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Mitochondrial fusion is increased by the nuclear coactivator PGC-1beta.

Liesa M, Borda-d'Agua B, Medina-Gómez G, Lelliott CJ, Paz JC, Rojo M, Palacín M, Vidal-Puig A, Zorzano A - PLoS ONE (2008)

Bottom Line: Here, we demonstrate that reduced mitochondrial size observed in knock-out mice for the transcriptional regulator PGC-1beta is associated with a selective reduction in Mitofusin 2 (Mfn2) expression, a mitochondrial fusion protein.This PGC-1beta-induced elongation specifically requires Mfn2 as this process is absent in Mfn2-ablated cells.Finally, we show that PGC-1beta increases Mfn2 promoter activity and transcription by coactivating the nuclear receptor Estrogen Related Receptor alpha (ERRalpha).

View Article: PubMed Central - PubMed

Affiliation: Institute for Research in Biomedicine (IRB Barcelona), Barcelona, Spain.

ABSTRACT

Background: There is no evidence to date on whether transcriptional regulators are able to shift the balance between mitochondrial fusion and fission events through selective control of gene expression.

Methodology/principal findings: Here, we demonstrate that reduced mitochondrial size observed in knock-out mice for the transcriptional regulator PGC-1beta is associated with a selective reduction in Mitofusin 2 (Mfn2) expression, a mitochondrial fusion protein. This decrease in Mfn2 is specific since expression of the remaining components of mitochondrial fusion and fission machinery were not affected. Furthermore, PGC-1beta increases mitochondrial fusion and elongates mitochondrial tubules. This PGC-1beta-induced elongation specifically requires Mfn2 as this process is absent in Mfn2-ablated cells. Finally, we show that PGC-1beta increases Mfn2 promoter activity and transcription by coactivating the nuclear receptor Estrogen Related Receptor alpha (ERRalpha).

Conclusions/significance: Taken together, our data reveal a novel mechanism by which mammalian cells control mitochondrial fusion. In addition, we describe a novel role of PGC-1beta in mitochondrial physiology, namely the control of mitochondrial fusion mainly through Mfn2.

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Related in: MedlinePlus

PGC-1β increases Mfn2 transcription through nuclear receptor ERRα.(A) Total RNA was obtained from C2C12 myotubes transduced with LacZ (white bars) or PGC-1β (black bars) adenovirus at a MOI 100 from 3 independent experiments in duplicate. Mfn2 and β-Actin mRNA levels were measured by real-time PCR. Results are mean±SEM and are expressed relative to β-Actin values, *, statistical difference at p<0.001. (B) Transcriptional activity of −1982/+45 human Mfn2 promoter was determined by luciferase activity (corrected by renilla activity) in 10T1/2 and HeLa cells. Results are mean±SEM from 3 independent experiments performed in triplicate. Cells were transfected with 200 ng of an irrelevant vector (Basal, white bars) or PGC-1β expression vector (PGC-1β, black bars) and expressed relative to the basal group; * statistical difference at p<0.05. (C) Luciferase activity (corrected by renilla) from 10T1/2 cells transfected with a −459/−352 Mfn2 promoter fragment containing a mutation that disrupted box 2 (−459/−352 Box 2 Mut, black bars) or with the wild-type fragment (−459/−352, white bars), together with an irrelevant vector (Basal), ERRα, 200 ng of PGC-1β or ERRα+PGC-1β expression plasmids. Mean±SEM values from 3 independent transfection experiments performed in triplicate are shown. *, statistical difference compared with basal groups, p<0.05. #, statistical difference compared with the wild-type promoter fragment activity, p<0.05.
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pone-0003613-g001: PGC-1β increases Mfn2 transcription through nuclear receptor ERRα.(A) Total RNA was obtained from C2C12 myotubes transduced with LacZ (white bars) or PGC-1β (black bars) adenovirus at a MOI 100 from 3 independent experiments in duplicate. Mfn2 and β-Actin mRNA levels were measured by real-time PCR. Results are mean±SEM and are expressed relative to β-Actin values, *, statistical difference at p<0.001. (B) Transcriptional activity of −1982/+45 human Mfn2 promoter was determined by luciferase activity (corrected by renilla activity) in 10T1/2 and HeLa cells. Results are mean±SEM from 3 independent experiments performed in triplicate. Cells were transfected with 200 ng of an irrelevant vector (Basal, white bars) or PGC-1β expression vector (PGC-1β, black bars) and expressed relative to the basal group; * statistical difference at p<0.05. (C) Luciferase activity (corrected by renilla) from 10T1/2 cells transfected with a −459/−352 Mfn2 promoter fragment containing a mutation that disrupted box 2 (−459/−352 Box 2 Mut, black bars) or with the wild-type fragment (−459/−352, white bars), together with an irrelevant vector (Basal), ERRα, 200 ng of PGC-1β or ERRα+PGC-1β expression plasmids. Mean±SEM values from 3 independent transfection experiments performed in triplicate are shown. *, statistical difference compared with basal groups, p<0.05. #, statistical difference compared with the wild-type promoter fragment activity, p<0.05.

Mentions: Our initial aim was to determine whether PGC-1β regulates the expression of Mfn2 in C2C12 muscle cells. Differentiated C2C12 cells show low levels of PGC-1β mRNA as assessed by Northern blot [18] and by real-time PCR (data not shown). To this end, C2C12 myotubes were transduced either with a mouse PGC-1β adenovirus or with a control LacZ adenovirus. Mfn2 mRNA levels doubled in PGC-1β-expressing muscle cells compared to control transduced myotubes (Fig. 1A). To demonstrate that PGC-1β directly increases Mfn2 transcription, we transfected 10T1/2 mouse fibroblasts or HeLa cells with a construct containing a 2-kb fragment (−1982/+45) of the Mfn2 promoter fused to a luciferase reporter gene, together with an irrelevant vector (Basal) or mouse PGC-1β expression vector (PGC-1β). PGC-1β markedly enhanced Mfn2 promoter activity (10.3±0.9- and 4.2±0.6-fold values over basal Mfn2 promoter activity in 10T1/2 and HeLa respectively, Fig. 1B). In a previous study, using electrophoretic mobility shift and chromatin immunoprecipitation assays, we showed that ERRα binds to Mfn2 promoter between nucleotides −459/−396 [27]. This DNA region contains three putative boxes with the capacity to bind nuclear receptors, where box 2 is critical for Mfn2 promoter response to PGC-1α coactivation of ERRα [27]. On the basis of these observations, we determined whether PGC-1β coactivated ERRα through box 2 in a similar way as PGC-1α. We transfected 10T1/2 cells with a construct containing a −459/−352 Mfn2 promoter fragment fused to a luciferase reporter gene or with a mutated version of the same fragment that disrupted box 2 [27]. We observed a marked coactivation of ERRα by PGC-1β in the −459/−352 Mfn2 promoter fragment (Fig. 1C). This effect was completely blunted when box 2 was disrupted (11.9±1.09 vs. 2.8±0.6-fold over basal promoter activity, p = 0.001, Fig. 1C). Furthermore, cancellation of box 2 markedly reduced the activation driven by PGC-1β (7.0±0.5 vs. 1.9±0.5, p = 0.001) or by ERRα (2.2±0.1 vs. 1.6±0.2, p = 0.04), although residual activation was still present. Similar results were obtained when the 2- kb Mfn2 promoter was cotransfected with ERRα or PGC-1β (Figure S1).


Mitochondrial fusion is increased by the nuclear coactivator PGC-1beta.

Liesa M, Borda-d'Agua B, Medina-Gómez G, Lelliott CJ, Paz JC, Rojo M, Palacín M, Vidal-Puig A, Zorzano A - PLoS ONE (2008)

PGC-1β increases Mfn2 transcription through nuclear receptor ERRα.(A) Total RNA was obtained from C2C12 myotubes transduced with LacZ (white bars) or PGC-1β (black bars) adenovirus at a MOI 100 from 3 independent experiments in duplicate. Mfn2 and β-Actin mRNA levels were measured by real-time PCR. Results are mean±SEM and are expressed relative to β-Actin values, *, statistical difference at p<0.001. (B) Transcriptional activity of −1982/+45 human Mfn2 promoter was determined by luciferase activity (corrected by renilla activity) in 10T1/2 and HeLa cells. Results are mean±SEM from 3 independent experiments performed in triplicate. Cells were transfected with 200 ng of an irrelevant vector (Basal, white bars) or PGC-1β expression vector (PGC-1β, black bars) and expressed relative to the basal group; * statistical difference at p<0.05. (C) Luciferase activity (corrected by renilla) from 10T1/2 cells transfected with a −459/−352 Mfn2 promoter fragment containing a mutation that disrupted box 2 (−459/−352 Box 2 Mut, black bars) or with the wild-type fragment (−459/−352, white bars), together with an irrelevant vector (Basal), ERRα, 200 ng of PGC-1β or ERRα+PGC-1β expression plasmids. Mean±SEM values from 3 independent transfection experiments performed in triplicate are shown. *, statistical difference compared with basal groups, p<0.05. #, statistical difference compared with the wild-type promoter fragment activity, p<0.05.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2570954&req=5

pone-0003613-g001: PGC-1β increases Mfn2 transcription through nuclear receptor ERRα.(A) Total RNA was obtained from C2C12 myotubes transduced with LacZ (white bars) or PGC-1β (black bars) adenovirus at a MOI 100 from 3 independent experiments in duplicate. Mfn2 and β-Actin mRNA levels were measured by real-time PCR. Results are mean±SEM and are expressed relative to β-Actin values, *, statistical difference at p<0.001. (B) Transcriptional activity of −1982/+45 human Mfn2 promoter was determined by luciferase activity (corrected by renilla activity) in 10T1/2 and HeLa cells. Results are mean±SEM from 3 independent experiments performed in triplicate. Cells were transfected with 200 ng of an irrelevant vector (Basal, white bars) or PGC-1β expression vector (PGC-1β, black bars) and expressed relative to the basal group; * statistical difference at p<0.05. (C) Luciferase activity (corrected by renilla) from 10T1/2 cells transfected with a −459/−352 Mfn2 promoter fragment containing a mutation that disrupted box 2 (−459/−352 Box 2 Mut, black bars) or with the wild-type fragment (−459/−352, white bars), together with an irrelevant vector (Basal), ERRα, 200 ng of PGC-1β or ERRα+PGC-1β expression plasmids. Mean±SEM values from 3 independent transfection experiments performed in triplicate are shown. *, statistical difference compared with basal groups, p<0.05. #, statistical difference compared with the wild-type promoter fragment activity, p<0.05.
Mentions: Our initial aim was to determine whether PGC-1β regulates the expression of Mfn2 in C2C12 muscle cells. Differentiated C2C12 cells show low levels of PGC-1β mRNA as assessed by Northern blot [18] and by real-time PCR (data not shown). To this end, C2C12 myotubes were transduced either with a mouse PGC-1β adenovirus or with a control LacZ adenovirus. Mfn2 mRNA levels doubled in PGC-1β-expressing muscle cells compared to control transduced myotubes (Fig. 1A). To demonstrate that PGC-1β directly increases Mfn2 transcription, we transfected 10T1/2 mouse fibroblasts or HeLa cells with a construct containing a 2-kb fragment (−1982/+45) of the Mfn2 promoter fused to a luciferase reporter gene, together with an irrelevant vector (Basal) or mouse PGC-1β expression vector (PGC-1β). PGC-1β markedly enhanced Mfn2 promoter activity (10.3±0.9- and 4.2±0.6-fold values over basal Mfn2 promoter activity in 10T1/2 and HeLa respectively, Fig. 1B). In a previous study, using electrophoretic mobility shift and chromatin immunoprecipitation assays, we showed that ERRα binds to Mfn2 promoter between nucleotides −459/−396 [27]. This DNA region contains three putative boxes with the capacity to bind nuclear receptors, where box 2 is critical for Mfn2 promoter response to PGC-1α coactivation of ERRα [27]. On the basis of these observations, we determined whether PGC-1β coactivated ERRα through box 2 in a similar way as PGC-1α. We transfected 10T1/2 cells with a construct containing a −459/−352 Mfn2 promoter fragment fused to a luciferase reporter gene or with a mutated version of the same fragment that disrupted box 2 [27]. We observed a marked coactivation of ERRα by PGC-1β in the −459/−352 Mfn2 promoter fragment (Fig. 1C). This effect was completely blunted when box 2 was disrupted (11.9±1.09 vs. 2.8±0.6-fold over basal promoter activity, p = 0.001, Fig. 1C). Furthermore, cancellation of box 2 markedly reduced the activation driven by PGC-1β (7.0±0.5 vs. 1.9±0.5, p = 0.001) or by ERRα (2.2±0.1 vs. 1.6±0.2, p = 0.04), although residual activation was still present. Similar results were obtained when the 2- kb Mfn2 promoter was cotransfected with ERRα or PGC-1β (Figure S1).

Bottom Line: Here, we demonstrate that reduced mitochondrial size observed in knock-out mice for the transcriptional regulator PGC-1beta is associated with a selective reduction in Mitofusin 2 (Mfn2) expression, a mitochondrial fusion protein.This PGC-1beta-induced elongation specifically requires Mfn2 as this process is absent in Mfn2-ablated cells.Finally, we show that PGC-1beta increases Mfn2 promoter activity and transcription by coactivating the nuclear receptor Estrogen Related Receptor alpha (ERRalpha).

View Article: PubMed Central - PubMed

Affiliation: Institute for Research in Biomedicine (IRB Barcelona), Barcelona, Spain.

ABSTRACT

Background: There is no evidence to date on whether transcriptional regulators are able to shift the balance between mitochondrial fusion and fission events through selective control of gene expression.

Methodology/principal findings: Here, we demonstrate that reduced mitochondrial size observed in knock-out mice for the transcriptional regulator PGC-1beta is associated with a selective reduction in Mitofusin 2 (Mfn2) expression, a mitochondrial fusion protein. This decrease in Mfn2 is specific since expression of the remaining components of mitochondrial fusion and fission machinery were not affected. Furthermore, PGC-1beta increases mitochondrial fusion and elongates mitochondrial tubules. This PGC-1beta-induced elongation specifically requires Mfn2 as this process is absent in Mfn2-ablated cells. Finally, we show that PGC-1beta increases Mfn2 promoter activity and transcription by coactivating the nuclear receptor Estrogen Related Receptor alpha (ERRalpha).

Conclusions/significance: Taken together, our data reveal a novel mechanism by which mammalian cells control mitochondrial fusion. In addition, we describe a novel role of PGC-1beta in mitochondrial physiology, namely the control of mitochondrial fusion mainly through Mfn2.

Show MeSH
Related in: MedlinePlus