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Sirtuin 3, a new target of PGC-1alpha, plays an important role in the suppression of ROS and mitochondrial biogenesis.

Kong X, Wang R, Xue Y, Liu X, Zhang H, Chen Y, Fang F, Chang Y - PLoS ONE (2010)

Bottom Line: Knockdown of ERRalpha reduced the induction of Sirt3 by PGC-1alpha in C(2)C(12) myotubes.Finally, SIRT3 stimulated mitochondrial biogenesis, and SIRT3 knockdown decreased the stimulatory effect of PGC-1alpha on mitochondrial biogenesis in C(2)C(12) myotubes.Our results indicate that Sirt3 functions as a downstream target gene of PGC-1alpha and mediates the PGC-1alpha effects on cellular ROS production and mitochondrial biogenesis.

View Article: PubMed Central - PubMed

Affiliation: The National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China.

ABSTRACT

Background: Sirtuin 3 (SIRT3) is one of the seven mammalian sirtuins, which are homologs of the yeast Sir2 gene. SIRT3 is the only sirtuin with a reported association with the human life span. Peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC-1alpha) plays important roles in adaptive thermogenesis, gluconeogenesis, mitochondrial biogenesis and respiration. PGC-1alpha induces several key reactive oxygen species (ROS)-detoxifying enzymes, but the molecular mechanism underlying this is not well understood.

Results: Here we show that PGC-1alpha strongly stimulated mouse Sirt3 gene expression in muscle cells and hepatocytes. Knockdown of PGC-1alpha led to decreased Sirt3 gene expression. PGC-1alpha activated the mouse SIRT3 promoter, which was mediated by an estrogen-related receptor (ERR) binding element (ERRE) (-407/-399) mapped to the promoter region. Chromatin immunoprecipitation and electrophoretic mobility shift assays confirmed that ERRalpha bound to the identified ERRE and PGC-1alpha co-localized with ERRalpha in the mSirt3 promoter. Knockdown of ERRalpha reduced the induction of Sirt3 by PGC-1alpha in C(2)C(12) myotubes. Furthermore, Sirt3 was essential for PGC-1alpha-dependent induction of ROS-detoxifying enzymes and several components of the respiratory chain, including glutathione peroxidase-1, superoxide dismutase 2, ATP synthase 5c, and cytochrome c. Overexpression of SIRT3 or PGC-1alpha in C(2)C(12) myotubes decreased basal ROS level. In contrast, knockdown of mSIRT3 increased basal ROS level and blocked the inhibitory effect of PGC-1alpha on cellular ROS production. Finally, SIRT3 stimulated mitochondrial biogenesis, and SIRT3 knockdown decreased the stimulatory effect of PGC-1alpha on mitochondrial biogenesis in C(2)C(12) myotubes.

Conclusion: Our results indicate that Sirt3 functions as a downstream target gene of PGC-1alpha and mediates the PGC-1alpha effects on cellular ROS production and mitochondrial biogenesis. Thus, SIRT3 integrates cellular energy metabolism and ROS generation. The elucidation of the molecular mechanisms of SIRT3 regulation and its physiological functions may provide a novel target for treating ROS-related disease.

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ERRα interacts with the mouse Sirt3 promoter in vitro and in vivo.A, Electrophoretic mobility shift assay was executed using a biotin probe. Biotin-labeled double-stranded oligonucleotides were incubated with or without nuclear extract containing ERRα protein. Approximately 200-fold excess wild-type and ERRE-mutated unlabeled double-stranded oligonucleotides were used for competitive inhibition. B, Primary hepatocytes were isolated, cultured in 100 mm dishes, and infected with adenoviruses expressing ERRα-FLAG and/or PGC-1α, or GFP as a control. For ChIP, protein-DNA complexes were immunoprecipitated with anti-FLAG or control IgG antibody. The mSirt3 promoter region harboring the ERRE site was amplified by PCR. C, Primary hepatocytes were isolated from mouse liver, and chromatin was immunoprecipitated with anti-PGC-1α antibody. Normal IgG was used as control. The mSirt3 promoter region harboring the ERRE (proximal region) could be amplified by PCR. However, the distal region of the mSirt3 promoter, having no ERRE and used as a negative control, could not be amplified.
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pone-0011707-g003: ERRα interacts with the mouse Sirt3 promoter in vitro and in vivo.A, Electrophoretic mobility shift assay was executed using a biotin probe. Biotin-labeled double-stranded oligonucleotides were incubated with or without nuclear extract containing ERRα protein. Approximately 200-fold excess wild-type and ERRE-mutated unlabeled double-stranded oligonucleotides were used for competitive inhibition. B, Primary hepatocytes were isolated, cultured in 100 mm dishes, and infected with adenoviruses expressing ERRα-FLAG and/or PGC-1α, or GFP as a control. For ChIP, protein-DNA complexes were immunoprecipitated with anti-FLAG or control IgG antibody. The mSirt3 promoter region harboring the ERRE site was amplified by PCR. C, Primary hepatocytes were isolated from mouse liver, and chromatin was immunoprecipitated with anti-PGC-1α antibody. Normal IgG was used as control. The mSirt3 promoter region harboring the ERRE (proximal region) could be amplified by PCR. However, the distal region of the mSirt3 promoter, having no ERRE and used as a negative control, could not be amplified.

Mentions: To determine whether ERRα binds to the potential binding element identified in the mSirt3 gene promoter in vitro, a DNA fragment encompassing the sequence −386/−419 was biotin-labeled, and EMSA was carried out using nuclear protein extracted from 293A cells transfected with ERRα expression vector. A retardation band (DNA-protein complex) was specifically detected, and it almost completely disappeared upon the use of 200-fold excess of unlabeled wild-type oligonucleotides. However, 200-fold excess of unlabeled mutant oligonucleotides did not affect the formation of the DNA-protein complex (Fig. 3A).


Sirtuin 3, a new target of PGC-1alpha, plays an important role in the suppression of ROS and mitochondrial biogenesis.

Kong X, Wang R, Xue Y, Liu X, Zhang H, Chen Y, Fang F, Chang Y - PLoS ONE (2010)

ERRα interacts with the mouse Sirt3 promoter in vitro and in vivo.A, Electrophoretic mobility shift assay was executed using a biotin probe. Biotin-labeled double-stranded oligonucleotides were incubated with or without nuclear extract containing ERRα protein. Approximately 200-fold excess wild-type and ERRE-mutated unlabeled double-stranded oligonucleotides were used for competitive inhibition. B, Primary hepatocytes were isolated, cultured in 100 mm dishes, and infected with adenoviruses expressing ERRα-FLAG and/or PGC-1α, or GFP as a control. For ChIP, protein-DNA complexes were immunoprecipitated with anti-FLAG or control IgG antibody. The mSirt3 promoter region harboring the ERRE site was amplified by PCR. C, Primary hepatocytes were isolated from mouse liver, and chromatin was immunoprecipitated with anti-PGC-1α antibody. Normal IgG was used as control. The mSirt3 promoter region harboring the ERRE (proximal region) could be amplified by PCR. However, the distal region of the mSirt3 promoter, having no ERRE and used as a negative control, could not be amplified.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2908542&req=5

pone-0011707-g003: ERRα interacts with the mouse Sirt3 promoter in vitro and in vivo.A, Electrophoretic mobility shift assay was executed using a biotin probe. Biotin-labeled double-stranded oligonucleotides were incubated with or without nuclear extract containing ERRα protein. Approximately 200-fold excess wild-type and ERRE-mutated unlabeled double-stranded oligonucleotides were used for competitive inhibition. B, Primary hepatocytes were isolated, cultured in 100 mm dishes, and infected with adenoviruses expressing ERRα-FLAG and/or PGC-1α, or GFP as a control. For ChIP, protein-DNA complexes were immunoprecipitated with anti-FLAG or control IgG antibody. The mSirt3 promoter region harboring the ERRE site was amplified by PCR. C, Primary hepatocytes were isolated from mouse liver, and chromatin was immunoprecipitated with anti-PGC-1α antibody. Normal IgG was used as control. The mSirt3 promoter region harboring the ERRE (proximal region) could be amplified by PCR. However, the distal region of the mSirt3 promoter, having no ERRE and used as a negative control, could not be amplified.
Mentions: To determine whether ERRα binds to the potential binding element identified in the mSirt3 gene promoter in vitro, a DNA fragment encompassing the sequence −386/−419 was biotin-labeled, and EMSA was carried out using nuclear protein extracted from 293A cells transfected with ERRα expression vector. A retardation band (DNA-protein complex) was specifically detected, and it almost completely disappeared upon the use of 200-fold excess of unlabeled wild-type oligonucleotides. However, 200-fold excess of unlabeled mutant oligonucleotides did not affect the formation of the DNA-protein complex (Fig. 3A).

Bottom Line: Knockdown of ERRalpha reduced the induction of Sirt3 by PGC-1alpha in C(2)C(12) myotubes.Finally, SIRT3 stimulated mitochondrial biogenesis, and SIRT3 knockdown decreased the stimulatory effect of PGC-1alpha on mitochondrial biogenesis in C(2)C(12) myotubes.Our results indicate that Sirt3 functions as a downstream target gene of PGC-1alpha and mediates the PGC-1alpha effects on cellular ROS production and mitochondrial biogenesis.

View Article: PubMed Central - PubMed

Affiliation: The National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China.

ABSTRACT

Background: Sirtuin 3 (SIRT3) is one of the seven mammalian sirtuins, which are homologs of the yeast Sir2 gene. SIRT3 is the only sirtuin with a reported association with the human life span. Peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC-1alpha) plays important roles in adaptive thermogenesis, gluconeogenesis, mitochondrial biogenesis and respiration. PGC-1alpha induces several key reactive oxygen species (ROS)-detoxifying enzymes, but the molecular mechanism underlying this is not well understood.

Results: Here we show that PGC-1alpha strongly stimulated mouse Sirt3 gene expression in muscle cells and hepatocytes. Knockdown of PGC-1alpha led to decreased Sirt3 gene expression. PGC-1alpha activated the mouse SIRT3 promoter, which was mediated by an estrogen-related receptor (ERR) binding element (ERRE) (-407/-399) mapped to the promoter region. Chromatin immunoprecipitation and electrophoretic mobility shift assays confirmed that ERRalpha bound to the identified ERRE and PGC-1alpha co-localized with ERRalpha in the mSirt3 promoter. Knockdown of ERRalpha reduced the induction of Sirt3 by PGC-1alpha in C(2)C(12) myotubes. Furthermore, Sirt3 was essential for PGC-1alpha-dependent induction of ROS-detoxifying enzymes and several components of the respiratory chain, including glutathione peroxidase-1, superoxide dismutase 2, ATP synthase 5c, and cytochrome c. Overexpression of SIRT3 or PGC-1alpha in C(2)C(12) myotubes decreased basal ROS level. In contrast, knockdown of mSIRT3 increased basal ROS level and blocked the inhibitory effect of PGC-1alpha on cellular ROS production. Finally, SIRT3 stimulated mitochondrial biogenesis, and SIRT3 knockdown decreased the stimulatory effect of PGC-1alpha on mitochondrial biogenesis in C(2)C(12) myotubes.

Conclusion: Our results indicate that Sirt3 functions as a downstream target gene of PGC-1alpha and mediates the PGC-1alpha effects on cellular ROS production and mitochondrial biogenesis. Thus, SIRT3 integrates cellular energy metabolism and ROS generation. The elucidation of the molecular mechanisms of SIRT3 regulation and its physiological functions may provide a novel target for treating ROS-related disease.

Show MeSH
Related in: MedlinePlus