Salcher Stefan, Hagenbuchner Judith, Geiger Kathrin, Seiter Maximilian A, Rainer Johannes, Kofler Reinhard, Hermann Martin, Kiechl-Kohlendorfer Ursula, Ausserlechner Michael J, Obexer Petra
Department of Pediatrics I, Medical University Innsbruck, Anichstraße 35, 6020 Innsbruck, Austria.
Mol Cancer. 2014 Sep 28;13:224. doi: 10.1186/1476-4598-13-224.
FOXO transcription factors control cellular levels of reactive oxygen species (ROS) which critically contribute to cell survival and cell death in neuroblastoma. In the present study we investigated the regulation of C10orf10/DEPP by the transcription factor FOXO3. As a physiological function of C10orf10/DEPP has not been described so far we analyzed its effects on cellular ROS detoxification and death sensitization in human neuroblastoma cells.
The effect of DEPP on cellular ROS was measured by catalase activity assay and live cell fluorescence microscopy using the ROS-sensitive dye reduced MitoTracker Red CM-H2XROS. The cellular localization of DEPP was determined by confocal microscopy of EYFP-tagged DEPP, fluorescent peroxisomal- and mitochondrial probes and co-immunoprecipitation of the PEX7 receptor.
We report for the first time that DEPP regulates ROS detoxification and localizes to peroxisomes and mitochondria in neuroblastoma cells. FOXO3-mediated apoptosis involves a biphasic ROS accumulation. Knockdown of DEPP prevented the primary and secondary ROS wave during FOXO3 activation and attenuated FOXO3- and H2O2-induced apoptosis. Conditional overexpression of DEPP elevates cellular ROS levels and sensitizes to H2O2 and etoposide-induced cell death. In neuronal cells, cellular ROS are mainly detoxified in peroxisomes by the enzyme CAT/catalase. As DEPP contains a peroxisomal-targeting-signal-type-2 (PTS2) sequence at its N-terminus that allows binding to the PEX7 receptor and import into peroxisomes, we analyzed the effect of DEPP on cellular detoxification by measuring enzyme activity of catalase. Catalase activity was reduced in DEPP-overexpressing cells and significantly increased in DEPP-knockdown cells. DEPP directly interacts with the PEX7 receptor and localizes to the peroxisomal compartment. In parallel, the expression of the transcription factor peroxisome proliferator-activated receptor gamma (PPARG), a critical regulator of catalase enzyme activity, was strongly upregulated in DEPP-knockdown cells.
The combined data indicate that in neuroblastoma DEPP localizes to peroxisomes and mitochondria and impairs cellular ROS detoxification, which sensitizes tumor cells to ROS-induced cell death.
FOXO转录因子控制细胞内活性氧（ROS）水平，这对神经母细胞瘤细胞的存活和死亡起着关键作用。在本研究中，我们调查了转录因子FOXO3对C10orf10/DEPP的调控作用。由于目前尚未描述C10orf10/DEPP的生理功能，我们分析了其对人神经母细胞瘤细胞内ROS解毒和死亡致敏的影响。
使用对ROS敏感的染料还原型MitoTracker Red CM-H2XROS，通过过氧化氢酶活性测定和活细胞荧光显微镜检测DEPP对细胞内ROS的影响。通过共聚焦显微镜观察EYFP标记的DEPP、荧光过氧化物酶体和线粒体探针以及PEX7受体的共免疫沉淀，确定DEPP的细胞定位。
我们首次报道，DEPP在神经母细胞瘤细胞中调节ROS解毒并定位于过氧化物酶体和线粒体。FOXO3介导的细胞凋亡涉及双相ROS积累。敲低DEPP可阻止FOXO3激活过程中的初级和次级ROS波，并减弱FOXO3和H2O2诱导的细胞凋亡。条件性过表达DEPP可提高细胞内ROS水平，并使细胞对H2O2和依托泊苷诱导的细胞死亡敏感。在神经元细胞中，细胞内ROS主要通过CAT/过氧化氢酶在过氧化物酶体中解毒。由于DEPP在其N端含有一个2型过氧化物酶体靶向信号（PTS2）序列，该序列允许与PEX7受体结合并导入过氧化物酶体，我们通过测量过氧化氢酶的酶活性分析了DEPP对细胞解毒的影响。在过表达DEPP的细胞中过氧化氢酶活性降低，在敲低DEPP的细胞中显著增加。DEPP直接与PEX7受体相互作用并定位于过氧化物酶体区室。同时，转录因子过氧化物酶体增殖物激活受体γ（PPARG）的表达，过氧化氢酶活性的关键调节因子，在敲低DEPP的细胞中强烈上调。
综合数据表明，在神经母细胞瘤中，DEPP定位于过氧化物酶体和线粒体并损害细胞内ROS解毒，从而使肿瘤细胞对ROS诱导的细胞死亡敏感。
