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Transcriptome analysis of human OXR1 depleted cells reveals its role in regulating the p53 signaling pathway.

Yang M, Lin X, Rowe A, Rognes T, Eide L, Bjørås M - Sci Rep (2015)

Bottom Line: The oxidation resistance gene 1 (OXR1) is crucial for protecting against oxidative stress; however, its molecular function is unknown.In total, in non-treated and hydrogen peroxide exposed HeLa cells, OXR1 depletion resulted in down-regulation of 554 genes and up-regulation of 253 genes.In summary, OXR1 may act as a sensor of cellular oxidative stress to regulate the transcriptional networks required to detoxify reactive oxygen species and modulate cell cycle and apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Oslo University Hospital and University of Oslo, Norway.

ABSTRACT
The oxidation resistance gene 1 (OXR1) is crucial for protecting against oxidative stress; however, its molecular function is unknown. We employed RNA sequencing to examine the role of human OXR1 for genome wide transcription regulation. In total, in non-treated and hydrogen peroxide exposed HeLa cells, OXR1 depletion resulted in down-regulation of 554 genes and up-regulation of 253 genes. These differentially expressed genes include transcription factors (i.e. HIF1A, SP6, E2F8 and TCF3), antioxidant genes (PRDX4, PTGS1 and CYGB) and numerous genes of the p53 signaling pathway involved in cell-cycle arrest (i.e. cyclin D, CDK6 and RPRM) and apoptosis (i.e. CytC and CASP9). We demonstrated that OXR1 depleted cells undergo cell cycle arrest in G2/M phase during oxidative stress and increase protein expression of the apoptosis initiator protease CASP9. In summary, OXR1 may act as a sensor of cellular oxidative stress to regulate the transcriptional networks required to detoxify reactive oxygen species and modulate cell cycle and apoptosis.

No MeSH data available.


Related in: MedlinePlus

(a) Caspase 9 protein level increased and was partly cleaved into active forms in hOXR1 depleted HeLa. Left panel: Western blot analysis. Actin was used as internal loading control. Right panel: quantification of protein bands. siCon: control siRNA; siOXR1: hOXR1 siRNA; NT: non-treatment; R0h: cells were treated with 0.5 mM H2O2 for 1 h and harvested immediately without recovery. The quantification is the average of two independent experiments. (b) Model of hOXR1 mediated regulation of antioxidant defense, early stress response, cell cycle and apoptosis. HOXR1 up regulates transcription of four antioxidant genes, resulting in suppression of ROS and modulation of early stress response genes. In hOXR1 depleted cells, increased ROS leads to increased oxidative damage (i.e. DNA damage) that triggers cell cycle arrest in G2/M and apoptosis via regulation of RPRM, CASP9 and several other genes in the p53 pathway. We postulate that hOXR1 may regulate the early stress response, cell cycle and apoptosis directly or indirectly by interaction with transcription factors such as SP1 (Sp1 transcription factor) and AP2 (activating enhancer binding protein 2). Genes labeled in red or green are up- or down-regulated, respectively, in hOXR1 depleted cells.
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f7: (a) Caspase 9 protein level increased and was partly cleaved into active forms in hOXR1 depleted HeLa. Left panel: Western blot analysis. Actin was used as internal loading control. Right panel: quantification of protein bands. siCon: control siRNA; siOXR1: hOXR1 siRNA; NT: non-treatment; R0h: cells were treated with 0.5 mM H2O2 for 1 h and harvested immediately without recovery. The quantification is the average of two independent experiments. (b) Model of hOXR1 mediated regulation of antioxidant defense, early stress response, cell cycle and apoptosis. HOXR1 up regulates transcription of four antioxidant genes, resulting in suppression of ROS and modulation of early stress response genes. In hOXR1 depleted cells, increased ROS leads to increased oxidative damage (i.e. DNA damage) that triggers cell cycle arrest in G2/M and apoptosis via regulation of RPRM, CASP9 and several other genes in the p53 pathway. We postulate that hOXR1 may regulate the early stress response, cell cycle and apoptosis directly or indirectly by interaction with transcription factors such as SP1 (Sp1 transcription factor) and AP2 (activating enhancer binding protein 2). Genes labeled in red or green are up- or down-regulated, respectively, in hOXR1 depleted cells.

Mentions: Previously, we showed that hOXR1 depletion induces apoptosis under oxidative stress10. The p53 signaling pathway plays an important role in induction of apoptosis, and three pro-apoptotic genes belonging to the p53 pathway were strongly induced by hOXR1 depletion: CYCS, caspase 9 (CASP9) and insulin-like growth factor binding protein 3 (IGFBP3). IGFBP3 stabilizes IGF (insulin-like growth factor) and alters its interaction with cell surface receptors. During oxidative stress, CYCS is released into the cytoplasm from mitochondria and activates Apaf1 (apoptotic peptidase activating factor 1), which in turn cleaves procaspase 9 into its active form of dimer p35/p12 or p35/p10. Another active dimer, p37/p10, is generated through cleavage of the procaspase 9 by caspase 318. To examine the effect of hOXR1 depletion on the protein level, we assessed CASP9 by western analysis and found that hOXR1 depletion resulted in a significant induction as well as processing of the cellular CASP9 protein. The CASP9 antibody was raised against amino acids 100–270, which is recognizing both p37 and p35. Three bands of CASP9 were detected, of which the largest band at 47 kDa (CASP_I) is the inactive form of procaspase 9 whereas the middle band at 37 kDa (CASP_II, corresponding to p37) and the lower band at 35 kDa (CASP_III, corresponding to p35) are the active forms after cleavage (Fig. 7a). The control cells only expressed the inactive form CASP9_I, while the hOXR1 depleted cells expressed both the inactive form I and the active form II/III both in non-treated cells and after H2O2 treatment. In addition, the protein level of CASP_I increased more than 2-fold in hOXR1 depleted cells in comparison to control cells. Thus, it appears that hOXR1 regulates apoptosis via the p53 pathway by controlling CytC and CASP9 expression and CASP9 activation.


Transcriptome analysis of human OXR1 depleted cells reveals its role in regulating the p53 signaling pathway.

Yang M, Lin X, Rowe A, Rognes T, Eide L, Bjørås M - Sci Rep (2015)

(a) Caspase 9 protein level increased and was partly cleaved into active forms in hOXR1 depleted HeLa. Left panel: Western blot analysis. Actin was used as internal loading control. Right panel: quantification of protein bands. siCon: control siRNA; siOXR1: hOXR1 siRNA; NT: non-treatment; R0h: cells were treated with 0.5 mM H2O2 for 1 h and harvested immediately without recovery. The quantification is the average of two independent experiments. (b) Model of hOXR1 mediated regulation of antioxidant defense, early stress response, cell cycle and apoptosis. HOXR1 up regulates transcription of four antioxidant genes, resulting in suppression of ROS and modulation of early stress response genes. In hOXR1 depleted cells, increased ROS leads to increased oxidative damage (i.e. DNA damage) that triggers cell cycle arrest in G2/M and apoptosis via regulation of RPRM, CASP9 and several other genes in the p53 pathway. We postulate that hOXR1 may regulate the early stress response, cell cycle and apoptosis directly or indirectly by interaction with transcription factors such as SP1 (Sp1 transcription factor) and AP2 (activating enhancer binding protein 2). Genes labeled in red or green are up- or down-regulated, respectively, in hOXR1 depleted cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4663793&req=5

f7: (a) Caspase 9 protein level increased and was partly cleaved into active forms in hOXR1 depleted HeLa. Left panel: Western blot analysis. Actin was used as internal loading control. Right panel: quantification of protein bands. siCon: control siRNA; siOXR1: hOXR1 siRNA; NT: non-treatment; R0h: cells were treated with 0.5 mM H2O2 for 1 h and harvested immediately without recovery. The quantification is the average of two independent experiments. (b) Model of hOXR1 mediated regulation of antioxidant defense, early stress response, cell cycle and apoptosis. HOXR1 up regulates transcription of four antioxidant genes, resulting in suppression of ROS and modulation of early stress response genes. In hOXR1 depleted cells, increased ROS leads to increased oxidative damage (i.e. DNA damage) that triggers cell cycle arrest in G2/M and apoptosis via regulation of RPRM, CASP9 and several other genes in the p53 pathway. We postulate that hOXR1 may regulate the early stress response, cell cycle and apoptosis directly or indirectly by interaction with transcription factors such as SP1 (Sp1 transcription factor) and AP2 (activating enhancer binding protein 2). Genes labeled in red or green are up- or down-regulated, respectively, in hOXR1 depleted cells.
Mentions: Previously, we showed that hOXR1 depletion induces apoptosis under oxidative stress10. The p53 signaling pathway plays an important role in induction of apoptosis, and three pro-apoptotic genes belonging to the p53 pathway were strongly induced by hOXR1 depletion: CYCS, caspase 9 (CASP9) and insulin-like growth factor binding protein 3 (IGFBP3). IGFBP3 stabilizes IGF (insulin-like growth factor) and alters its interaction with cell surface receptors. During oxidative stress, CYCS is released into the cytoplasm from mitochondria and activates Apaf1 (apoptotic peptidase activating factor 1), which in turn cleaves procaspase 9 into its active form of dimer p35/p12 or p35/p10. Another active dimer, p37/p10, is generated through cleavage of the procaspase 9 by caspase 318. To examine the effect of hOXR1 depletion on the protein level, we assessed CASP9 by western analysis and found that hOXR1 depletion resulted in a significant induction as well as processing of the cellular CASP9 protein. The CASP9 antibody was raised against amino acids 100–270, which is recognizing both p37 and p35. Three bands of CASP9 were detected, of which the largest band at 47 kDa (CASP_I) is the inactive form of procaspase 9 whereas the middle band at 37 kDa (CASP_II, corresponding to p37) and the lower band at 35 kDa (CASP_III, corresponding to p35) are the active forms after cleavage (Fig. 7a). The control cells only expressed the inactive form CASP9_I, while the hOXR1 depleted cells expressed both the inactive form I and the active form II/III both in non-treated cells and after H2O2 treatment. In addition, the protein level of CASP_I increased more than 2-fold in hOXR1 depleted cells in comparison to control cells. Thus, it appears that hOXR1 regulates apoptosis via the p53 pathway by controlling CytC and CASP9 expression and CASP9 activation.

Bottom Line: The oxidation resistance gene 1 (OXR1) is crucial for protecting against oxidative stress; however, its molecular function is unknown.In total, in non-treated and hydrogen peroxide exposed HeLa cells, OXR1 depletion resulted in down-regulation of 554 genes and up-regulation of 253 genes.In summary, OXR1 may act as a sensor of cellular oxidative stress to regulate the transcriptional networks required to detoxify reactive oxygen species and modulate cell cycle and apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Oslo University Hospital and University of Oslo, Norway.

ABSTRACT
The oxidation resistance gene 1 (OXR1) is crucial for protecting against oxidative stress; however, its molecular function is unknown. We employed RNA sequencing to examine the role of human OXR1 for genome wide transcription regulation. In total, in non-treated and hydrogen peroxide exposed HeLa cells, OXR1 depletion resulted in down-regulation of 554 genes and up-regulation of 253 genes. These differentially expressed genes include transcription factors (i.e. HIF1A, SP6, E2F8 and TCF3), antioxidant genes (PRDX4, PTGS1 and CYGB) and numerous genes of the p53 signaling pathway involved in cell-cycle arrest (i.e. cyclin D, CDK6 and RPRM) and apoptosis (i.e. CytC and CASP9). We demonstrated that OXR1 depleted cells undergo cell cycle arrest in G2/M phase during oxidative stress and increase protein expression of the apoptosis initiator protease CASP9. In summary, OXR1 may act as a sensor of cellular oxidative stress to regulate the transcriptional networks required to detoxify reactive oxygen species and modulate cell cycle and apoptosis.

No MeSH data available.


Related in: MedlinePlus