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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 mediates the PGC-1α activation of the Sirt3 gene promoter in HepG2 cells.A, Schematic representation of the promoter sequence of mSirt3 (Luc-2036). Exon 1A (E1A) and exon 1B (E1B) of mSirt3 are alternatively spliced according to previous report [34]. Thus, mSirt3 has two different transcriptional start sites. However, E1A does not encode amino acids. B, 5′-Deletion series of the mSirt3 promoter fused to luciferase reporter gene were cotransfected into HepG2 cells together with pcDNA3.1 (control) or PGC-1α in the presence or absence of ERRα expression plasmids. Two days later, cells were harvested and the relative luciferase activity (RLA) was corrected for Renilla luciferase activity and normalized to the control activity. C, The nucleotide sequence from −424 to −366 of the mouse (Mou) Sirt3 gene promoter was aligned with corresponding sequences from different species, including rat, humans (Hum), and chimpanzee (Chp). Evolutionarily conserved elements are indicated in large, bold, italic type. D, left panel, Wild-type putative ERRα binding element and its mutant sequence; right panel, Reporter gene plasmid containing 2.2 kb of the wild-type (WT) Sirt3 promoter (Luc-2036) or ERRE mutant (mut Luc-2036) were transfected into HepG2 cells together with PGC-1α in the presence or absence of ERRα expression plasmids or empty plasmid (pcDNA3.1). The graph depicts RLA corrected for Renilla luciferase activity and normalized to the control activity of mSirt3. All values represent at least three independent transfections, each conducted in triplicate. *, P<0.05.
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pone-0011707-g002: ERRα mediates the PGC-1α activation of the Sirt3 gene promoter in HepG2 cells.A, Schematic representation of the promoter sequence of mSirt3 (Luc-2036). Exon 1A (E1A) and exon 1B (E1B) of mSirt3 are alternatively spliced according to previous report [34]. Thus, mSirt3 has two different transcriptional start sites. However, E1A does not encode amino acids. B, 5′-Deletion series of the mSirt3 promoter fused to luciferase reporter gene were cotransfected into HepG2 cells together with pcDNA3.1 (control) or PGC-1α in the presence or absence of ERRα expression plasmids. Two days later, cells were harvested and the relative luciferase activity (RLA) was corrected for Renilla luciferase activity and normalized to the control activity. C, The nucleotide sequence from −424 to −366 of the mouse (Mou) Sirt3 gene promoter was aligned with corresponding sequences from different species, including rat, humans (Hum), and chimpanzee (Chp). Evolutionarily conserved elements are indicated in large, bold, italic type. D, left panel, Wild-type putative ERRα binding element and its mutant sequence; right panel, Reporter gene plasmid containing 2.2 kb of the wild-type (WT) Sirt3 promoter (Luc-2036) or ERRE mutant (mut Luc-2036) were transfected into HepG2 cells together with PGC-1α in the presence or absence of ERRα expression plasmids or empty plasmid (pcDNA3.1). The graph depicts RLA corrected for Renilla luciferase activity and normalized to the control activity of mSirt3. All values represent at least three independent transfections, each conducted in triplicate. *, P<0.05.

Mentions: To demonstrate that PGC-1α-dependent stimulation of Sirt3 gene expression occurs at the transcriptional level, we transfected a promoter reporter into HepG2 cells. The first construct contained the approximately 2.2-kb fragment (−2036, +146) of the mSirt3 promoter fused to a luciferase reporter gene (Luc-2036) (Fig. 2A). Overexpression of PGC-1α caused a 4-fold activation of Luc-2036 (Fig. 2B). Next, a series of truncated segments of the promoter fused to the luciferase gene (Luc-491, Luc-242, and Luc-161) were transfected into HepG2 cells to map the cis-acting element conferring the PGC-1α-dependent activation of luciferase. PGC-1α-mediated reporter activation was not significantly compromised upon deletion of the −2036 bp to −491 bp region of the mSirt3 promoter. However, further deletion of −491 bp to −242 bp abolished the PGC-1α effect on the mSIRT3 promoter (Fig. 2B). Further truncation of the promoter from −242 bp to −161 bp did not accentuate the process (Fig. 2B).


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 mediates the PGC-1α activation of the Sirt3 gene promoter in HepG2 cells.A, Schematic representation of the promoter sequence of mSirt3 (Luc-2036). Exon 1A (E1A) and exon 1B (E1B) of mSirt3 are alternatively spliced according to previous report [34]. Thus, mSirt3 has two different transcriptional start sites. However, E1A does not encode amino acids. B, 5′-Deletion series of the mSirt3 promoter fused to luciferase reporter gene were cotransfected into HepG2 cells together with pcDNA3.1 (control) or PGC-1α in the presence or absence of ERRα expression plasmids. Two days later, cells were harvested and the relative luciferase activity (RLA) was corrected for Renilla luciferase activity and normalized to the control activity. C, The nucleotide sequence from −424 to −366 of the mouse (Mou) Sirt3 gene promoter was aligned with corresponding sequences from different species, including rat, humans (Hum), and chimpanzee (Chp). Evolutionarily conserved elements are indicated in large, bold, italic type. D, left panel, Wild-type putative ERRα binding element and its mutant sequence; right panel, Reporter gene plasmid containing 2.2 kb of the wild-type (WT) Sirt3 promoter (Luc-2036) or ERRE mutant (mut Luc-2036) were transfected into HepG2 cells together with PGC-1α in the presence or absence of ERRα expression plasmids or empty plasmid (pcDNA3.1). The graph depicts RLA corrected for Renilla luciferase activity and normalized to the control activity of mSirt3. All values represent at least three independent transfections, each conducted in triplicate. *, P<0.05.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2908542&req=5

pone-0011707-g002: ERRα mediates the PGC-1α activation of the Sirt3 gene promoter in HepG2 cells.A, Schematic representation of the promoter sequence of mSirt3 (Luc-2036). Exon 1A (E1A) and exon 1B (E1B) of mSirt3 are alternatively spliced according to previous report [34]. Thus, mSirt3 has two different transcriptional start sites. However, E1A does not encode amino acids. B, 5′-Deletion series of the mSirt3 promoter fused to luciferase reporter gene were cotransfected into HepG2 cells together with pcDNA3.1 (control) or PGC-1α in the presence or absence of ERRα expression plasmids. Two days later, cells were harvested and the relative luciferase activity (RLA) was corrected for Renilla luciferase activity and normalized to the control activity. C, The nucleotide sequence from −424 to −366 of the mouse (Mou) Sirt3 gene promoter was aligned with corresponding sequences from different species, including rat, humans (Hum), and chimpanzee (Chp). Evolutionarily conserved elements are indicated in large, bold, italic type. D, left panel, Wild-type putative ERRα binding element and its mutant sequence; right panel, Reporter gene plasmid containing 2.2 kb of the wild-type (WT) Sirt3 promoter (Luc-2036) or ERRE mutant (mut Luc-2036) were transfected into HepG2 cells together with PGC-1α in the presence or absence of ERRα expression plasmids or empty plasmid (pcDNA3.1). The graph depicts RLA corrected for Renilla luciferase activity and normalized to the control activity of mSirt3. All values represent at least three independent transfections, each conducted in triplicate. *, P<0.05.
Mentions: To demonstrate that PGC-1α-dependent stimulation of Sirt3 gene expression occurs at the transcriptional level, we transfected a promoter reporter into HepG2 cells. The first construct contained the approximately 2.2-kb fragment (−2036, +146) of the mSirt3 promoter fused to a luciferase reporter gene (Luc-2036) (Fig. 2A). Overexpression of PGC-1α caused a 4-fold activation of Luc-2036 (Fig. 2B). Next, a series of truncated segments of the promoter fused to the luciferase gene (Luc-491, Luc-242, and Luc-161) were transfected into HepG2 cells to map the cis-acting element conferring the PGC-1α-dependent activation of luciferase. PGC-1α-mediated reporter activation was not significantly compromised upon deletion of the −2036 bp to −491 bp region of the mSirt3 promoter. However, further deletion of −491 bp to −242 bp abolished the PGC-1α effect on the mSIRT3 promoter (Fig. 2B). Further truncation of the promoter from −242 bp to −161 bp did not accentuate the process (Fig. 2B).

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