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NRF2 Regulates PINK1 Expression under Oxidative Stress Conditions.

Murata H, Takamatsu H, Liu S, Kataoka K, Huh NH, Sakaguchi M - PLoS ONE (2015)

Bottom Line: In this study, we found that nuclear factor (erythroid-derived 2)-like 2 (NRF2), an antioxidant transcription factor, regulates PINK1 expression under oxidative stress conditions.Transcriptionally up-regulated PINK1 diminished oxidative stress-associated cell death.The results indicate that PINK1 expression is positively regulated by NRF2 and that the NRF2-PINK1 signaling axis is deeply involved in cell survival.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Kita-ku, Okayama 700-8558, Japan.

ABSTRACT
Mutations of the PTEN-induced putative kinase 1 (PINK1) gene are a cause of autosomal recessive forms of Parkinson's disease. Recent studies have revealed that PINK1 is an essential factor for controlling mitochondrial quality, and that it protects cells from oxidative stresses. Although there has been considerable progress in the elucidation of various aspects of PINK1 protein regulation such as activation, stability and degradation, the transcriptional regulation of PINK1 mRNA under stress conditions remains unclear. In this study, we found that nuclear factor (erythroid-derived 2)-like 2 (NRF2), an antioxidant transcription factor, regulates PINK1 expression under oxidative stress conditions. Damaged mitochondria arising from stress conditions induced NRF2-dependent transcription of the PINK1 gene through production of reactive oxygen species (ROS). Either an ROS scavenger or forced expression of KEAP1, a potent inhibitory partner to NRF2, restricted PINK1 expression induced by activated NRF2. Transcriptionally up-regulated PINK1 diminished oxidative stress-associated cell death. The results indicate that PINK1 expression is positively regulated by NRF2 and that the NRF2-PINK1 signaling axis is deeply involved in cell survival.

No MeSH data available.


Related in: MedlinePlus

Induction of PINK1 expression by oxidative stress inducers.(A-D) Luciferase assay using the PINK1 promoter. SH-SY5Y cells were transfected with the PINK1 promoter-luciferase reporter construct (PINK1 pro-luc) and GFP in parallel for 24 h followed by treatment with 0–20 μM 6-OHDA for 48 h (A), 0–20 μM CdCl2 for 48 h (B), 0–10 nM rotenone for 48 h (C) or 0–100 μM paraquat for 48 h (D). The luciferase activity was normalized to the fluorescence of GFP in each sample. (E-H) Real-time PCR analysis of PINK1 mRNA expression. SH-SY5Y cells were exposed to 20 μM 6-OHDA (E), 20 μM CdCl2 (F), 10 nM rotenone (G) or 100 μM paraquat (H) at different times. PINK1 mRNA levels were measured by real-time PCR. Relative expression was determined using GAPDH as a housekeeping gene. *, significantly different from the non-treated cells (p < 0.01); **, p < 0.05.
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pone.0142438.g001: Induction of PINK1 expression by oxidative stress inducers.(A-D) Luciferase assay using the PINK1 promoter. SH-SY5Y cells were transfected with the PINK1 promoter-luciferase reporter construct (PINK1 pro-luc) and GFP in parallel for 24 h followed by treatment with 0–20 μM 6-OHDA for 48 h (A), 0–20 μM CdCl2 for 48 h (B), 0–10 nM rotenone for 48 h (C) or 0–100 μM paraquat for 48 h (D). The luciferase activity was normalized to the fluorescence of GFP in each sample. (E-H) Real-time PCR analysis of PINK1 mRNA expression. SH-SY5Y cells were exposed to 20 μM 6-OHDA (E), 20 μM CdCl2 (F), 10 nM rotenone (G) or 100 μM paraquat (H) at different times. PINK1 mRNA levels were measured by real-time PCR. Relative expression was determined using GAPDH as a housekeeping gene. *, significantly different from the non-treated cells (p < 0.01); **, p < 0.05.

Mentions: To examine the effect of oxidative stress on expression of PINK1, a reporter assay was carried out using the PINK1 promoter (3 kbp)-luciferase-reporter construct (PINK1 pro-luc). We used human neuroblastoma SH-SY5Y cells for this assay because the cells are often used as in vitro models to analyze neuronal function and Parkinson’s disease. SH-SY5Y cells were transfected with PINK1 pro-luc for 24 h and then treated with a neurotoxic reagent, 6-hydroxydopamine (6-OHDA); a metal-stress inducer, CdCl2; an inhibitor of mitochondrial respiratory complex I, rotenone; and an ROS inducer, paraquat for 48 h. As shown in Fig 1A–1D, these reagents increased the luciferase activity of PINK1 pro-luc in a dose-dependent manner. We next examined effects of the reagents on expression of endogenous PINK1 mRNA at regular time intervals. These reagents increased PINK1 mRNA levels at 12–48 h (Fig 1E–1H). These results indicate that transcriptional expression of PINK1 is positively regulated under oxidative stress conditions.


NRF2 Regulates PINK1 Expression under Oxidative Stress Conditions.

Murata H, Takamatsu H, Liu S, Kataoka K, Huh NH, Sakaguchi M - PLoS ONE (2015)

Induction of PINK1 expression by oxidative stress inducers.(A-D) Luciferase assay using the PINK1 promoter. SH-SY5Y cells were transfected with the PINK1 promoter-luciferase reporter construct (PINK1 pro-luc) and GFP in parallel for 24 h followed by treatment with 0–20 μM 6-OHDA for 48 h (A), 0–20 μM CdCl2 for 48 h (B), 0–10 nM rotenone for 48 h (C) or 0–100 μM paraquat for 48 h (D). The luciferase activity was normalized to the fluorescence of GFP in each sample. (E-H) Real-time PCR analysis of PINK1 mRNA expression. SH-SY5Y cells were exposed to 20 μM 6-OHDA (E), 20 μM CdCl2 (F), 10 nM rotenone (G) or 100 μM paraquat (H) at different times. PINK1 mRNA levels were measured by real-time PCR. Relative expression was determined using GAPDH as a housekeeping gene. *, significantly different from the non-treated cells (p < 0.01); **, p < 0.05.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4640816&req=5

pone.0142438.g001: Induction of PINK1 expression by oxidative stress inducers.(A-D) Luciferase assay using the PINK1 promoter. SH-SY5Y cells were transfected with the PINK1 promoter-luciferase reporter construct (PINK1 pro-luc) and GFP in parallel for 24 h followed by treatment with 0–20 μM 6-OHDA for 48 h (A), 0–20 μM CdCl2 for 48 h (B), 0–10 nM rotenone for 48 h (C) or 0–100 μM paraquat for 48 h (D). The luciferase activity was normalized to the fluorescence of GFP in each sample. (E-H) Real-time PCR analysis of PINK1 mRNA expression. SH-SY5Y cells were exposed to 20 μM 6-OHDA (E), 20 μM CdCl2 (F), 10 nM rotenone (G) or 100 μM paraquat (H) at different times. PINK1 mRNA levels were measured by real-time PCR. Relative expression was determined using GAPDH as a housekeeping gene. *, significantly different from the non-treated cells (p < 0.01); **, p < 0.05.
Mentions: To examine the effect of oxidative stress on expression of PINK1, a reporter assay was carried out using the PINK1 promoter (3 kbp)-luciferase-reporter construct (PINK1 pro-luc). We used human neuroblastoma SH-SY5Y cells for this assay because the cells are often used as in vitro models to analyze neuronal function and Parkinson’s disease. SH-SY5Y cells were transfected with PINK1 pro-luc for 24 h and then treated with a neurotoxic reagent, 6-hydroxydopamine (6-OHDA); a metal-stress inducer, CdCl2; an inhibitor of mitochondrial respiratory complex I, rotenone; and an ROS inducer, paraquat for 48 h. As shown in Fig 1A–1D, these reagents increased the luciferase activity of PINK1 pro-luc in a dose-dependent manner. We next examined effects of the reagents on expression of endogenous PINK1 mRNA at regular time intervals. These reagents increased PINK1 mRNA levels at 12–48 h (Fig 1E–1H). These results indicate that transcriptional expression of PINK1 is positively regulated under oxidative stress conditions.

Bottom Line: In this study, we found that nuclear factor (erythroid-derived 2)-like 2 (NRF2), an antioxidant transcription factor, regulates PINK1 expression under oxidative stress conditions.Transcriptionally up-regulated PINK1 diminished oxidative stress-associated cell death.The results indicate that PINK1 expression is positively regulated by NRF2 and that the NRF2-PINK1 signaling axis is deeply involved in cell survival.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Kita-ku, Okayama 700-8558, Japan.

ABSTRACT
Mutations of the PTEN-induced putative kinase 1 (PINK1) gene are a cause of autosomal recessive forms of Parkinson's disease. Recent studies have revealed that PINK1 is an essential factor for controlling mitochondrial quality, and that it protects cells from oxidative stresses. Although there has been considerable progress in the elucidation of various aspects of PINK1 protein regulation such as activation, stability and degradation, the transcriptional regulation of PINK1 mRNA under stress conditions remains unclear. In this study, we found that nuclear factor (erythroid-derived 2)-like 2 (NRF2), an antioxidant transcription factor, regulates PINK1 expression under oxidative stress conditions. Damaged mitochondria arising from stress conditions induced NRF2-dependent transcription of the PINK1 gene through production of reactive oxygen species (ROS). Either an ROS scavenger or forced expression of KEAP1, a potent inhibitory partner to NRF2, restricted PINK1 expression induced by activated NRF2. Transcriptionally up-regulated PINK1 diminished oxidative stress-associated cell death. The results indicate that PINK1 expression is positively regulated by NRF2 and that the NRF2-PINK1 signaling axis is deeply involved in cell survival.

No MeSH data available.


Related in: MedlinePlus