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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 a direct transcriptional target of FOXO3. a) Quantitative RT-PCR of DEPP expression in SH-EP/FOXO3 cells, treated with 75 nM 4OHT for 2 hours to activate FOXO3(A3)ERtm or a combination of 4OHT and cycloheximide (CHX; 10 μg/ml), which was used to block protein synthesis. Shown are mean values ± s.e.m. of three independent experiments, each performed in triplicates. b) The DEPP gene promoter (-1116 bp relative to the transcription start) contains three putative binding sites for FOXO3, which are highlighted in the schematic representation. A DEPP wild type promoter reporter plasmid and DEPP promoter reporter plasmids containing mutations of the three FOXO3-binding sites (B1, B2 and B3; indicated as bold in sequence) were transfected into SH-EP/FOXO3 cells. The cells were treated with 100 nM 4OHT for 4 hours to activate FOXO3(A3)ERtm and a luciferase-assay was performed. Direct binding of FOXO3 to the DEPP promoter leads to increased luciferase activity. The increase of the luciferase signal was calculated as fold over untreated controls. Shown are mean values ± s.e.m. of three independent experiments, each performed in duplicates; statistical analysis was done with the Student’s unpaired t-test, *P < 0.05; **P < 0.025 compared to corresponding controls. c) ChIP analysis on the interaction between FOXO3 and the FOXO3 binding sites B1 + 2 and B3 of the DEPP promoter in SH-EP/FOXO3 cells treated with 100 nM 4OHT for 3 hours. Quantification was performed by quantitative RT-PCR with specific primers for B1 + 2 and B3. Shown are mean values of two independent experiments, each performed in duplicates.
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Fig2: DEPP is a direct transcriptional target of FOXO3. a) Quantitative RT-PCR of DEPP expression in SH-EP/FOXO3 cells, treated with 75 nM 4OHT for 2 hours to activate FOXO3(A3)ERtm or a combination of 4OHT and cycloheximide (CHX; 10 μg/ml), which was used to block protein synthesis. Shown are mean values ± s.e.m. of three independent experiments, each performed in triplicates. b) The DEPP gene promoter (-1116 bp relative to the transcription start) contains three putative binding sites for FOXO3, which are highlighted in the schematic representation. A DEPP wild type promoter reporter plasmid and DEPP promoter reporter plasmids containing mutations of the three FOXO3-binding sites (B1, B2 and B3; indicated as bold in sequence) were transfected into SH-EP/FOXO3 cells. The cells were treated with 100 nM 4OHT for 4 hours to activate FOXO3(A3)ERtm and a luciferase-assay was performed. Direct binding of FOXO3 to the DEPP promoter leads to increased luciferase activity. The increase of the luciferase signal was calculated as fold over untreated controls. Shown are mean values ± s.e.m. of three independent experiments, each performed in duplicates; statistical analysis was done with the Student’s unpaired t-test, *P < 0.05; **P < 0.025 compared to corresponding controls. c) ChIP analysis on the interaction between FOXO3 and the FOXO3 binding sites B1 + 2 and B3 of the DEPP promoter in SH-EP/FOXO3 cells treated with 100 nM 4OHT for 3 hours. Quantification was performed by quantitative RT-PCR with specific primers for B1 + 2 and B3. Shown are mean values of two independent experiments, each performed in duplicates.

Mentions: To study whether DEPP is a direct target of FOXO3 in neuroblastoma cells, quantitative RT-PCR analysis of SH-EP/FOXO3 cells treated with 75 nM 4OHT and with 10 μg/ml of the protein biosynthesis inhibitor cycloheximide (CHX) for 2 hours was performed. Treatment with CHX did not prevent the induction of DEPP after FOXO3 activation, which implies that induction of DEPP by FOXO3 does not depend on de novo synthesis of additional proteins, but is due to direct transcriptional regulation (Figure 2a). To further test this hypothesis, a DEPP promoter reporter luciferase assay was performed in SH-EP/FOXO3 cells using a 1116 bp genomic fragment of the promoter cloned upstream of firefly luciferase. The DEPP promoter contains three putative binding sites for FOXO3 (Figure 2b), which were mutated for this experiment. The first binding site for FOXO3 is located at -537 (B1), the second at -179 (B2), and the third at -151 (B3) relative to the start of the DEPP mRNA[26].


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 a direct transcriptional target of FOXO3. a) Quantitative RT-PCR of DEPP expression in SH-EP/FOXO3 cells, treated with 75 nM 4OHT for 2 hours to activate FOXO3(A3)ERtm or a combination of 4OHT and cycloheximide (CHX; 10 μg/ml), which was used to block protein synthesis. Shown are mean values ± s.e.m. of three independent experiments, each performed in triplicates. b) The DEPP gene promoter (-1116 bp relative to the transcription start) contains three putative binding sites for FOXO3, which are highlighted in the schematic representation. A DEPP wild type promoter reporter plasmid and DEPP promoter reporter plasmids containing mutations of the three FOXO3-binding sites (B1, B2 and B3; indicated as bold in sequence) were transfected into SH-EP/FOXO3 cells. The cells were treated with 100 nM 4OHT for 4 hours to activate FOXO3(A3)ERtm and a luciferase-assay was performed. Direct binding of FOXO3 to the DEPP promoter leads to increased luciferase activity. The increase of the luciferase signal was calculated as fold over untreated controls. Shown are mean values ± s.e.m. of three independent experiments, each performed in duplicates; statistical analysis was done with the Student’s unpaired t-test, *P < 0.05; **P < 0.025 compared to corresponding controls. c) ChIP analysis on the interaction between FOXO3 and the FOXO3 binding sites B1 + 2 and B3 of the DEPP promoter in SH-EP/FOXO3 cells treated with 100 nM 4OHT for 3 hours. Quantification was performed by quantitative RT-PCR with specific primers for B1 + 2 and B3. Shown are mean values of two independent experiments, each performed in duplicates.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4197242&req=5

Fig2: DEPP is a direct transcriptional target of FOXO3. a) Quantitative RT-PCR of DEPP expression in SH-EP/FOXO3 cells, treated with 75 nM 4OHT for 2 hours to activate FOXO3(A3)ERtm or a combination of 4OHT and cycloheximide (CHX; 10 μg/ml), which was used to block protein synthesis. Shown are mean values ± s.e.m. of three independent experiments, each performed in triplicates. b) The DEPP gene promoter (-1116 bp relative to the transcription start) contains three putative binding sites for FOXO3, which are highlighted in the schematic representation. A DEPP wild type promoter reporter plasmid and DEPP promoter reporter plasmids containing mutations of the three FOXO3-binding sites (B1, B2 and B3; indicated as bold in sequence) were transfected into SH-EP/FOXO3 cells. The cells were treated with 100 nM 4OHT for 4 hours to activate FOXO3(A3)ERtm and a luciferase-assay was performed. Direct binding of FOXO3 to the DEPP promoter leads to increased luciferase activity. The increase of the luciferase signal was calculated as fold over untreated controls. Shown are mean values ± s.e.m. of three independent experiments, each performed in duplicates; statistical analysis was done with the Student’s unpaired t-test, *P < 0.05; **P < 0.025 compared to corresponding controls. c) ChIP analysis on the interaction between FOXO3 and the FOXO3 binding sites B1 + 2 and B3 of the DEPP promoter in SH-EP/FOXO3 cells treated with 100 nM 4OHT for 3 hours. Quantification was performed by quantitative RT-PCR with specific primers for B1 + 2 and B3. Shown are mean values of two independent experiments, each performed in duplicates.
Mentions: To study whether DEPP is a direct target of FOXO3 in neuroblastoma cells, quantitative RT-PCR analysis of SH-EP/FOXO3 cells treated with 75 nM 4OHT and with 10 μg/ml of the protein biosynthesis inhibitor cycloheximide (CHX) for 2 hours was performed. Treatment with CHX did not prevent the induction of DEPP after FOXO3 activation, which implies that induction of DEPP by FOXO3 does not depend on de novo synthesis of additional proteins, but is due to direct transcriptional regulation (Figure 2a). To further test this hypothesis, a DEPP promoter reporter luciferase assay was performed in SH-EP/FOXO3 cells using a 1116 bp genomic fragment of the promoter cloned upstream of firefly luciferase. The DEPP promoter contains three putative binding sites for FOXO3 (Figure 2b), which were mutated for this experiment. The first binding site for FOXO3 is located at -537 (B1), the second at -179 (B2), and the third at -151 (B3) relative to the start of the DEPP mRNA[26].

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