Regulation of Proline Oxidase for Bioenergetics during Nutrient Stress

我们之前的研究表明，噻唑烷二酮是2型糖尿病的常用药物，通过PPARgamma是痘启动子的有效激活剂（Pandhare J, et al， J. Biol）。化学。中文信息学报，28 (1):2044,2006；Phang J. et al., PPAR Res., 2008；2008: 542694)。POX与pppargamma的偶联强烈提示POX参与了生物能量学的调节和对营养胁迫的反应，这一发现促使我们考虑mTOR-AMPK信号通路。该通路整合来自生长因子、营养物质、能量水平和细胞应激的信号，调节蛋白质翻译和细胞生长。这一途径的突变与许多肿瘤表型有关。我们测试了雷帕霉素（mTOR抑制剂）、LY 294002 （PI3-K/Akt抑制剂）和5-氨基-4-carboxamide核糖呋喃苷（AICAR）（一种激活amp定向蛋白激酶（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信号的药物都显著激活了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，同时增加细胞ATP水平，可能维持植物生存状态。有趣的是，用痘siRNA阻断痘表达或用脱氢脯氨酸抑制痘的催化活性可显著抑制雷帕霉素诱导的细胞ATP的增加。这些研究表明脯氨酸可以在PPARgamma和mTOR/AMPK信号通路的调控下作为应激底物发挥作用(Pandhare J, et al.， J. Cell。物化学。中文信息学报，107:759,2009；彭建明，等。中华医学杂志。138:2008年5月，2008)。虽然我们发现在营养胁迫条件下，痘表达产生ATP，但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，我们检测了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3H2O测量的糖酵解并没有随着POX过表达而改变。与此相反，经痘诱导后，戊糖磷酸分流（PPS）增加了5倍以上。此外，限制葡萄糖(。0.05 mM)， ATP水平逐渐下降。然而，当诱导痘时，ATP水平在添加脯氨酸或不添加脯氨酸的情况下保持不变。据推测，内源性脯氨酸的循环可能介导了这种效应。这些发现表明，当葡萄糖限制时，POX通过PPS促进葡萄糖代谢，而脯氨酸的循环将由分流产生的NADPH转移到减少ATP生成的潜在来源（Pandhare J, et al.， J. Cell）。物化学。， 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治疗结直肠癌细胞显著诱导痘，这种诱导依赖于PPARgamma，因为oxLDL颗粒中含有的氧化代谢物7-酮胆固醇是PPARgamma的有效激活剂。OxLDL可以刺激细胞自噬和细胞凋亡。有趣的是，siRNA敲低POX可部分阻断自噬，而oxldl激活的细胞凋亡则不能。因此，POX似乎参与了oxLDL的自噬激活。为了直接评估这种联系，我们使用了dld -off-POX细胞，这是一种稳定的转染物，当多西环素从培养基中去除时，可以诱导POX。我们发现LC3-I与LC3-II结合，LC3-II被整合到自噬体中。重要的是，自噬的关键基因beclin-1的表达被痘的表达诱导。因此，由oxLDL通过PPARgamma诱导的POX表达是激活自噬的信号机制。这是有趣的，因为我们已经提出痘启动生态吞噬，消耗底物，如胶原蛋白，在微环境中。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。根据上述证据，痘在营养应激期间提供ATP的来源，我们也解决了缺氧引起的代谢应激的问题。通过实时PCR或荧光素酶检测痘启动子活性，多种缺氧培养细胞的痘表达均有所增加。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水平在缺氧时降低，在POX敲低时进一步降低。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。综上所述，这些研究表明，通过AMPK磷酸化级联反应对缺氧的反应涉及诱导痘，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.