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C10ORF10/DEPP, a transcriptional target of FOXO3, regulates ROS-sensitivity in human neuroblastoma.

Salcher S, Hagenbuchner J, Geiger K, Seiter MA, Rainer J, Kofler R, Hermann M, Kiechl-Kohlendorfer U, Ausserlechner MJ, Obexer P - Mol. Cancer (2014)

Bottom Line: 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.Catalase activity was reduced in DEPP-overexpressing cells and significantly increased in DEPP-knockdown cells.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics I, Medical University Innsbruck, Anichstraße 35, 6020 Innsbruck, Austria. michael.j.ausserlechner@i-med.ac.at.

ABSTRACT

Background: 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.

Methods: 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.

Results: 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.

Conclusion: 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.

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DEPP is located in mitochondria and peroxisomes. a) SH-EP/tetEGFP and SH-EP/tetEYFP-DEPP cells were treated with 200 ng/ml doxy for 24 hours and subjected to subcellular fractionation. The protein levels of DEPP, GFP and catalase in the cytosolic and the nuclear fraction were determined by immunoblot. Alpha-Tubulin (cytosolic) and Lamin (nuclear) were used to control the purity of the fractions (left panel). The peroxisomal fraction of SH-EP/tetEYFP-DEPP cells treated with 200 ng/ml doxy for 24 hours was separated using a peroxisome isolation kit (PEROX1, Sigma). DEPP protein expression was determined by immunoblot. Catalase (peroxisomes) and CoxIV (mitochondria) were used to control the purity of the peroxisomal fraction (right panel). b) SH-EP/tetEYFP-DEPP cells were grown on eight-well chambered cover glasses, treated with 200 ng/ml doxy for 24 hours and analyzed by live confocal microscopy. Mitochondria were stained using the specific mitochondrial probe MitoTrackerRed/CMXRos (30 nM). Peroxisomal staining was performed with a CellLight® Peroxisomes-RFP fusion construct. c) SH-EP/tetDEPP cells treated with 200 ng/ml doxy for 24 hours were subjected to CoIP analysis with a Pierce Crosslink Magnetic IP/Co-IP kit to investigate whether the DEPP protein co-immunopurifies with PEX7-cross-linked beads. The expression of PEX7 and DEPP was determined by immunoblot. Alpha-Tubulin served as loading control.
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Fig7: DEPP is located in mitochondria and peroxisomes. a) SH-EP/tetEGFP and SH-EP/tetEYFP-DEPP cells were treated with 200 ng/ml doxy for 24 hours and subjected to subcellular fractionation. The protein levels of DEPP, GFP and catalase in the cytosolic and the nuclear fraction were determined by immunoblot. Alpha-Tubulin (cytosolic) and Lamin (nuclear) were used to control the purity of the fractions (left panel). The peroxisomal fraction of SH-EP/tetEYFP-DEPP cells treated with 200 ng/ml doxy for 24 hours was separated using a peroxisome isolation kit (PEROX1, Sigma). DEPP protein expression was determined by immunoblot. Catalase (peroxisomes) and CoxIV (mitochondria) were used to control the purity of the peroxisomal fraction (right panel). b) SH-EP/tetEYFP-DEPP cells were grown on eight-well chambered cover glasses, treated with 200 ng/ml doxy for 24 hours and analyzed by live confocal microscopy. Mitochondria were stained using the specific mitochondrial probe MitoTrackerRed/CMXRos (30 nM). Peroxisomal staining was performed with a CellLight® Peroxisomes-RFP fusion construct. c) SH-EP/tetDEPP cells treated with 200 ng/ml doxy for 24 hours were subjected to CoIP analysis with a Pierce Crosslink Magnetic IP/Co-IP kit to investigate whether the DEPP protein co-immunopurifies with PEX7-cross-linked beads. The expression of PEX7 and DEPP was determined by immunoblot. Alpha-Tubulin served as loading control.

Mentions: DEPP was described to reside exclusively in the nucleus of HEK293 cells[3], whereas Stepp et al. described DEPP as an unstable protein located in aggresomes in Vero cells[31]. To analyze the localization of DEPP in neuroblastoma cells we performed subcellular fractionation and immunoblot analyses of SH-EP/tetEGFP and SH-EP/tetEYFP-DEPP cells treated with 200 ng/ml doxy for 24 hours. As shown in Figure 7a (left panel) we found DEPP mainly present in the cytoplasmic and not in the nuclear fraction. Catalase, which is located in the peroxisomes and the mitochondria, was also mainly present in the cytoplasmic fraction. In a next step we treated SH-EP/tetEYFP-DEPP cells with 200 ng/ml doxy for 24 hours, isolated the peroxisomes and performed immunoblot analysis of DEPP expression (Figure 7a, right panel). By this subcellular fractionation experiment we clearly demonstrated that a significant proportion of DEPP is located in peroxisomes. To further analyze the localization of DEPP we performed live cell confocal imaging of SH-EP/tetEYFP-DEPP cells treated with 200 ng/ml doxy for 24 hours. Staining of the mitochondria with MitotrackerRed/CMXRos and labeling of peroxisomes by expression of a CellLight® Peroxisomes-RFP marker protein clearly showed that the DEPP protein co-localizes with both, mitochondria and peroxisomes in neuroblastoma cells (Figure 7b). We detected DEPP in SH-EP/EYFP-DEPP cells exclusively in the mitochondria (Figure 7b, top two lines) or in the peroxisomes (Figure 7b, bottom line) and also simultaneously in both organelles (Figure 7b, third line). Therefore the live cell confocal imaging analyses indicate that the DEPP protein is present in both organelles and may translocate between mitochondria and peroxisomes, which are cooperating and cross-talking[32] and are both critical for the control of cellular ROS[15, 33, 34]. As DEPP lacks a mitochondrial targeting sequence but contains a PTS2-signal at its N-terminus we next studied whether DEPP is targeted to peroxisomes in a PTS2-dependent manner via interaction with the PEX7 receptor[27]. As shown in Figure 7c, DEPP co-immunopurifies with the peroxisomal PEX7 receptor in SH-EP/tetDEPP cells treated with 200 ng/ml doxy for 24 hours. This is in line with the subcellular fractionation assays (Figure 7a) and the live cell imaging experiments (Figure 7b) demonstrating that DEPP is present in peroxisomes.Figure 7


C10ORF10/DEPP, a transcriptional target of FOXO3, regulates ROS-sensitivity in human neuroblastoma.

Salcher S, Hagenbuchner J, Geiger K, Seiter MA, Rainer J, Kofler R, Hermann M, Kiechl-Kohlendorfer U, Ausserlechner MJ, Obexer P - Mol. Cancer (2014)

DEPP is located in mitochondria and peroxisomes. a) SH-EP/tetEGFP and SH-EP/tetEYFP-DEPP cells were treated with 200 ng/ml doxy for 24 hours and subjected to subcellular fractionation. The protein levels of DEPP, GFP and catalase in the cytosolic and the nuclear fraction were determined by immunoblot. Alpha-Tubulin (cytosolic) and Lamin (nuclear) were used to control the purity of the fractions (left panel). The peroxisomal fraction of SH-EP/tetEYFP-DEPP cells treated with 200 ng/ml doxy for 24 hours was separated using a peroxisome isolation kit (PEROX1, Sigma). DEPP protein expression was determined by immunoblot. Catalase (peroxisomes) and CoxIV (mitochondria) were used to control the purity of the peroxisomal fraction (right panel). b) SH-EP/tetEYFP-DEPP cells were grown on eight-well chambered cover glasses, treated with 200 ng/ml doxy for 24 hours and analyzed by live confocal microscopy. Mitochondria were stained using the specific mitochondrial probe MitoTrackerRed/CMXRos (30 nM). Peroxisomal staining was performed with a CellLight® Peroxisomes-RFP fusion construct. c) SH-EP/tetDEPP cells treated with 200 ng/ml doxy for 24 hours were subjected to CoIP analysis with a Pierce Crosslink Magnetic IP/Co-IP kit to investigate whether the DEPP protein co-immunopurifies with PEX7-cross-linked beads. The expression of PEX7 and DEPP was determined by immunoblot. Alpha-Tubulin served as loading control.
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Related In: Results  -  Collection

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Fig7: DEPP is located in mitochondria and peroxisomes. a) SH-EP/tetEGFP and SH-EP/tetEYFP-DEPP cells were treated with 200 ng/ml doxy for 24 hours and subjected to subcellular fractionation. The protein levels of DEPP, GFP and catalase in the cytosolic and the nuclear fraction were determined by immunoblot. Alpha-Tubulin (cytosolic) and Lamin (nuclear) were used to control the purity of the fractions (left panel). The peroxisomal fraction of SH-EP/tetEYFP-DEPP cells treated with 200 ng/ml doxy for 24 hours was separated using a peroxisome isolation kit (PEROX1, Sigma). DEPP protein expression was determined by immunoblot. Catalase (peroxisomes) and CoxIV (mitochondria) were used to control the purity of the peroxisomal fraction (right panel). b) SH-EP/tetEYFP-DEPP cells were grown on eight-well chambered cover glasses, treated with 200 ng/ml doxy for 24 hours and analyzed by live confocal microscopy. Mitochondria were stained using the specific mitochondrial probe MitoTrackerRed/CMXRos (30 nM). Peroxisomal staining was performed with a CellLight® Peroxisomes-RFP fusion construct. c) SH-EP/tetDEPP cells treated with 200 ng/ml doxy for 24 hours were subjected to CoIP analysis with a Pierce Crosslink Magnetic IP/Co-IP kit to investigate whether the DEPP protein co-immunopurifies with PEX7-cross-linked beads. The expression of PEX7 and DEPP was determined by immunoblot. Alpha-Tubulin served as loading control.
Mentions: DEPP was described to reside exclusively in the nucleus of HEK293 cells[3], whereas Stepp et al. described DEPP as an unstable protein located in aggresomes in Vero cells[31]. To analyze the localization of DEPP in neuroblastoma cells we performed subcellular fractionation and immunoblot analyses of SH-EP/tetEGFP and SH-EP/tetEYFP-DEPP cells treated with 200 ng/ml doxy for 24 hours. As shown in Figure 7a (left panel) we found DEPP mainly present in the cytoplasmic and not in the nuclear fraction. Catalase, which is located in the peroxisomes and the mitochondria, was also mainly present in the cytoplasmic fraction. In a next step we treated SH-EP/tetEYFP-DEPP cells with 200 ng/ml doxy for 24 hours, isolated the peroxisomes and performed immunoblot analysis of DEPP expression (Figure 7a, right panel). By this subcellular fractionation experiment we clearly demonstrated that a significant proportion of DEPP is located in peroxisomes. To further analyze the localization of DEPP we performed live cell confocal imaging of SH-EP/tetEYFP-DEPP cells treated with 200 ng/ml doxy for 24 hours. Staining of the mitochondria with MitotrackerRed/CMXRos and labeling of peroxisomes by expression of a CellLight® Peroxisomes-RFP marker protein clearly showed that the DEPP protein co-localizes with both, mitochondria and peroxisomes in neuroblastoma cells (Figure 7b). We detected DEPP in SH-EP/EYFP-DEPP cells exclusively in the mitochondria (Figure 7b, top two lines) or in the peroxisomes (Figure 7b, bottom line) and also simultaneously in both organelles (Figure 7b, third line). Therefore the live cell confocal imaging analyses indicate that the DEPP protein is present in both organelles and may translocate between mitochondria and peroxisomes, which are cooperating and cross-talking[32] and are both critical for the control of cellular ROS[15, 33, 34]. As DEPP lacks a mitochondrial targeting sequence but contains a PTS2-signal at its N-terminus we next studied whether DEPP is targeted to peroxisomes in a PTS2-dependent manner via interaction with the PEX7 receptor[27]. As shown in Figure 7c, DEPP co-immunopurifies with the peroxisomal PEX7 receptor in SH-EP/tetDEPP cells treated with 200 ng/ml doxy for 24 hours. This is in line with the subcellular fractionation assays (Figure 7a) and the live cell imaging experiments (Figure 7b) demonstrating that DEPP is present in peroxisomes.Figure 7

Bottom Line: 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.Catalase activity was reduced in DEPP-overexpressing cells and significantly increased in DEPP-knockdown cells.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics I, Medical University Innsbruck, Anichstraße 35, 6020 Innsbruck, Austria. michael.j.ausserlechner@i-med.ac.at.

ABSTRACT

Background: 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.

Methods: 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.

Results: 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.

Conclusion: 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.

Show MeSH
Related in: MedlinePlus