ATP来源的证据，我们还解决了由于缺氧引起的代谢应激的问题。通过真实的时间PCR或荧光素酶测定POX启动子活性，在mRNA水平上监测到各种缺氧培养细胞显示POX表达增加。Western blot检测POX mRNA和POX蛋白表达随缺氧（5%、0.5%、0.05%氧）和缺氧时间的增加而增加。有趣的是，POX的增加不是由HIF-1 α介导的。相反，POX反应由AMPK介导。与对AMPK的依赖性一致，我们发现ATP水平随着缺氧而降低，随着POX敲低而进一步降低。重要的是，由于缺氧导致的细胞增殖的降低通过用siRNA敲低POX而加剧。虽然POX是负责，在很大程度上，在缺氧的ROS的增加，它没有诱导细胞凋亡所测量的PARP裂解。相反，缺氧诱导自噬，并且这种自噬通过siRNA敲低POX而减少。总之，这些研究表明，通过AMPK磷酸化级联反应对缺氧的反应涉及POX的诱导，POX激活自噬以维持ATP和细胞存活。

We previously showed that thiazolidinediones, commonly-used drugs for type 2 diabetes, were potent activators of the POX promoter through PPARgamma (Pandhare J, et al., J. Biol. Chem., 281:2044, 2006; Phang J. et al., PPAR Res. , 2008;2008: 542694). The coupling of POX to PPARgamma strongly suggests that POX is involved in regulation of bioenergetics and responses to nutrient stress, a finding which led us to consider the mTOR-AMPK signaling pathway. This pathway integrates signals from growth factors, nutrients, energy levels and cellular stress to regulate protein translation and cell growth. Mutations in this pathway have been associated with a number of neoplastic phenotypes. We tested the effects of rapamycin, an inhibitor of mTOR, LY 294002, an inhibitor of PI3-K/Akt, and 5-amino-4-carboxamide ribofuranoside (AICAR), a purine analog which activates AMP-directed protein kinase (AMPK). We found that these agents which block mTOR signaling at 3 different sites, all markedly activated POX activity. Additionally, rapamycin, by blocking mTOR, inhibited protein translation and cell growth and concomitantly increased cellular ATP levels, presumably to sustain a vegetative survival state. Interestingly, blockade of POX expression by POX siRNA or inhibiting POX catalytic activity with dehydroproline markedly inhibited the rapamycin-induced increase in cellular ATP. These studies suggest that proline can function as a stress substrate under the regulation of PPARgamma and the mTOR/AMPK signaling pathways (Pandhare J, et al., J. Cell. Biochem., 107:759, 2009; Phang JM less than I.et al., J. Nutr.138:2008S, 2008). Although we showed that POX expression generated ATP under conditions of nutrient stress, the biochemical source for the ATP required elucidation. Glucose is the main source for ATP in cultured cells; therefore we tested whether glycolysis was increased by POX overexpression. Surprisingly, glycolysis measured by the conversion of (5)-3H-glucose to 3H2O was not changed with POX overexpression. In contrast, the pentose phosphate shunt (PPS) was increased more than 5-fold when POX was induced. Furthermore, with limiting glucose (.05 mM), ATP levels progressively fell. However, when POX was induced, ATP levels were maintained in the presence or absence of added proline. Presumably, the cycling of endogenous proline could mediate the effect. These findings suggest that when glucose is limiting, POX promotes the metabolism of glucose through the PPS and the cycling of proline shuttles the NADPH generated from the shunt into a source of reducing potential for ATP generation (Pandhare J, et al., J. Cell. Biochem., 107:759, 2009). Another seminal finding is that POX-mediated signaling is involved in lipid metabolism and signaling initiated by oxidized low-density lipoprotein (oxLDL). Treatment of colorectal cancer cells with oxLDL markedly induced POX, and this induction was dependent on PPARgamma because 7-ketocholesterol, an oxidized metabolite contained in the oxLDL particle is a potent activator of PPARgamma. OxLDL can stimulate both autophagy and apoptosis. Interestingly, autophagy was partially blocked by knockdown of POX by siRNA, whereas oxLDL-activated apoptosis was not. Thus, POX appears to be involved in the autophagic activation by oxLDL. To directly assess this linkage, we used DLD-tet-off-POX cells, stable transfectants in which POX is induced when doxycycline is removed from the medium. We found that LC3-I is cleaved to LC3-II with POX expression, and LC3-II is incorporated into autophagosomes. Importantly the expression of beclin-1, a critical gene for autophagy, was induced by the expression of POX. Thus, the expression of POX , induced by oxLDL through PPARgamma, is a signaling mechanism for the activation of autophagy. This is of interest because we have proposed that POX initiates ecophagy, the consumption of substrates, e.g. collagen, in the microenvironment. Our studies suggested that ecophagy not only precedes but also may activate autophagy. With the aforementioned evidence that POX provides a source of ATP during nutrient stress, we also addressed the question of metabolic stress due to hypoxia. 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 acdtivity. 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 cleavage. Instead, Hypoxia induced autophagy and this autophagy was decreased by the knockdown of POX by siRNA. Taken together, these studies suggested that the response to hypoxia through the AMPK phosphorylation cascade involved the induction of POX which activated autophagy to maintain ATP and cell survival.