PGC-1α buffers ROS-mediated removal of mitochondria during myogenesis
Identifieur interne : 000005 ( Pmc/Curation ); précédent : 000004; suivant : 000006PGC-1α buffers ROS-mediated removal of mitochondria during myogenesis
Auteurs : S. Baldelli [Italie] ; K. Aquilano [Italie] ; M R Ciriolo [Italie]Source :
- Cell Death & Disease [ 2041-4889 ] ; 2014.
Abstract
Mitochondrial biogenesis and mitophagy are recognized as critical processes underlying mitochondrial homeostasis. However, the molecular pathway(s) coordinating the balance between these cellular programs is still poorly investigated. Here, we show an induction of the nuclear and mitochondrial peroxisome proliferator-activated receptor gamma, coactivator 1 alpha (PGC-1
Url:
DOI: 10.1038/cddis.2014.458
PubMed: 25375380
PubMed Central: 4260723
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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">PGC-1<italic>α</italic> buffers ROS-mediated removal of mitochondria during myogenesis</title><author><name sortKey="Baldelli, S" sort="Baldelli, S" uniqKey="Baldelli S" first="S" last="Baldelli">S. Baldelli</name><affiliation wicri:level="1"><nlm:aff id="aff1"><institution>Scientific Institute for Research, Hospitalization and Health Care, Università Telematica San Raffaele Roma</institution>, Via di Val Cannuta 247, Rome,<country>Italy</country></nlm:aff><country xml:lang="fr">Italie</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Aquilano, K" sort="Aquilano, K" uniqKey="Aquilano K" first="K" last="Aquilano">K. Aquilano</name><affiliation wicri:level="1"><nlm:aff id="aff2"><institution>Department of Biology, University of Rome “Tor Vergata”</institution>, Via della Ricerca Scientifica 1, Rome,<country>Italy</country></nlm:aff><country xml:lang="fr">Italie</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Ciriolo, M R" sort="Ciriolo, M R" uniqKey="Ciriolo M" first="M R" last="Ciriolo">M R Ciriolo</name><affiliation wicri:level="1"><nlm:aff id="aff2"><institution>Department of Biology, University of Rome “Tor Vergata”</institution>, Via della Ricerca Scientifica 1, Rome,<country>Italy</country></nlm:aff><country xml:lang="fr">Italie</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation><affiliation wicri:level="1"><nlm:aff id="aff3"><institution>IRCCS San Raffaele</institution>, Via di Val Cannuta 247, Rome,<country>Italy</country></nlm:aff><country xml:lang="fr">Italie</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">25375380</idno><idno type="pmc">4260723</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4260723</idno><idno type="RBID">PMC:4260723</idno><idno type="doi">10.1038/cddis.2014.458</idno><date when="2014">2014</date><idno type="wicri:Area/Pmc/Corpus">000005</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000005</idno><idno type="wicri:Area/Pmc/Curation">000005</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Curation">000005</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">PGC-1<italic>α</italic> buffers ROS-mediated removal of mitochondria during myogenesis</title><author><name sortKey="Baldelli, S" sort="Baldelli, S" uniqKey="Baldelli S" first="S" last="Baldelli">S. Baldelli</name><affiliation wicri:level="1"><nlm:aff id="aff1"><institution>Scientific Institute for Research, Hospitalization and Health Care, Università Telematica San Raffaele Roma</institution>, Via di Val Cannuta 247, Rome,<country>Italy</country></nlm:aff><country xml:lang="fr">Italie</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Aquilano, K" sort="Aquilano, K" uniqKey="Aquilano K" first="K" last="Aquilano">K. Aquilano</name><affiliation wicri:level="1"><nlm:aff id="aff2"><institution>Department of Biology, University of Rome “Tor Vergata”</institution>, Via della Ricerca Scientifica 1, Rome,<country>Italy</country></nlm:aff><country xml:lang="fr">Italie</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Ciriolo, M R" sort="Ciriolo, M R" uniqKey="Ciriolo M" first="M R" last="Ciriolo">M R Ciriolo</name><affiliation wicri:level="1"><nlm:aff id="aff2"><institution>Department of Biology, University of Rome “Tor Vergata”</institution>, Via della Ricerca Scientifica 1, Rome,<country>Italy</country></nlm:aff><country xml:lang="fr">Italie</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation><affiliation wicri:level="1"><nlm:aff id="aff3"><institution>IRCCS San Raffaele</institution>, Via di Val Cannuta 247, Rome,<country>Italy</country></nlm:aff><country xml:lang="fr">Italie</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author></analytic><series><title level="j">Cell Death & Disease</title><idno type="eISSN">2041-4889</idno><imprint><date when="2014">2014</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>Mitochondrial biogenesis and mitophagy are recognized as critical processes underlying mitochondrial homeostasis. However, the molecular pathway(s) coordinating the balance between these cellular programs is still poorly investigated. Here, we show an induction of the nuclear and mitochondrial peroxisome proliferator-activated receptor gamma, coactivator 1 alpha (PGC-1<italic>α</italic>) during myogenesis, which in turn co-activates the transcription of nuclear and mtDNA-encoded mitochondrial genes. We demonstrate that PGC-1<italic>α</italic> also buffers oxidative stress occurring during differentiation by promoting the expression of antioxidant enzymes. Indeed, by downregulating PGC-1<italic>α,</italic> we observed an impairment of antioxidants expression, which was accompanied by a significant reactive oxygen species (ROS) burst and increase of oxidative damage to proteins. In parallel, we detected a decrease of mitochondrial mass and function as well as increased mitophagy through the ROS/FOXO1 pathway. Upon PGC-1<italic>α</italic> downregulation, we found ROS-dependent nuclear translocation of FOXO1 and transcription of its downstream targets including mitophagic genes such as LC3 and PINK1. Such events were significantly reverted after treatment with the antioxidant Trolox, suggesting that PGC-1<italic>α</italic> assures mitochondrial integrity by indirectly buffering ROS. Finally, the lack of PGC-1<italic>α</italic> gave rise to a decrease in MYOG and a strong induction of atrophy-related ubiquitin ligases FBXO32 (FBXO32), indicative of a degenerative process. Overall, our results reveal that in myotubes, PGC-1<italic>α</italic> takes center place in mitochondrial homeostasis during differentiation because of its ability to avoid ROS-mediated removal of mitochondria.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Liesa, M" uniqKey="Liesa M">M Liesa</name></author><author><name sortKey="Palacin, M" uniqKey="Palacin M">M Palacin</name></author><author><name sortKey="Zorzano, A" uniqKey="Zorzano A">A Zorzano</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Romanello, V" uniqKey="Romanello V">V Romanello</name></author><author><name sortKey="Sandri, M" uniqKey="Sandri M">M Sandri</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Benard, G" uniqKey="Benard G">G Benard</name></author><author><name sortKey="Bellance, N" uniqKey="Bellance N">N Bellance</name></author><author><name sortKey="Jose, C" uniqKey="Jose C">C 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<front><journal-meta><journal-id journal-id-type="nlm-ta">Cell Death Dis</journal-id><journal-id journal-id-type="iso-abbrev">Cell Death Dis</journal-id><journal-title-group><journal-title>Cell Death & Disease</journal-title></journal-title-group><issn pub-type="epub">2041-4889</issn><publisher><publisher-name>Nature Publishing Group</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">25375380</article-id><article-id pub-id-type="pmc">4260723</article-id><article-id pub-id-type="pii">cddis2014458</article-id><article-id pub-id-type="doi">10.1038/cddis.2014.458</article-id><article-categories><subj-group subj-group-type="heading"><subject>Original Article</subject></subj-group></article-categories><title-group><article-title>PGC-1<italic>α</italic> buffers ROS-mediated removal of mitochondria during myogenesis</article-title><alt-title alt-title-type="running">PGC-1<italic>α</italic> maintains basal flux of mitophagy</alt-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Baldelli</surname><given-names>S</given-names></name><xref ref-type="aff" rid="aff1">1</xref></contrib><contrib contrib-type="author"><name><surname>Aquilano</surname><given-names>K</given-names></name><xref ref-type="aff" rid="aff2">2</xref><xref ref-type="author-notes" rid="note1"><sup>4</sup></xref></contrib><contrib contrib-type="author"><name><surname>Ciriolo</surname><given-names>M R</given-names></name><xref ref-type="aff" rid="aff2">2</xref><xref ref-type="aff" rid="aff3">3</xref><xref ref-type="author-notes" rid="note1"><sup>4</sup></xref><xref ref-type="corresp" rid="caf1">*</xref></contrib><aff id="aff1"><label>1</label><institution>Scientific Institute for Research, Hospitalization and Health Care, Università Telematica San Raffaele Roma</institution>, Via di Val Cannuta 247, Rome,<country>Italy</country></aff><aff id="aff2"><label>2</label><institution>Department of Biology, University of Rome “Tor Vergata”</institution>, Via della Ricerca Scientifica 1, Rome,<country>Italy</country></aff><aff id="aff3"><label>3</label><institution>IRCCS San Raffaele</institution>, Via di Val Cannuta 247, Rome,<country>Italy</country></aff></contrib-group><author-notes><corresp id="caf1"><label>*</label><institution>Department of Biology, University of Rome ‘Tor Vergata'</institution>, Via della Ricerca Scientifica, Rome 00133, <country>Italy</country>. Tel: +39 06 72594369; Fax: +39 06 72594311; E-mail: <email>ciriolo@bio.uniroma2.it</email></corresp><fn fn-type="present-address" id="note1"><label>4</label><p>These authors contribute equally to this work.</p></fn></author-notes><pub-date pub-type="ppub"><month>11</month><year>2014</year></pub-date><pub-date pub-type="epub"><day>06</day><month>11</month><year>2014</year></pub-date><pub-date pub-type="pmc-release"><day>1</day><month>11</month><year>2014</year></pub-date><volume>5</volume><issue>11</issue><fpage>e1515</fpage><lpage></lpage><history><date date-type="received"><day>04</day><month>04</month><year>2014</year></date><date date-type="rev-recd"><day>02</day><month>09</month><year>2014</year></date><date date-type="accepted"><day>17</day><month>09</month><year>2014</year></date></history><permissions><copyright-statement>Copyright © 2014 Macmillan Publishers Limited</copyright-statement><copyright-year>2014</copyright-year><copyright-holder>Macmillan Publishers Limited</copyright-holder><license license-type="open-access" xlink:href="http://creativecommons.org/licenses/by/4.0/"><pmc-comment>author-paid</pmc-comment>
<license-p><italic>Cell Death and Disease</italic> is an open-access journal published by <italic>Nature Publishing Group</italic>. This work is licensed under a Creative Commons Attribution 4.0 International Licence. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons licence, users will need to obtain permission from the licence holder to reproduce the material. To view a copy of this licence, visit <ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by/4.0">http://creativecommons.org/licenses/by/4.0</ext-link></license-p></license></permissions><abstract><p>Mitochondrial biogenesis and mitophagy are recognized as critical processes underlying mitochondrial homeostasis. However, the molecular pathway(s) coordinating the balance between these cellular programs is still poorly investigated. Here, we show an induction of the nuclear and mitochondrial peroxisome proliferator-activated receptor gamma, coactivator 1 alpha (PGC-1<italic>α</italic>) during myogenesis, which in turn co-activates the transcription of nuclear and mtDNA-encoded mitochondrial genes. We demonstrate that PGC-1<italic>α</italic> also buffers oxidative stress occurring during differentiation by promoting the expression of antioxidant enzymes. Indeed, by downregulating PGC-1<italic>α,</italic> we observed an impairment of antioxidants expression, which was accompanied by a significant reactive oxygen species (ROS) burst and increase of oxidative damage to proteins. In parallel, we detected a decrease of mitochondrial mass and function as well as increased mitophagy through the ROS/FOXO1 pathway. Upon PGC-1<italic>α</italic> downregulation, we found ROS-dependent nuclear translocation of FOXO1 and transcription of its downstream targets including mitophagic genes such as LC3 and PINK1. Such events were significantly reverted after treatment with the antioxidant Trolox, suggesting that PGC-1<italic>α</italic> assures mitochondrial integrity by indirectly buffering ROS. Finally, the lack of PGC-1<italic>α</italic> gave rise to a decrease in MYOG and a strong induction of atrophy-related ubiquitin ligases FBXO32 (FBXO32), indicative of a degenerative process. Overall, our results reveal that in myotubes, PGC-1<italic>α</italic> takes center place in mitochondrial homeostasis during differentiation because of its ability to avoid ROS-mediated removal of mitochondria.</p></abstract></article-meta></front><floats-group><fig id="fig1"><label>Figure 1</label><caption><p>Nuclear and mitochondrial PGC-1<italic>α</italic> induction is necessary to mitochondrial functionality during myogenesis. C2C12 cells were differentiated in DM for the indicated days. (<bold>a</bold>) Twenty micrograms of total proteins were loaded for western blot analysis of PGC-1<italic>α</italic>, TFAM and COX4I1. TUBB was used as the loading control. (<bold>b</bold>) Total RNA was isolated, and relative mRNA level of PGC-1<italic>α</italic> was analyzed by RT-qPCR. Data are expressed as means±S.D. (<italic>n</italic>=6, *<italic>P</italic><0.001). (<bold>c</bold>) Twenty micrograms of mitochondrial proteins extracts were loaded for western blot analysis of PGC-1<italic>α</italic> and TFAM. TOMM20 was used as the loading control. To exclude the presence of nuclear contaminants, the nitrocellulose was probed with H2B antibody. Numbers indicate the density of immunoreactive bands calculated using the Software Quantity one (Bio-Rad) and reported as the ratio of PGC-1<italic>α</italic>/TOMM20 and TFAM/TOMM20. (<bold>d</bold>) mtDNA-IP assay was carried out on cross-linked mitochondria using PGC-1<italic>α</italic> (<italic>left panel</italic>) or TFAM (<italic>right panel</italic>) antibody followed by qPCR analysis of D-Loop TFAM <italic>consensus</italic> sequence. Data are expressed as means±S.D. (<italic>n</italic>=4, *<italic>P</italic><0.001). (<bold>e</bold>) C2C12 cells were transfected with scramble (scr) or PGC-1<italic>α</italic> siRNA (PGC-1<italic>α</italic>(−)), differentiated in DM for the indicated days. DNA was extracted and relative mtDNA content was assayed by analyzing D-Loop level through qPCR. D-Loop value was normalized to ribosomal protein large subunit (RPL). Data are expressed as means±S.D. (<italic>n</italic>=5, *<italic>P</italic><0.001 <italic>versus</italic> day 0 scr cells; °<italic>P</italic><0.001 <italic>versus</italic> scr cells). (<bold>f</bold>) Cells were grown on glass coverslips, differentiated until day 2 and incubated for 30 min with 1 <italic>μ</italic>g/ml JC-1. Paraformaldehyde-fixed cells were stained with Hoechst 33342 and subjected to fluorescence microscopy. Magnitude insets are shown at the bottom and top of each picture. Images reported are from one experiment representative of three that gave similar results. Immunoblots reported in the figures are representative of at least four experiments that gave similar results</p></caption><graphic xlink:href="cddis2014458f1"></graphic></fig><fig id="fig2"><label>Figure 2</label><caption><p>PGC-1<italic>α</italic> buffers mitophagy during myogenesis. C2C12 cells were transfected with scramble (scr) or PGC-1<italic>α</italic> siRNA (PGC-1<italic>α</italic>(−)), differentiated in DM for the indicated days. (<bold>a</bold>) C2C12 cells were treated with 5 <italic>μ</italic>M CQ and 20 <italic>μ</italic>g of total proteins extracts were loaded for western blot analysis of LC3I-II and SQSTM1. TUBB was used as the loading control. Numbers indicate the density of immunoreactive bands calculated using the Software Quantity one (Bio-Rad) and reported as the ratio of LC3II/TUBB and SQSTM1/TUBB. (<bold>b</bold>) C2C12 cells were treated with 5 mM 3MA and 20 <italic>μ</italic>g of total proteins extracts were loaded for western blot analysis of LC3I-II and SQSTM1. TUBB was used as the loading control. Numbers indicate the density of immunoreactive bands calculated using the Software Quantity one (Bio-Rad) and reported as the ratio of LC3II/TUBB and SQSTM1/TUBB. (<bold>c</bold>) Total RNA was isolated, and relative mRNA level of LC3 and BNIP3 were analyzed by RT-qPCR. Data are expressed as means±S.D. (<italic>n</italic>=6, *<italic>P</italic><0.001 <italic>versus</italic> day 0 scr cells). (<bold>d</bold>) Twenty micrograms of total proteins extracts were loaded for western blot analysis of pPRKAA2, PRKAA2 and pRPS6KB1. TUBB was used as the loading control. (<bold>e</bold>) Twenty micrograms of total proteins were loaded for western blot analysis of PINK1 and PARK2. TUBB was used as the loading control. (<bold>f</bold>) Twenty micrograms of total proteins were loaded for western blot analysis of PINK1, PARK2, BNIP3 and MFN2. TUBB was used as the loading control. (<bold>g</bold>) Total RNA was isolated, and relative mRNA level of PARK2 were analyzed by RT-qPCR. Data are expressed as means±S.D. (<italic>n</italic>=4, *<italic>P</italic><0.001 <italic>versus</italic> day 0 scr cells). (<bold>h</bold>) Twenty micrograms of mitochondrial proteins extracts were loaded for western blot analysis of PINK1, PARK2 and BNIP3. TOMM20 was used as the loading control. (<bold>i</bold>) Twenty micrograms of mitochondrial and cytoplasmatic proteins extracts were loaded for western blot analysis of PARK2 and BNIP3. TOMM20 and LDH were used as the loading control. Immunoblots reported in the figures are representative of at least four experiments that gave similar results</p></caption><graphic xlink:href="cddis2014458f2"></graphic></fig><fig id="fig3"><label>Figure 3</label><caption><p>PGC-1<italic>α</italic> deficiency induces mitophagy in C2C12 cells. C2C12 cells were co-transfected with scramble (scr) or PGC-1<italic>α</italic> siRNA (PGC-1<italic>α</italic>(−)) and with EGFP-LC3. At day 0, C2C12 cells were fixed with paraformaldehyde and immunostained with mouse anti-HSPA9, Hoechst 33342 and then visualized by confocal microscopy. Scale bar 7 <italic>μ</italic>m. Overlap and Pearson's coefficients were calculated by JACoP (plugin of ImageJ Software) in at least 10 different images. Data are expressed as means±S.D. (<italic>n</italic>=4, *<italic>P</italic><0.001, <sup>#</sup><italic>P</italic><0.01 <italic>versus</italic> scr cells). Yellow in the merged image indicates co-localization of EGFP-LC3 with mitochondria (HSPA9). Magnitude insets are shown at the bottom and top of each picture. Images reported are from one experiment representative of three that gave similar results</p></caption><graphic xlink:href="cddis2014458f3"></graphic></fig><fig id="fig4"><label>Figure 4</label><caption><p>PGC-1<italic>α</italic> downregulation is associated with an increase of oxidative stress and alterations of mitochondrial homeostasis. C2C12 cells were transfected with scramble (scr) or PGC-1<italic>α</italic> siRNA (PGC-1<italic>α</italic>(−)) and differentiated in DM for the indicated days. (<bold>a</bold>) Twenty micrograms of total proteins were derivatized with DNP and carbonylation was detected by western blot with DNP antibody. TUBB was used as the loading control. (<bold>b</bold>) Twenty micrograms of total proteins were loaded for western blot analysis of SOD2 and TRX1. TUBB was used as the loading control. (<bold>c</bold>) C2C12 cells were treated with 200 <italic>μ</italic>M Trolox and maintained throughout the experiment. ROS production was assayed by cytofluorimetric analysis after DCF-DA staining. ROS level was reported as the percentage of DCF-positive cells and expressed as means ±S.D. (<italic>n</italic>=3; *<italic>P</italic><0.001 <italic>versus</italic> scr cells; °<italic>P</italic><0.01 <italic>versus</italic> PGC-1<italic>α</italic>(−) cells). (<bold>d</bold>) Twenty micrograms of total proteins were derivatized with DNP and carbonylation was detected by western blot with DNP antibody. TUBB was used as the loading control. (<bold>e</bold>) C2C12 cells were incubated with MitoTrackerRed for 30 min and mitochondrial content was assayed by cytofluorimetric analysis. Data are expressed as percentage of MitoTrackerRed-positive cells (<italic>n</italic>=4; *<italic>P</italic><0.001 <italic>versus</italic> scr cells; °<italic>P</italic><0.01 <italic>versus</italic> PGC-1<italic>α</italic>(−) cells). (<bold>f</bold>) DNA was extracted and relative mtDNA content was assayed by analyzing the D-Loop level through qPCR. D-Loop value was normalized to RPL. Data are expressed as means±S.D. (<italic>n</italic>=5, *<italic>P</italic><0.001 <italic>versus</italic> scr cells; °<italic>P</italic><0.001 <italic>versus</italic> PGC-1<italic>α</italic>(−) cells). (<bold>g</bold>) Twenty micrograms of total proteins were loaded for western blot analysis of LC3I-II and PINK1. TUBB was used as the loading control. Immunoblots reported in the figures are representative of at least four experiments that gave similar results. (<bold>h</bold>) C2C12 cells were transfected with empty vector (Mock) or pSV-PGC-1<italic>α</italic> vector (PGC-1<italic>α</italic>(+)) and differentiated in DM for the indicated days. Twenty micrograms of total proteins were loaded for western blot analysis of PGC-1<italic>α</italic>, PARK2, PINK1 and BNIP3. TUBB was used as the loading control</p></caption><graphic xlink:href="cddis2014458f4"></graphic></fig><fig id="fig5"><label>Figure 5</label><caption><p>FOXO1 promotes mitophagy in a ROS-dependent manner during myogenesis. C2C12 cells were transfected with scramble (scr) or PGC-1<italic>α</italic> siRNA (PGC-1<italic>α</italic>(−)) and differentiated in DM for the indicated days. (<bold>a</bold>) Twenty micrograms of total proteins were loaded for western blot analysis of FOXO1. TUBB was used as the loading control. (<bold>b</bold>) Twenty micrograms of nuclear and cytoplasmatic proteins were loaded for western blot analysis of FOXO1. Sp1 and LDH were used as markers of fraction purity and as the loading control. (<bold>c</bold>, <bold>d</bold>) At day 2 of myogenesis, ChIP assay was carried out on cross-linked nuclei using LC3II (<italic>upper panel</italic>) or PINK1 (<italic>bottom panel</italic>) antibody followed by qPCR analysis of FOXO1 <italic>consensus</italic> sequence. Data are expressed as means±S.D. (<italic>n</italic>=4, *<italic>P</italic><0.001 <italic>versus</italic> scr cells; °<italic>P</italic><0.001 <italic>versus</italic> day 0 PGC-1<italic>α</italic>(−) cells). (<bold>e</bold>) C2C12 cells were treated with 200 <italic>μ</italic>M Trolox and maintained throughout the experiment (2 days). Twenty micrograms of nuclear and cytoplasmatic proteins were loaded for western blot analysis of FOXO1. Sp1 and LDH were used as markers of fraction purity and as the loading control. (<bold>f</bold>, <bold>g</bold>) C2C12 cells were treated with 200 <italic>μ</italic>M Trolox and maintained throughout the experiment (2 days). ChIP assay was carried out on cross-linked nuclei using LC3II (<italic>upper panel</italic>) or PINK1 (<italic>bottom panel</italic>) antibody followed by qPCR analysis of FOXO1 <italic>consensus</italic> sequence. Data are expressed as means±S.D. (<italic>n</italic>=6, *<italic>P</italic><0.001 <italic>versus</italic> scr cells; °<italic>P</italic><0.001 <italic>versus</italic> untreated day 2 PGC-1<italic>α</italic>(−) cells). Immunoblots reported in the figures are representative of at least four experiments that gave similar results</p></caption><graphic xlink:href="cddis2014458f5"></graphic></fig><fig id="fig6"><label>Figure 6</label><caption><p>Downregulation of FOXO1 enables mitophagy induction. C2C12 cells were transfected with scramble (scr), PGC-1<italic>α</italic> and/or FOXO1 siRNA (PGC-1<italic>α</italic>(−), FOXO1(−) and PGC-1<italic>α</italic>/FOXO1(−)) and differentiated in DM for the indicated days. (<bold>a</bold>) Twenty micrograms of total proteins were loaded for western blot analysis of FOXO1, PGC-1<italic>α</italic>, SQSTM1 and PINK1. TUBB was used as the loading control. Numbers indicate the density of immunoreactive bands calculated using the Software Quantity one (Bio-Rad) and reported as the ratio of SQSTM1/TUBB and PINK1/TUBB. (<bold>b</bold>) Twenty micrograms of total proteins were derivatized with DNP and carbonylation was detected by western blot with DNP antibody. TUBB was used as the loading control. (<bold>c</bold>) Twenty micrograms of total proteins were loaded for western blot analysis of FOXO1, PGC-1<italic>α</italic>, PINK1 and BNIP3. TUBB was used as the loading control. Numbers indicate the density of immunoreactive bands calculated using the Software Quantity one (Bio-Rad) and reported as the ratio of proteins/TUBB. Immunoblots reported in the figures are representative of at least four experiments that gave similar results</p></caption><graphic xlink:href="cddis2014458f6"></graphic></fig><fig id="fig7"><label>Figure 7</label><caption><p>Depletion of PGC-1<italic>α</italic> inhibits myogenin and induces FBXO32 expression. C2C12 cells were transfected with scramble (scr) or PGC-1<italic>α</italic> siRNA (PGC-1<italic>α</italic>(−)) and differentiated in DM for the indicated days. (<bold>a</bold>, <bold>b</bold>) Total RNA was isolated, and relative mRNA levels of MYOG (<bold>a</bold>) and FBXO32 (<bold>b</bold>) were analyzed by RT-qPCR. Data are expressed as means±S.D. (<italic>n</italic>=4, *<italic>P</italic><0.001 <italic>versus</italic> day 0 scr cells; °<italic>P</italic><0.001 <italic>versus</italic> scr cells). (<bold>c</bold>, <bold>d</bold>) C2C12 cells were transfected with scramble (scr), PGC-1<italic>α</italic> and/or PINK1 siRNA (PGC-1<italic>α</italic>(−), PINK1(−) and PGC-1<italic>α</italic>/PINK1(−)) and differentiated for 2 days. Total RNA was isolated, and relative mRNA levels of MYOG (<bold>c</bold>) and FBXO32 (<bold>d</bold>) were analyzed by RT-qPCR. Data are expressed as means±S.D. (<italic>n</italic>=4, *<italic>P</italic><0.001 <italic>versus</italic> scr cells; °<italic>P</italic><0.001 <italic>versus</italic> PGC-1<italic>α</italic>(−) cells, PGC-1<italic>α</italic>(−)/PINK1(−) cells and <sup>#</sup><italic>P</italic><0.001 <italic>versus</italic> PINK1(−) cells)</p></caption><graphic xlink:href="cddis2014458f7"></graphic></fig><fig id="fig8"><label>Figure 8</label><caption><p>Schematic model of PGC-1<italic>α</italic> function in regulation of mitochondrial content during myogenesis. During myogenesis, PGC-1<italic>α</italic> maintains mitochondrial homeostasis ensuring mitochondrial biogenesis, counteracting oxidative stress and buffering mitophagy. Through this regulation, PGC-1<italic>α</italic> functions as a positive regulator of mitochondrial biogenesis moving the skeletal muscle balance towards the production of new mitochondria. Conversely, the downregulation of PGC-1<italic>α</italic> leads to alteration of mitochondrial network promoting the induction of mitophagy through a ROS-dependent binding of FOXO1 transcription factor on LC3 and PINK1 gene promoter</p></caption><graphic xlink:href="cddis2014458f8"></graphic></fig><table-wrap id="tbl1"><label>Table 1</label><caption><title>List of primers used for RT-qPCR and ChIP analysis</title></caption><table frame="hsides" rules="groups" border="1"><colgroup><col align="left"></col><col align="left"></col></colgroup><thead valign="bottom"><tr><th align="left" valign="top" charoff="50"><bold>Gene</bold></th><th align="left" valign="top" charoff="50"><bold>Sequences</bold></th></tr></thead><tbody valign="top"><tr><td align="left" valign="top" charoff="50">COX4I1 (mouse)</td><td align="left" valign="top" charoff="50">RV 5′-TCACAACACTCCCATGTGCT-3′
FW 5′-GAATGTTGGCTTCCAGAGCG-3′</td></tr><tr><td align="left" valign="top" charoff="50">PGC-1<italic>α</italic> (mouse)</td><td align="left" valign="top" charoff="50">RV 5′-ACTCGGATTGCTCCGGCCCT-3′
FW 5′-ACTGACGGCCTAACTCCACCCA-3′</td></tr><tr><td align="left" valign="top" charoff="50">MT-CO1 (mouse)</td><td align="left" valign="top" charoff="50">RV 5′-ACCTATTCGTACTCCGGCCT-3′
FW 5′-ATGTGACCCGATTTGGCGTT-3′</td></tr><tr><td align="left" valign="top" charoff="50">MT-ATP6 (mouse)</td><td align="left" valign="top" charoff="50">RV 5′-GTGGAAGGAAGTGGGCAAGTGAGC-3′
FW 5′-GCCATTCCACTATGAGCTGGAGCC-3′</td></tr><tr><td align="left" valign="top" charoff="50">TFAM (mouse)</td><td align="left" valign="top" charoff="50">RV 5′-TCCGCCCTATAAGCATCTTG-3′
FW 5′-CCGAGGTGGTTTTCATCTGT-3′</td></tr><tr><td align="left" valign="top" charoff="50">PARK2 (mouse)</td><td align="left" valign="top" charoff="50">RV 5′-ACAGGGCTCCTGACATCTG-3′
FW 5′-CAAGGACACGTCGGTAGCTT-3′</td></tr><tr><td align="left" valign="top" charoff="50">LC3 (mouse)</td><td align="left" valign="top" charoff="50">RV 5′-CCACTCTTTGTTCAAAGCTCCGGC-3′
FW 5′-CGTCGCCGGAGTCAGATGTC-3′</td></tr><tr><td align="left" valign="top" charoff="50">NRF1 (mouse)</td><td align="left" valign="top" charoff="50">RV 5′-ATGGGCGGCAGCTTGACTGT-3′
FW 5′-GCGCAGCCGCTCTGAGAACTTAT-3′</td></tr><tr><td align="left" valign="top" charoff="50">NRF2 (mouse)</td><td align="left" valign="top" charoff="50">RV 5′-TGGGCCCTGATGAGGGGCAGTG-3′
FW 5′-TCCGCCAGCTACTCCAGGTTGG-3′</td></tr><tr><td align="left" valign="top" charoff="50">POLRMT (mouse)</td><td align="left" valign="top" charoff="50">RV 5′-GACGGCGTTAGGTTGACTGA-3′
FW 5′-CCAGTTCACCAGGATGGCTC-3′</td></tr><tr><td align="left" valign="top" charoff="50">POLG (mouse)</td><td align="left" valign="top" charoff="50">RV 5′-ACAAGTCCTCGGTCCGCTTC-3′
FW 5′-CACCCTGAGGCTGCGTG-3′</td></tr><tr><td align="left" valign="top" charoff="50">RPL (mouse)</td><td align="left" valign="top" charoff="50">RV 5′-ATGGCGGAGGGGCAGGTTCTG-3′
FW 5′-GTACGACCACCACCTTCCGGC-3′</td></tr><tr><td align="left" valign="top" charoff="50">BNIP3 (mouse)</td><td align="left" valign="top" charoff="50">RV 5′-GGTCGACTTGACCAATCCCAT-3′
FW 5′-ACAGCACTCTGTCTGAGGAA-3′</td></tr><tr><td align="left" valign="top" charoff="50">D-Loop <italic>consensus sequence</italic></td><td align="left" valign="top" charoff="50">RV 5′-CATGAATAATTAGCCTTAGGTGAT-3′
FW 5′-TCAGACATCTGGTTCTTCTTACTTCAG-3′</td></tr><tr><td align="left" valign="top" charoff="50">FOXO1 <italic>consensus sequence</italic> on LC3 promoter</td><td align="left" valign="top" charoff="50">RV 5′-CCCAAGGATCTCAACCAAAC-3′
FW 5′-CCTCAGCTGGTAAGAGCAT-3′</td></tr><tr><td align="left" valign="top" charoff="50">FOXO1 <italic>consensus sequence</italic> on PINK1 promoter</td><td align="left" valign="top" charoff="50">RV 5′-CTGTCGACCGCCATGGTGGCGCGGTGACC-3′
FW 5′-TGAGAGCACTTGGGAGTGGGGGAGAAGAG-3′</td></tr></tbody></table></table-wrap></floats-group></pmc></record>Pour manipuler ce document sous Unix (Dilib)
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