FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2788097887> ?p ?o ?g. }

• W2788097887 endingPage "609.e8" @default.
• W2788097887 startingPage "594" @default.
• W2788097887 abstract "•The MDM2 oncoprotein localizes to the mitochondrial matrix independently of p53•Mitochondrial MDM2 inhibits transcription of the complex I (CI) subunit MT-ND6•MDM2 deficiency in skeletal muscles increases CI activity and muscular endurance•Mitochondrial MDM2 increases cancer cell migration and invasion Accumulating evidence indicates that the MDM2 oncoprotein promotes tumorigenesis beyond its canonical negative effects on the p53 tumor suppressor, but these p53-independent functions remain poorly understood. Here, we show that a fraction of endogenous MDM2 is actively imported in mitochondria to control respiration and mitochondrial dynamics independently of p53. Mitochondrial MDM2 represses the transcription of NADH-dehydrogenase 6 (MT-ND6) in vitro and in vivo, impinging on respiratory complex I activity and enhancing mitochondrial ROS production. Recruitment of MDM2 to mitochondria increases during oxidative stress and hypoxia. Accordingly, mice lacking MDM2 in skeletal muscles exhibit higher MT-ND6 levels, enhanced complex I activity, and increased muscular endurance in mild hypoxic conditions. Furthermore, increased mitochondrial MDM2 levels enhance the migratory and invasive properties of cancer cells. Collectively, these data uncover a previously unsuspected function of the MDM2 oncoprotein in mitochondria that play critical roles in skeletal muscle physiology and may contribute to tumor progression. Accumulating evidence indicates that the MDM2 oncoprotein promotes tumorigenesis beyond its canonical negative effects on the p53 tumor suppressor, but these p53-independent functions remain poorly understood. Here, we show that a fraction of endogenous MDM2 is actively imported in mitochondria to control respiration and mitochondrial dynamics independently of p53. Mitochondrial MDM2 represses the transcription of NADH-dehydrogenase 6 (MT-ND6) in vitro and in vivo, impinging on respiratory complex I activity and enhancing mitochondrial ROS production. Recruitment of MDM2 to mitochondria increases during oxidative stress and hypoxia. Accordingly, mice lacking MDM2 in skeletal muscles exhibit higher MT-ND6 levels, enhanced complex I activity, and increased muscular endurance in mild hypoxic conditions. Furthermore, increased mitochondrial MDM2 levels enhance the migratory and invasive properties of cancer cells. Collectively, these data uncover a previously unsuspected function of the MDM2 oncoprotein in mitochondria that play critical roles in skeletal muscle physiology and may contribute to tumor progression. The p53 pathway is functionally inactivated in most, if not all, cancers. Somatic mutations in TP53 occur in about 50% of human tumors, and many cancers retaining wild-type TP53 display functional inactivation of the p53 pathway by viral oncoproteins or alterations of p53 regulators, including the Mouse Double Minute 2 (MDM2) oncogene (Wade et al., 2013Wade M. Li Y.-C. Wahl G.M. MDM2, MDMX and p53 in oncogenesis and cancer therapy.Nat. Rev. Cancer. 2013; 13: 83-96Crossref PubMed Scopus (854) Google Scholar). MDM2 is a major negative regulator of the p53 pathway through its well-documented E3 ligase activity that targets p53 protein for proteasomal degradation. MDM2 also contributes to the regulation of p53 target genes by directly inhibiting p53 transactivation domains and by ubiquitylating histones at p53-responsive genes (Momand et al., 1992Momand J. Zambetti G.P. Olson D.C. George D. Levine A.J. The mdm-2 oncogene product forms a complex with the p53 protein and inhibits p53-mediated transactivation.Cell. 1992; 69: 1237-1245Abstract Full Text PDF PubMed Scopus (2787) Google Scholar, Oliner et al., 1993Oliner J.D. Pietenpol J.A. Thiagalingam S. Gyuris J. Kinzler K.W. Vogelstein B. Oncoprotein MDM2 conceals the activation domain of tumour suppressor p53.Nature. 1993; 362: 857-860Crossref PubMed Scopus (1303) Google Scholar, Minsky and Oren, 2004Minsky N. Oren M. The RING domain of Mdm2 mediates histone ubiquitylation and transcriptional repression.Mol. Cell. 2004; 16: 631-639Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). However, it is now recognized that MDM2 oncogenic activities extend beyond the direct control of p53. Thus, MDM2-mediated ubiquitylation of several transcription factors or transcriptional co-regulators contributes to the regulation of p53-independent programs (Biderman et al., 2012Biderman L. Manley J.L. Prives C. Mdm2 and MdmX as regulators of gene expression.Genes Cancer. 2012; 3: 264-273Crossref PubMed Scopus (37) Google Scholar). Recent findings also identified a p53-independent role for chromatin-bound MDM2 in the transcriptional control of genes involved in cell fate and metabolism (Wienken et al., 2016Wienken M. Dickmanns A. Nemajerova A. Kramer D. Najafova Z. Weiss M. Karpiuk O. Kassem M. Zhang Y. Lozano G. et al.MDM2 associates with Polycomb Repressor Complex 2 and enhances stemness-promoting chromatin modifications independent of p53.Mol. Cell. 2016; 61: 68-83Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, Riscal et al., 2016Riscal R. Schrepfer E. Arena G. Cissé M.Y. Bellvert F. Heuillet M. Rambow F. Bonneil E. Sabourdy F. Vincent C. et al.Chromatin-bound MDM2 regulates serine metabolism and redox homeostasis independently of p53.Mol. Cell. 2016; 62: 890-902Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). Moreover, the RING domain of MDM2 is required for its binding to the mRNAs of several cancer-related genes to control their stability or translation, including those encoding p53 itself, the X-linked inhibitor of apoptosis (XIAP), N-MYC, and vascular endothelial growth factor (VEGF) (Fåhraeus and Olivares-Illana, 2014Fåhraeus R. Olivares-Illana V. MDM2’s social network.Oncogene. 2014; 33: 4365-4376Crossref PubMed Scopus (71) Google Scholar). Here, we show that a fraction of MDM2 protein localizes in the mitochondrial matrix independently of p53 and regulates the transcription of the mitochondrial genome to control respiration in both normal and cancer cells. We assessed MDM2 subcellular localization in several cancer cell lines by immunoblotting following biochemical fractionation of various cellular compartments and isolation of purified organelles. As anticipated, MDM2 was detected in fractions enriched in cytosolic or nuclear proteins prepared from p53 null H1299 lung cancer cells. Unexpectedly, significant fractions of both ectopic FLAG-tagged MDM2 and endogenous MDM2 were detected in protein extracts prepared from purified mitochondria. The specificity of MDM2 immunoreactivity was confirmed using extracts prepared from MDM2-depleted H1299 cells, and the quality of the fractionation was verified by measuring the levels of cytosolic tubulin (TUB), nuclear TATA binding protein (TBP), and mitochondrial TIM23 proteins (Figures 1A and 1B ). Next, we evaluated the amount of mitochondrial MDM2 in a panel of human cancer cell lines. When detectable by immunoblotting, a variable amount of endogenous MDM2, ranging from 2% to 5% of total MDM2, localized to mitochondria in these cells (Figures 1C, 1D, and S1A). The amount of mitochondrial MDM2 (mtMDM2) did not correlate with the p53 status of these cell lines, suggesting that MDM2 localized to mitochondria in a p53-independent manner. Consistent with this notion, short hairpin RNA (shRNA)-mediated depletion of wild-type (WT) p53 in MCF7 or of mutant p53 in T47D breast cancer cells did not alter the relative proportion of mtMDM2, indicating that neither WT nor mutant p53 was required for the localization of endogenous MDM2 to mitochondria (Figures 1E and S1B). Confocal microscopy analysis showed that FLAG-tagged full-length (FL) MDM2 (residues 1 to 491) exhibited different subcellular localization in H1299 cells. Approximately 35% of H1299 cells displayed a strictly nuclear pattern for ectopic FL-MDM2, 55% of them exhibited both nuclear and cytoplasmic MDM2, and 10% of them displayed a staining pattern that was restricted to the cytoplasm (Figure S1C). Super-resolution microscopy performed at a resolution of 100 nm indicated that approximately 8% of FL-MDM2 co-localized with the mitochondrial marker TFAM (Figure 1F). Mitochondrial localization of ectopic FL-MDM2 in H1299 cells was further confirmed by immunogold labeling and transmission electron microscopy (TEM). Consistent with MDM2 localization in different subcellular compartments, gold particles were detected in the nucleus and the cytoplasm, but also inside the mitochondrial matrix, at the edge of mitochondrial cristae (Figures 1G and S1D). We next performed protease protection assays on purified mitochondria isolated from H1299 cells expressing FL-MDM2. At concentrations of 0.5 and 1 μg/mL Proteinase K (PK), the outer mitochondrial membrane (OMM) protein TOM20 and the inner mitochondrial membrane (IMM) protein TIM23 were progressively degraded. In contrast, MDM2 and the mitochondrial matrix protein TFAM were both resistant to mild protease digestion. Complete PK-mediated digestion of TFAM and MDM2 proteins occurred only after treatment with Triton X-100, indicating that both proteins localized in the same mitochondrial compartment (Figure 1H). These data demonstrate that a fraction of MDM2 protein localizes to the mitochondrial matrix. Mitochondrial proteins often contain an N-terminal leader peptide that is cleaved in the IMM before they can be released in the matrix (Dudek et al., 2013Dudek J. Rehling P. van der Laan M. Mitochondrial protein import: common principles and physiological networks.Biochim. Biophys. Acta. 2013; 1833: 274-285Crossref PubMed Scopus (185) Google Scholar). Analysis of the MDM2 protein sequence failed to identify a canonical mitochondrial localization signal. In order to ascertain which domain of MDM2 was required for its recruitment to mitochondria, we performed subcellular fractionation assays and immunogold staining for TEM in H1299 cells expressing FL-MDM2 or different MDM2 deletion mutants. Deletion of its N-terminal region increased the total levels of MDM2 but did not change its relative subcellular localization, whereas the deletion of the C-terminal region (amino acids [aa] 292–491) impacted on its localization in mitochondria (Figures 2A–2C and S2A–S2C). Smaller deletions of the C-terminal region indicated that the last 61 aa (aa 430–491) of MDM2, and a more central region (aa 291–391) played a significant role in MDM2 localization in mitochondria (Figures S2D and S2E). We next asked whether MDM2 recruitment to this organelle was dependent on mitochondrial transporters. FL-MDM2 co-immunoprecipitated with endogenous TOM20 and TIM23 proteins, two components of the mitochondrial translocator complexes located in the OMM and the IMM, respectively (Figure 2D). Transport through these mitochondrial translocator complexes depends on the membrane potential (ΔΨ) across the IMM (Geissler et al., 2000Geissler A. Krimmer T. Bömer U. Guiard B. Rassow J. Pfanner N. Membrane potential-driven protein import into mitochondria. The sorting sequence of cytochrome b(2) modulates the deltapsi-dependence of translocation of the matrix-targeting sequence.Mol. Biol. Cell. 2000; 11: 3977-3991Crossref PubMed Scopus (103) Google Scholar). Accordingly, the amount of mtMDM2 in H1299 cells decreased upon treatment with carbonyl cyanide4-(trifuoromethoxy) phenylhydrazone (FCCP), a protonophore that disrupts mitochondrial membrane potential (Figure 2E). Moreover, MDM2 co-immunoprecipitated with the mitochondrial 70-kDa heat shock protein (mtHsp70), also known as mortalin, as well as with its direct partner TID1 (Figures 2D and S2F). These two proteins are central components of the mitochondrial protein import motor that play a key role in the import and proper folding of proteins localized in mitochondrial matrix (Syken et al., 1999Syken J. De-Medina T. Münger K. TID1, a human homolog of the Drosophila tumor suppressor l(2)tid, encodes two mitochondrial modulators of apoptosis with opposing functions.Proc. Natl. Acad. Sci. USA. 1999; 96: 8499-8504Crossref PubMed Scopus (124) Google Scholar, Schneider et al., 1994Schneider H.C. Berthold J. Bauer M.F. Dietmeier K. Guiard B. Brunner M. Neupert W. Mitochondrial Hsp70/MIM44 complex facilitates protein import.Nature. 1994; 371: 768-774Crossref PubMed Scopus (333) Google Scholar). In H1299 cells, mtHsp70/mortalin and TID1 co-immunoprecipitated with FL-MDM2 but not with the MDM2 1-291 mutant that displayed impaired mitochondrial localization (Figure 2F). Importantly, pharmacological inhibition (by treatment with MKT-077) or shRNA-mediated depletion of mtHsp70/mortalin decreased the basal levels of mtMDM2 (Figures 2G and 2H). Thus, our data indicate that MDM2 is actively imported into the mitochondrial matrix by specific mitochondrial transporters. Next, we assessed by confocal microscopy whether modulating mtMDM2 levels influenced the organization of the mitochondrial network. Immunostaining of the ATP5A subunit of the ATP synthase complex or staining with the mitochondrial dye MitoTracker Red indicated that MDM2-depleted cells exhibited a more fragmented mitochondrial network than control cells (Figures 3A, 3B, S3A, and S3B). MDM2 depletion also resulted in enhanced phosphorylation of dynamin-related protein 1 (DRP1) on serine 637, an event previously associated with increased mitochondrial fission (Wang et al., 2012Wang W. Wang Y. Long J. Wang J. Haudek S.B. Overbeek P. Chang B.H.J. Schumacker P.T. Danesh F.R. Mitochondrial fission triggered by hyperglycemia is mediated by ROCK1 activation in podocytes and endothelial cells.Cell Metab. 2012; 15: 186-200Abstract Full Text Full Text PDF PubMed Scopus (342) Google Scholar), but had no significant effect on the levels of Mitofusin 2 (MFN2) and Optic Atrophy 1 (OPA1) proteins, which promote fusion (Figure 3C). Strikingly, H1299 cells expressing FL-MDM2 displayed an asymmetrical perinuclear clustering of mitochondria (Figures 3D and 3E). This spatial redistribution of mitochondria was observed only in cells exhibiting cytoplasmic staining of FL-MDM2 but not in cells exhibiting exclusively nuclear MDM2 localization or in cells expressing MDM2 1-291 (Figures 3D, 3E, and S3C), suggesting that the mitochondrial pool of MDM2 was directly implicated in this clustering of mitochondria. To confirm this notion, we generated a chimeric MDM2 protein fused to the N-terminal mitochondrial targeting sequence (MTS) of the E1 subunit of the pyruvate dehydrogenase complex. Biochemical fractionation and immunofluorescence (IF) assays indicated that MTS-MDM2 localized predominantly to mitochondria and that its expression further increased the percentage of cells exhibiting perinuclear clustering of mitochondria (Figures 3D–3E and S3D). A similar phenotype was observed upon expression of MTS-MDM2 in U2OS and MCF7 cells (Figures S3E and S3F), and videomicroscopy indicated that this reorganization of the mitochondrial network occurred as soon as 6 hr after transfecting H1299 cells with a vector encoding MTS-MDM2 (mv1). To investigate whether alterations in mitochondrial dynamics were also accompanied by changes in ultrastructure, we performed conventional TEM analysis in H1299 cells expressing control or MDM2 shRNAs and in cells expressing FL-MDM2, MTS-MDM2, or the MDM2 1-291 mutant. MDM2-depleted H1299 cells displayed more fragmented mitochondria than control cells but no significant abnormalities of mitochondrial ultrastructure (Figure 3F). In contrast, H1299 cells expressing ectopic FL-MDM2 exhibited loss of mitochondrial matrix electron density, and cristae appeared narrower in these cells, misoriented, and/or reduced in number. These morphological changes were significantly amplified and more frequent upon expression of MTS-MDM2, whereas cells expressing MDM2 1-291 contained mitochondria with an electron-dense matrix and well-organized intact cristae that were indistinguishable from those of control cells (Figure 3G). Importantly, changes of mitochondrial ultrastructure triggered by higher mtMDM2 levels were not associated with increased apoptosis, as shown by the lack of mitochondrial swelling and the absence of detectable cytochrome c release or caspase-3 cleavage (Figures 3G and S3G–S3I). The mitochondrial localization of MDM2 prompted us to investigate its role in respiration. First, we analyzed the consequences of MDM2 deficiency on oxygen consumption and respiratory chain enzymatic activities. State III respiration linked to the electron transport chain (ETC) complex I (CI), measured in the presence of glutamine, malate, and pyruvate (EIII GMP) as substrates, increased significantly upon MDM2 depletion in H1299 cells, as well as after Cre-mediated inactivation of murine Mdm2 in Mdm2flox/flox; p53KO primary mouse embryonic fibroblasts (MEFs) (Figures 4A, 4B, and S4A). In contrast, oxygen consumption driven by complex II (CII), measured in the presence of the CI inhibitor rotenone and succinate (EIII SR) as a substrate, was not affected by MDM2 deficiency (Figure S4B). Of note, increased oxygen consumption in MDM2-depleted H1299 cells was abolished upon incubation with the CI inhibitor metformin, further supporting the role of mtMDM2 in CI-driven respiration (Figure S4C). In line with these results, CI enzymatic activity significantly increased upon shRNA-mediated depletion of MDM2, whereas CII and IV activities remained unchanged (Figure 4C). Next, we measured mitochondrial respiration in cells expressing MTS-MDM2 before these cells exhibited morphological changes in their mitochondrial ultrastructure. CI-driven oxygen consumption was reduced in H1299 cells expressing MTS-MDM2, whereas CII-driven respiration was only marginally affected (Figures 4D and S4D). Consistently, CI activity decreased, whereas that of CII and IV was unaffected in these cells (Figure 4E). Moreover, expression of the MDM2 1-291 that was marginally detected in mitochondria failed to impact on oxygen consumption (Figure 4D). Finally, we evaluated whether the E3 ligase function of MDM2 was implicated in the regulation of mitochondrial respiration. An MDM2 mutant harboring the C464A mutation that abolishes its E3 ligase activity (MDM2-C464A) was efficiently recruited to mitochondria and inhibited CI-driven respiration to an extent similar to that of FL-MDM2 following transient transfection in H1299 cells (Figures S4E and S4F). Inhibition of mitochondrial respiration was also observed in H1299 cells transduced with a lentivirus encoding MTS-MDM2 harboring the C464A mutation (MTS-MDM2-C464A) but not in cells expressing an MDM2 E3 ligase-deficient isoform that predominantly localized to chromatin due to the deletion of its central acidic domain (ΔAD-MDM2-C464A) (Riscal et al., 2016Riscal R. Schrepfer E. Arena G. Cissé M.Y. Bellvert F. Heuillet M. Rambow F. Bonneil E. Sabourdy F. Vincent C. et al.Chromatin-bound MDM2 regulates serine metabolism and redox homeostasis independently of p53.Mol. Cell. 2016; 62: 890-902Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar) (Figure S4G). Since abnormal ETC CI activity is often associated with oxidative stress, we then measured the effect of mtMDM2 on reactive oxygen species (ROS) levels by IF, using the MitoSox probe that specifically detects mitochondrial superoxide ions. H1299 cells expressing MTS-MDM2 exhibited increased mitochondrial ROS levels when compared to control cells (Figure 4F). Conversely, MCF7-sh p53 cells exhibited decreased ROS levels upon acute depletion of endogenous MDM2 (Figure 4G). Therefore, these data indicate that mtMDM2 interferes with ETC CI-driven respiration, leading to increased mitochondrial ROS production. Next, we explored the mechanism by which mtMDM2 controls ETC CI activity. The role of chromatin-bound MDM2 in the transcription of metabolic genes encoded by the nuclear genome (Riscal et al., 2016Riscal R. Schrepfer E. Arena G. Cissé M.Y. Bellvert F. Heuillet M. Rambow F. Bonneil E. Sabourdy F. Vincent C. et al.Chromatin-bound MDM2 regulates serine metabolism and redox homeostasis independently of p53.Mol. Cell. 2016; 62: 890-902Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar) prompted us to evaluate whether MDM2 also regulates transcription of the mitochondrial genome independently of p53. Supporting this hypothesis, quantitative chromatin immunoprecipitation (qChIP) assays showed that MTS-MDM2, but not MDM2 1-291, associated with the light strand promoter (LSP) that drives the transcription of the light strand of the mitochondrial genome (Figure 5A). No binding of MTS-MDM2 on the heavy strand promoter (HSP) was detected under the same experimental conditions (Figure S5A). Importantly, binding of endogenous MDM2 to the LSP promoter was also detected in parental but not in MDM2-depleted T47D cells (Figure 5B). Interestingly, these qChIP assays indicated that increased mtMDM2 levels correlated with decreased association of mitochondrial transcription factor A (TFAM) with the LSP, despite total TFAM protein levels remaining constant (Figures 5C and S5B). Consistent with the absence of MDM2 on the HSP, TFAM binding to HSP was not affected by MTS-MDM2 (Figure S5C). The impact of mtMDM2 on TFAM binding to the LSP was confirmed by mTRIP, a single-cell confocal imaging technique combining fluorescence in situ hybridization (FISH) performed with an LSP-specific probe with TFAM IF (Figures 5D and S5D) (Chatre and Ricchetti, 2013Chatre L. Ricchetti M. Large heterogeneity of mitochondrial DNA transcription and initiation of replication exposed by single-cell imaging.J. Cell Sci. 2013; 126: 914-926Crossref PubMed Scopus (30) Google Scholar). Moreover, atomic force microscopy (AFM) showed that purified recombinant MDM2 protein distributed preferentially to the control region of mtDNA containing the LSP but failed to bind to an irrelevant DNA probe in vitro (Figures 5E and S5E). To further assess the selective inhibitory activity of MDM2 at the LSP, we evaluated the ability of recombinant MDM2 to inhibit mitochondrial transcription in vitro using a dual-promoter template containing both LSP and HSP1 (Uchida et al., 2017Uchida A. Murugesapillai D. Kastner M. Wang Y. Lodeiro M.F. Prabhakar S. Oliver G.V. Arnold J.J. Maher L.J. Williams M.C. Cameron C.E. Unexpected sequences and structures of mtDNA required for efficient transcription from the first heavy-strand promoter.eLife. 2017; 6: e27283Crossref PubMed Scopus (19) Google Scholar). At TFAM concentrations in which both promoters were maximally activated, MDM2 reduced LSP transcription in a dose-dependent manner, whereas it had a much lesser impact on HSP-driven transcription (Figures 5F and S5F). Transcription from the LSP and HSP generates long polycistronic transcripts encoding rRNAs, tRNAs, and structural subunits of respiratory complexes. The LSP-driven transcript encodes only one structural component of the ETC, NADH-dehydrogenase 6 (MT-ND6), an important subunit of the ETC CI (Montoya et al., 1982Montoya J. Christianson T. Levens D. Rabinowitz M. Attardi G. Identification of initiation sites for heavy-strand and light-strand transcription in human mitochondrial DNA.Proc. Natl. Acad. Sci. USA. 1982; 79: 7195-7199Crossref PubMed Scopus (203) Google Scholar). RNA-FISH/mTRIP analyses showed that MT-ND6 RNA levels were lower in H1299 cells expressing MTS-MDM2 than in control cells (Figure 5G). Conversely, shRNA-mediated depletion of endogenous MDM2 resulted in increased MT-ND6 RNA levels (Figure 5H). qRT-PCR analyses showed that MDM2 depletion increased the RNA level of MT-ND6 in H1299 cells but not that of MT-ND2, MT-ND4, and MT-ND5 (Figures 5I and S5G). Moreover, expression of MTS-MDM2-C464A in MDM2-depleted cells still repressed MT-ND6 expression, confirming that the E3 ligase activity of MDM2 was dispensable for the transcriptional repression of MT-ND6 (Figure 5J). Consistent with that repressive function, increased MT-ND6 protein levels were detected both in MDM2-depleted H1299 cells and in Mdm2KO; p53KO MEFs, whereas expression of MTS-MDM2 in H1299 cells decreased MT-ND6 protein levels (Figures 5K, 5L, and S5H). Importantly, the specificity of MT-ND6 immunoblots was confirmed in cell lines harboring a frameshift mutation in MT-ND6 abolishing its expression (Figure S5I) (Perales-Clemente et al., 2010Perales-Clemente E. Fernández-Vizarra E. Acín-Pérez R. Movilla N. Bayona-Bafaluy M.P. Moreno-Loshuertos R. Pérez-Martos A. Fernández-Silva P. Enríquez J.A. Five entry points of the mitochondrially encoded subunits in mammalian complex I assembly.Mol. Cell. Biol. 2010; 30: 3038-3047Crossref PubMed Scopus (56) Google Scholar). These data demonstrate that mtMDM2 directly represses the expression of MT-ND6, a key subunit of ETC CI. While screening for MDM2 subcellular localization in H1299 cells exposed to different stress conditions, we found that oxidative stress led to the rapid accumulation of MDM2 in mitochondria. The 4-fold increase of endogenous mtMDM2 levels induced by menadione was abrogated by the addition of the ROS scavenger N-acetyl-cysteine (NAC). MtMDM2 levels also increased upon treatment with rotenone or the pyruvate kinase M2 inhibitor shikonin, likely reflecting the ability of these compounds to induce oxidative stress. In contrast, UV irradiation, the genotoxic agents SN38 and etoposide, or the endoplasmic reticulum (ER)-stress inducer thapsigargin, had no major effect on the amount of mtMDM2 (Figures S6A–S6C). Interestingly, shifting H1299 cells from a 21% O2 to a 1% O2 atmosphere, or treatment with the hypoxia-mimetic cobalt chloride (CoCl2), led to a 3-fold increase of mitochondrial, but not of total, MDM2 levels (Figures 6A and S6D). Co-immunoprecipitation experiments showed that hypoxia increased the binding of endogenous MDM2 to mtHsp70/mortalin and TID1, suggesting that it stimulated the active import of MDM2 into mitochondria (Figures 6B and S6E). Mild PK digestion of purified mitochondria prepared from hypoxic H1299 cells was sufficient to degrade the OMM protein TOM20, but not MDM2 or TFAM, supporting the notion that both basal and hypoxia-induced mtMDM2 are located in the mitochondrial matrix (Figure S6F). Moreover, qChIP assays indicated that hypoxia increased the recruitment of endogenous MDM2 to the LSP and significantly decreased TFAM association with the LSP (Figure 6C). Accumulation of endogenous MDM2 in mitochondria also occurred in NAC-treated H1299 cells cultured in low-oxygen conditions, indicating that increased recruitment of MDM2 into mitochondria during hypoxia was not an indirect effect of electron leakage through defective ETC that leads to superoxide production (Figure S6G). In line with our findings, increased levels of mtMDM2 during hypoxia or oxidative stress correlated with decreased MT-ND6 protein levels, and hypoxia-induced repression of MT-ND6 was abolished upon MDM2 depletion (Figures 6D and S6C). Importantly, MDM2 deficiency altered neither HIF1α stabilization nor the induction of its target genes Lactate dehydrogenase A (LDHA) and Pyruvate Dehydrogenase Kinase 1 (PDK1) during hypoxia, excluding the possibility that the impact of MDM2 depletion on MT-ND6 regulation was indirectly linked to alterations in HIF-mediated responses (Figures S6H and S6I). Consistent with a previous report, hypoxia induced a rapid perinuclear aggregation of mitochondria in H1299 cells (Al-Mehdi et al., 2012Al-Mehdi A.B. Pastukh V.M. Swiger B.M. Reed D.J. Patel M.R. Bardwell G.C. Pastukh V.V. Alexeyev M.F. Gillespie M.N. Perinuclear mitochondrial clustering creates an oxidant-rich nuclear domain required for hypoxia-induced transcription.Sci. Signal. 2012; 5: ra47Crossref PubMed Scopus (249) Google Scholar) that was similar to that observed upon expression of MTS-MDM2. Notably, shRNA-mediated depletion of endogenous MDM2 impaired this hypoxia-induced perinuclear clustering of mitochondria (Figure 6E). Thus, we conclude that in cancer cells, both ROS and hypoxia trigger MDM2 recruitment to mitochondria to repress expression of MT-ND6, control respiration, and regulate mitochondrial network dynamics. To investigate the relevance of MDM2-mediated control of the ETC CI activity in vivo, we first analyzed the impact of hypoxia in murine skeletal muscles. As soon as 3 hr after exposing WT C57BL/6 mice to an atmosphere composed of 15% O2, mtMDM2 levels in skeletal muscles increased, whereas those of MT-ND6 decreased (Figure 6F). Next, we evaluated the consequences of Mdm2 inactivation in striated muscle cells by crossing Mdm2 conditional knockout (Mdm2 flox) animals (Grier et al., 2002Grier J.D. Yan W. Lozano G. Conditional allele of mdm2 which encodes a p53 inhibitor.Genesis. 2002; 32: 145-147Crossref PubMed Scopus (39) Google Scholar) with Acta1-Cre transgenic (Acta) mice that express the Cre recombinase under the control of the skeletal α-actin promoter (Miniou et al., 1999Miniou P. Tiziano D. Frugier T. Roblot N. Le Meur M. Melki J. Gene targeting restricted to mouse striated muscle lineage.Nucleic Acids Res. 1999; 27: e27Crossref PubMed Scopus (155) Google Scholar). To confirm that the phenotypes resulting from Mdm2 inactivation in striated muscles were p53 independent, these animals were also crossed with p53 knockout (KO) mice. We then monitored Cre-driven deletion of the Mdm2flox allele in different tissues harvested from Mdm2flox/flox; ActaCreTg; p53−/− and Mdm2+/flox or +/+; ActaCreTg; p53−/− mice (hereinafter referred to as Mdm2KO(ACTA); p53KO and Mdm2CTR(ACTA); p53KO mice, respectively). In this mouse model, Mdm2 inactivation was restricted to striated skeletal muscles, as evidenced by the strong reduction of Mdm2 mRNA levels in the gastrocnemius, tibialis, and soleus but not in the heart, lungs, liver, and brain (Figure S6J). Mdm2 inactivation had no impact on the mRNA levels of the muscular differentiation markers Myf6, Mef2c, MyoD, an" @default.
• W2788097887 created "2018-03-06" @default.
• W2788097887 creator A5001881330 @default.
• W2788097887 creator A5008853880 @default.
• W2788097887 creator A5014535333 @default.
• W2788097887 creator A5021526153 @default.
• W2788097887 creator A5023743313 @default.
• W2788097887 creator A5025891501 @default.
• W2788097887 creator A5027051175 @default.
• W2788097887 creator A5028963650 @default.
• W2788097887 creator A5029966397 @default.
• W2788097887 creator A5030226248 @default.
• W2788097887 creator A5031909044 @default.
• W2788097887 creator A5034491707 @default.
• W2788097887 creator A5037188019 @default.
• W2788097887 creator A5047029179 @default.
• W2788097887 creator A5047733868 @default.
• W2788097887 creator A5051158107 @default.
• W2788097887 creator A5058903432 @default.
• W2788097887 creator A5059471594 @default.
• W2788097887 creator A5060768772 @default.
• W2788097887 creator A5061417948 @default.
• W2788097887 creator A5066063809 @default.
• W2788097887 creator A5073501871 @default.
• W2788097887 creator A5075495173 @default.
• W2788097887 creator A5079830304 @default.
• W2788097887 creator A5086096635 @default.
• W2788097887 creator A5090251960 @default.
• W2788097887 creator A5090782738 @default.
• W2788097887 date "2018-02-01" @default.
• W2788097887 modified "2023-10-17" @default.
• W2788097887 title "Mitochondrial MDM2 Regulates Respiratory Complex I Activity Independently of p53" @default.
• W2788097887 cites W1578847946 @default.
• W2788097887 cites W1965173366 @default.
• W2788097887 cites W1971008105 @default.
• W2788097887 cites W1976295398 @default.
• W2788097887 cites W1978668631 @default.
• W2788097887 cites W1987395344 @default.
• W2788097887 cites W1998974825 @default.
• W2788097887 cites W1999310200 @default.
• W2788097887 cites W1999752916 @default.
• W2788097887 cites W2008971389 @default.
• W2788097887 cites W2013939152 @default.
• W2788097887 cites W2020871623 @default.
• W2788097887 cites W2023699381 @default.
• W2788097887 cites W2037464535 @default.
• W2788097887 cites W2042000925 @default.
• W2788097887 cites W2042103669 @default.
• W2788097887 cites W2044079387 @default.
• W2788097887 cites W2054257071 @default.
• W2788097887 cites W2055917628 @default.
• W2788097887 cites W2070143299 @default.
• W2788097887 cites W2088740611 @default.
• W2788097887 cites W2093361250 @default.
• W2788097887 cites W2099907467 @default.
• W2788097887 cites W2103858001 @default.
• W2788097887 cites W2105618103 @default.
• W2788097887 cites W2109123740 @default.
• W2788097887 cites W2110048031 @default.
• W2788097887 cites W2110253595 @default.
• W2788097887 cites W2123603473 @default.
• W2788097887 cites W2135426193 @default.
• W2788097887 cites W2141333442 @default.
• W2788097887 cites W2147443365 @default.
• W2788097887 cites W2148332345 @default.
• W2788097887 cites W2157926893 @default.
• W2788097887 cites W2160345685 @default.
• W2788097887 cites W2195022070 @default.
• W2788097887 cites W2219655163 @default.
• W2788097887 cites W2410882188 @default.
• W2788097887 cites W2606610030 @default.
• W2788097887 cites W2609757284 @default.
• W2788097887 doi "https://doi.org/10.1016/j.molcel.2018.01.023" @default.
• W2788097887 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/8215449" @default.
• W2788097887 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/29452639" @default.
• W2788097887 hasPublicationYear "2018" @default.
• W2788097887 type Work @default.
• W2788097887 sameAs 2788097887 @default.
• W2788097887 citedByCount "56" @default.
• W2788097887 countsByYear W27880978872018 @default.
• W2788097887 countsByYear W27880978872019 @default.
• W2788097887 countsByYear W27880978872020 @default.
• W2788097887 countsByYear W27880978872021 @default.
• W2788097887 countsByYear W27880978872022 @default.
• W2788097887 countsByYear W27880978872023 @default.
• W2788097887 crossrefType "journal-article" @default.
• W2788097887 hasAuthorship W2788097887A5001881330 @default.
• W2788097887 hasAuthorship W2788097887A5008853880 @default.
• W2788097887 hasAuthorship W2788097887A5014535333 @default.
• W2788097887 hasAuthorship W2788097887A5021526153 @default.
• W2788097887 hasAuthorship W2788097887A5023743313 @default.
• W2788097887 hasAuthorship W2788097887A5025891501 @default.
• W2788097887 hasAuthorship W2788097887A5027051175 @default.
• W2788097887 hasAuthorship W2788097887A5028963650 @default.
• W2788097887 hasAuthorship W2788097887A5029966397 @default.
• W2788097887 hasAuthorship W2788097887A5030226248 @default.
• W2788097887 hasAuthorship W2788097887A5031909044 @default.
• W2788097887 hasAuthorship W2788097887A5034491707 @default.

• next