Mitochondrial respiration reduces exposure of the nucleus to oxygen
2023; Elsevier BV; Volume: 299; Issue: 3 Linguagem: Inglês
10.1016/j.jbc.2023.103018
ISSN1083-351X
AutoresMateus Prates Mori, Rozhin Penjweini, Jin Ma, Greg Alspaugh, Alessio Andreoni, Young‐Chae Kim, Pingyuan Wang, Jay R. Knutson, Paul M. Hwang,
Tópico(s)Genetics, Aging, and Longevity in Model Organisms
ResumoThe endosymbiotic theory posits that ancient eukaryotic cells engulfed O2-consuming prokaryotes, which protected them against O2 toxicity. Previous studies have shown that cells lacking cytochrome c oxidase (COX), required for respiration, have increased DNA damage and reduced proliferation, which could be improved by reducing O2 exposure. With recently developed fluorescence lifetime microscopy–based probes demonstrating that the mitochondrion has lower [O2] than the cytosol, we hypothesized that the perinuclear distribution of mitochondria in cells may create a barrier for O2 to access the nuclear core, potentially affecting cellular physiology and maintaining genomic integrity. To test this hypothesis, we utilized myoglobin-mCherry fluorescence lifetime microscopy O2 sensors without subcellular targeting ("cytosol") or with targeting to the mitochondrion or nucleus for measuring their localized O2 homeostasis. Our results showed that, similar to the mitochondria, the nuclear [O2] was reduced by ∼20 to 40% compared with the cytosol under imposed O2 levels of ∼0.5 to 18.6%. Pharmacologically inhibiting respiration increased nuclear O2 levels, and reconstituting O2 consumption by COX reversed this increase. Similarly, genetic disruption of respiration by deleting SCO2, a gene essential for COX assembly, or restoring COX activity in SCO2−/− cells by transducing with SCO2 cDNA replicated these changes in nuclear O2 levels. The results were further supported by the expression of genes known to be affected by cellular O2 availability. Our study reveals the potential for dynamic regulation of nuclear O2 levels by mitochondrial respiratory activity, which in turn could affect oxidative stress and cellular processes such as neurodegeneration and aging. The endosymbiotic theory posits that ancient eukaryotic cells engulfed O2-consuming prokaryotes, which protected them against O2 toxicity. Previous studies have shown that cells lacking cytochrome c oxidase (COX), required for respiration, have increased DNA damage and reduced proliferation, which could be improved by reducing O2 exposure. With recently developed fluorescence lifetime microscopy–based probes demonstrating that the mitochondrion has lower [O2] than the cytosol, we hypothesized that the perinuclear distribution of mitochondria in cells may create a barrier for O2 to access the nuclear core, potentially affecting cellular physiology and maintaining genomic integrity. To test this hypothesis, we utilized myoglobin-mCherry fluorescence lifetime microscopy O2 sensors without subcellular targeting ("cytosol") or with targeting to the mitochondrion or nucleus for measuring their localized O2 homeostasis. Our results showed that, similar to the mitochondria, the nuclear [O2] was reduced by ∼20 to 40% compared with the cytosol under imposed O2 levels of ∼0.5 to 18.6%. Pharmacologically inhibiting respiration increased nuclear O2 levels, and reconstituting O2 consumption by COX reversed this increase. Similarly, genetic disruption of respiration by deleting SCO2, a gene essential for COX assembly, or restoring COX activity in SCO2−/− cells by transducing with SCO2 cDNA replicated these changes in nuclear O2 levels. The results were further supported by the expression of genes known to be affected by cellular O2 availability. Our study reveals the potential for dynamic regulation of nuclear O2 levels by mitochondrial respiratory activity, which in turn could affect oxidative stress and cellular processes such as neurodegeneration and aging. Molecular oxygen (O2) has a dual nature. Its oxidative potential permits efficient aerobic metabolism for beneficial cellular energy production, whereas its unstable nature can result in the generation of toxic reactive oxygen species (ROS) that cause oxidative damage and serve as the basis for the free radical theory on aging (1Harman D. Aging: a theory based on free radical and radiation chemistry.J. Gerontol. 1956; 11: 298-300Crossref PubMed Scopus (6703) Google Scholar, 2McCord J.M. Fridovich I. Superoxide dismutase. An enzymic function for erythrocuprein (hemocuprein).J. Biol. Chem. 1969; 244: 6049-6055Abstract Full Text PDF PubMed Google Scholar, 3Wickens A.P. Ageing and the free radical theory.Respir. Physiol. 2001; 128: 379-391Crossref PubMed Scopus (417) Google Scholar). In developing a mechanistic explanation for this original theory of aging, mitochondria were proposed to be the major source of ROS responsible for oxidative damage, although ROS can also serve important signaling functions (4Harman D. The biologic clock: the mitochondria?.J. Am. Geriatr. Soc. 1972; 20: 145-147Crossref PubMed Scopus (1582) Google Scholar, 5Lennicke C. Cocheme H.M. Redox metabolism: ROS as specific molecular regulators of cell signaling and function.Mol. Cell. 2021; 81: 3691-3707Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). This theory, however, has been beset with some inconsistent results in models ranging from yeast to human, and the concept of the mitochondrion as the major source of ROS has been questioned (6Hekimi S. Lapointe J. Wen Y. Taking a "good" look at free radicals in the aging process.Trends Cell Biol. 2011; 21: 569-576Abstract Full Text Full Text PDF PubMed Scopus (452) Google Scholar, 7Brown G.C. Borutaite V. There is no evidence that mitochondria are the main source of reactive oxygen species in mammalian cells.Mitochondrion. 2012; 12: 1-4Crossref PubMed Scopus (212) Google Scholar, 8Zsurka G. Peeva V. Kotlyar A. Kunz W.S. Is there still any role for oxidative stress in mitochondrial DNA-dependent aging?.Genes (Basel). 2018; 9: 175Crossref PubMed Scopus (38) Google Scholar). Mitochondria have multiple sites from which ROS may be generated, but its production in vivo is dependent on a number of important factors, such as the substrate, mitochondrial membrane potential, matrix pH, and intracellular O2 availability (9Starkov A.A. The role of mitochondria in reactive oxygen species metabolism and signaling.Ann. N. Y Acad. Sci. 2008; 1147: 37-52Crossref PubMed Scopus (601) Google Scholar). These factors, which may be interdependent in vivo, are difficult to control under experimental conditions to permit an accurate assessment of in vivo mitochondrial ROS production. Even the increase in ROS production associated with hypoxia has been suggested to be an experimental phenomenon (9Starkov A.A. The role of mitochondria in reactive oxygen species metabolism and signaling.Ann. N. Y Acad. Sci. 2008; 1147: 37-52Crossref PubMed Scopus (601) Google Scholar). From another perspective, the mitochondria could have net antioxidant effects by consuming O2, the essential substrate for ROS production, and thereby preventing its genotoxicity (10Bruyninckx W.J. Mason H.S. Morse S.A. Are physiological oxygen concentrations mutagenic?.Nature. 1978; 274: 606-607Crossref PubMed Scopus (52) Google Scholar). We previously showed that disrupting SCO2 (SCO2−/−), a gene regulated by tumor suppressor p53 and essential for respiration, results in higher intracellular levels of O2, increased oxidative DNA damage, and decreased proliferation under normal ambient O2 levels (∼20% O2) (11Sung H.J. Ma W. Wang P.-y. Hynes J. O'Riordan T.C. Combs C.A. et al.Mitochondrial respiration protects against oxygen-associated DNA damage.Nat. Commun. 2010; 1: 1-8Crossref PubMed Scopus (95) Google Scholar). Exposing these nonrespiring SCO2−/− cells to lower O2 levels (≤5%) decreased DNA damage and improved their growth; but other factors such as their propensity for regenerating NAD+ as also contributing to this improvement cannot be ruled out (11Sung H.J. Ma W. Wang P.-y. Hynes J. O'Riordan T.C. Combs C.A. et al.Mitochondrial respiration protects against oxygen-associated DNA damage.Nat. Commun. 2010; 1: 1-8Crossref PubMed Scopus (95) Google Scholar). Cellular O2 is mostly consumed by mitochondria; therefore, their dysfunction could result in elevated tissue O2 levels. In accord with this notion, more recent studies have shown increased brain tissue O2 levels in a mouse model of mitochondrial disease, and the neurodegeneration and shortened life span of these mice were ameliorated by decreasing ambient O2 exposure (12Ferrari M. Jain I.H. Goldberger O. Rezoagli E. Thoonen R. Cheng K.H. et al.Hypoxia treatment reverses neurodegenerative disease in a mouse model of Leigh syndrome.Proc. Natl. Acad. Sci. U. S. A. 2017; 114: E4241-E4250Crossref PubMed Scopus (87) Google Scholar, 13Jain I.H. Zazzeron L. Goldberger O. Marutani E. Wojtkiewicz G.R. Ast T. et al.Leigh syndrome mouse model can Be rescued by Interventions that normalize brain hyperoxia, but not HIF activation.Cell Metab. 2019; 30: 824-832.e823Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). The development of various probes and techniques for measuring O2 levels in cultured cells and tissues have contributed to understanding O2 homeostasis under normal and abnormal conditions (14Dmitriev R.I. Papkovsky D.B. Optical probes and techniques for O2 measurement in live cells and tissue.Cell Mol. Life Sci. 2012; 69: 2025-2039Crossref PubMed Scopus (187) Google Scholar, 15Mirabello V. Cortezon-Tamarit F. Pascu S.I. Oxygen sensing, hypoxia tracing and in vivo imaging with functional metalloprobes for the early detection of non-communicable diseases.Front. Chem. 2018; 6: 27Crossref PubMed Scopus (29) Google Scholar, 16Kostyuk A.I. Kokova A.D. Podgorny O.V. Kelmanson I.V. Fetisova E.S. Belousov V.V. et al.Genetically encoded tools for research of cell signaling and metabolism under brain hypoxia.Antioxidants (Basel). 2020; 9: 516Crossref PubMed Scopus (7) Google Scholar, 17Mori M.P. Penjweini R. Knutson J.R. Wang P.Y. Hwang P.M. Mitochondria and oxygen homeostasis.FEBS J. 2021; 289: 6959-6968Crossref PubMed Scopus (10) Google Scholar). Previous O2 homeostasis studies suggested the existence of substantial intracellular gradients under some O2-limited conditions (18Jones D.P. Intracellular diffusion gradients of O2 and ATP.Am. J. Physiol. 1986; 250: C663-675Crossref PubMed Google Scholar). Consistent with the view of mitochondria as O2 sinks capable of creating intracellular O2 gradients (19Gnaiger E. Steinlechner-Maran R. Mendez G. Eberl T. Margreiter R. Control of mitochondrial and cellular respiration by oxygen.J. Bioenerg. Biomembr. 1995; 27: 583-596Crossref PubMed Scopus (255) Google Scholar, 20Wenger R.H. Mitochondria: oxygen sinks rather than sensors?.Med. Hypotheses. 2006; 66: 380-383Crossref PubMed Scopus (30) Google Scholar), recent advances in fluorescence lifetime microscopy (FLIM) probes have permitted the measurement of O2 concentration ([O2]) within the mitochondria and confirmed that it is lower relative to the rest of the cytosol (21Penjweini R. Andreoni A. Rosales T. Kim J. Brenner M.D. Sackett D.L. et al.Intracellular oxygen mapping using a myoglobin-mCherry probe with fluorescence lifetime imaging.J. Biomed. Opt. 2018; 23: 1-14PubMed Google Scholar). Notably, this phenomenon was observed to be dependent on active respiration. Because mitochondria are observed to have a perinuclear localization, we hypothesized that the nucleus would also have low [O2] relative to the cytosol, secondary to the hypoxic microenvironment of the surrounding mitochondria. In the current study, we report that the subcellular compartment of the nucleus is indeed capable of being maintained at lower O2 levels by mitochondrial respiration. This dependence of nuclear [O2] on the functional state of the mitochondria may have important implications, such as adding another dimension to the metabolic control of epigenetics or genomic stability in cancer. The red fluorescent protein mCherry was fused to the O2-binding protein myoglobin (MB) to create the O2 sensor MB-mCherry as previously described (21Penjweini R. Andreoni A. Rosales T. Kim J. Brenner M.D. Sackett D.L. et al.Intracellular oxygen mapping using a myoglobin-mCherry probe with fluorescence lifetime imaging.J. Biomed. Opt. 2018; 23: 1-14PubMed Google Scholar). The FRET-induced changes in the lifetime of MB-mCherry have been shown to be dependent on the O2-bound or O2-free (oxy/deoxy) state of MB and carefully validated as reflecting intracellular O2 levels (Fig. 1) (21Penjweini R. Andreoni A. Rosales T. Kim J. Brenner M.D. Sackett D.L. et al.Intracellular oxygen mapping using a myoglobin-mCherry probe with fluorescence lifetime imaging.J. Biomed. Opt. 2018; 23: 1-14PubMed Google Scholar, 22Penjweini R. Roarke B. Alspaugh G. Gevorgyan A. Andreoni A. Pasut A. et al.Single cell-based fluorescence lifetime imaging of intracellular oxygenation and metabolism.Redox Biol. 2020; 34101549Crossref PubMed Scopus (21) Google Scholar, 23Sedlack A.J.H. Penjweini R. Link K.A. Brown A. Kim J. Park S.J. et al.Computational modeling and imaging of the intracellular oxygen gradient.Int. J. Mol. Sci. 2022; 23: 12597Crossref PubMed Scopus (1) Google Scholar). To test the idea that the perinuclear localization of mitochondria may deplete nuclear O2 levels, we transiently transfected human HCT116 cells with MB-mCherry constructs targeted to the different subcellular compartments: cytosol, nontargeted MB-mCherry; mitochondrion, mtMB-mCherry; or nucleus, nMB-mCherry. Generally, these three constructs revealed mCherry red fluorescence signals in their respective subcellular compartments by confocal fluorescence microscopy (Fig. 2A).Figure 2Respiring mitochondria surround the nucleus and decrease nuclear O2 levels. The O2 sensor myoglobin (MB)-mCherry was expressed in different subcellular compartments of the HCT116 cell line by transfecting with nontargeted MB-mCherry or MB-mCherry fused to either the mitochondrial targeting sequence of TFAM (transcription factor A, mitochondrial; mtMB-mCherry, Mito) or SV40 nuclear localization signal (nMB-mCherry, Nuc). A, HCT116 cells were transfected with the three different MB-mCherry (red) constructs and stained with the mitochondrial dye MitoTracker Green (green) and nuclear DNA dye DAPI (blue) to confirm their specific subcellular localization. Note that the diffuse red fluorescence in the nuclei of nontargeted MB-mCherry-transfected cells does not result in significant purple color compared with that of nuclear nMB-mCherry when merged with the blue of nuclear Hoechst. Scale bar reprsents 5 μm. B, HCT116 cells were transfected with plasmids containing the indicated probes for 24 h, subcellularly fractionated to isolate the cytosolic and nuclear compartments, and the resulting samples were immunoblotted using anti-mCherry antibody. Tubulin and lamin serve as subcellular compartment markers for the cytosol and nucleus, respectively; all molecular weight markers indicated in kilodalton. C, the total cell lysates of HCT116 cells transfected with the indicated plasmids were immunoblotted using mCherry antibody. The predicted size of the nontargeted MB-mCherry construct is ∼44 kDa, but a smaller immunoreactive fragment at ∼15 kDa is evident. Note the presence of a nonspecific band at ∼48 kDa present in nontransfected control cells. All molecular weight markers are indicated in kilodalton. D, Z-stack images of a single cell transfected with nMB-mCherry and costained with MitoTracker Green from top (height ∼25 μm) to bottom (height ∼5 μm) of the well slide reveals a 3-dimensional shell of mitochondria around the nucleus. Scale bar represents 5 μm. E, representative pseudocolor FLIM images of HCT116 cells transfected with nontargeted MB-mCherry, mitochondrial mtMB-mCherry, and nuclear nMB-mCherry. The cells were incubated under different O2 concentrations ranging from 0.5% (∼2.8 mm Hg) to 18.6% (∼130 mm Hg) for lifetime measurements. Shorter lifetime values indicate lower [O2] (red), whereas longer lifetime indicates higher [O2] (green–blue). Note that for E and F, a threshold was introduced during the data analyses to remove the background noise. Dashed white lines have been placed around cells to indicate those with higher levels of probe expression and excluded background debris. Also, note that the FLIM SPCImage software does not provide scale bars. F, corresponding pseudo-color images of apparent cytosolic, mitochondrial, and nuclear [O2]. The lifetime value for each pixel in FLIM images shown in C and the calibration curve (obtained for nonrespiring rotenone/antimycin treated or SCO2−/− cells) were used to estimate apparent compartmental [O2]. The summary of data used in E and F is provided in Tables S1 and S2. In the color bars, red indicates lower values, whereas green–blue indicates higher values. Except at the lowest imposed [O2] (∼2.8 mm Hg), Mann–Whitney tests showed a significant difference between [O2] obtained for nontargeted versus mitochondria- or nuclear-localized probes. The differences between the mitochondrial and nuclear [O2] were not statistically significant (n = 33–53). Statistical difference by two-way ANOVA with Tukey's post-test. Comparison was performed between mean values of the different compartments. Values are mean ± SD. ∗p < 0.01. DAPI, 4′,6-diamidino-2-phenylindole; FLIM, fluorescence lifetime microscopy.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The nontargeted MB-mCherry showed a diffuse pattern of red fluorescence throughout the cell, which contrasted with the more prominent perinuclear appearance of the mitochondrial targeted mtMB-mCherry construct nontargeted versus mito (Fig. 2A). The mtMB-mCherry red colocalized with the mitochondrial-specific dye MitoTracker Green to produce yellow merge signals, whereas the nuclear nMB-mCherry colocalized with the blue Hoechst DNA dye to result in purple merge color (mito versus nuc, Fig. 2A). Notably, the nontargeted MB-mCherry-transfected cells displayed some red mCherry fluorescence in their nuclei although immunoblotting of their subcellular fractions using anti-mCherry antibody showed nontargeted probe only in the cytosolic fraction but not nuclear fraction (Fig. 2, A and B). On the other hand, nMB-mCherry-transfected cells showed mCherry immunoreactivity only in the nuclear fraction confirming the specific targeting of this probe (Fig. 2B). Further investigation of nontargeted MB-mCherry-transfected cells revealed the presence of two mCherry immunoreactive bands in their whole cell lysates, a ∼44 kDa band corresponding to the predicted size of the fusion protein and a smaller fragment at ∼15 kDa (Fig. 2C). The mCherry sequence containing fragment derived from the nontargeted MB-mCherry construct may be able to diffuse into the nuclear compartment of intact cells but wash out during subcellular fractionation, potentially explaining our current observation. However, the mCherry fragment is too small to contain MB; therefore, it would not result in O2-sensitive FLIM measurements. Two-dimensional fluorescence images of HCT116 cells transfected with nMB-mCherry and stained with MitoTracker Green revealed that the mitochondria are localized around the nucleus in a perinuclear pattern. Confocal fluorescence imaging using Z-stack technique in a single cell further revealed that the mitochondria form a 3-dimensional network encasing the nucleus, similar to a shell. This configuration may serve as a protective barrier for the nucleus by preventing exposure to high levels of oxygen (Fig. 2D). FLIM measurements of cells transfected with these three MB-mCherry O2 probes were performed under different imposed O2 levels to demonstrate the dependence of O2 on its availability and consumption by mitochondria. The lifetime values of MB-mCherry were examined for any refractive index (or possible pH) changes in the intracellular environment by transfecting with control sensors: nontargeted, mitochondrial-targeted, or nuclear-targeted mCherry (alone, without an O2-responsive MB component) (Fig. S1). Lifetime measurements of all three MB-containing probes showed the characteristic hyperbolic O2-binding curve of MB when the transfected cells were exposed to controlled ambient O2 levels in tissue culture slide wells from ∼0.5% to 18.6% (taking into account water vapor and 5% CO2), corresponding to media [O2] of ∼2.8 mm Hg to 130 mm Hg (Fig. 2E). The shorter lifetimes observed for the mitochondrial- and nuclear-targeted probes across the range of imposed [O2] were consistent with "cytosolic" [O2] being higher compared with that of the mitochondrial and nuclear compartments. The overlapping FLIM values for the mtMB-mCherry and nMB-mCherry probes indicated that the mitochondrion and nucleus have similarly low [O2] levels and supported an association between these two compartments, as initially hypothesized (Fig. 2E). The subcellular FLIM measurements were converted to apparent compartmental [O2] (mm Hg) by using OxyLite Pro 1 point measurements of [O2] in the chamber culture medium exposed to 5% CO2 and appropriate N2 to decrease O2 levels as previously described (21Penjweini R. Andreoni A. Rosales T. Kim J. Brenner M.D. Sackett D.L. et al.Intracellular oxygen mapping using a myoglobin-mCherry probe with fluorescence lifetime imaging.J. Biomed. Opt. 2018; 23: 1-14PubMed Google Scholar). Likely because of diffusion limitations of O2 above the cell layer, even within low confluency cell culture conditions (24Place T.L. Domann F.E. Case A.J. Limitations of oxygen delivery to cells in culture: an underappreciated problem in basic and translational research.Free Radic. Biol. Med. 2017; 113: 311-322Crossref PubMed Scopus (216) Google Scholar), the apparent [O2] levels of all three subcellular compartments were lower than that of the culture medium determined by the imposed ambient O2 level (Fig. 2F). To ensure that these findings were not limited to the HCT116 cell line, FLIM measurements were repeated in human embryonic kidney 293T (HEK293T) cells transfected with the three O2 sensor constructs, and similar results were observed (Fig. S2A). Taken together, these results supported the hypothesis that mitochondria act as O2 sinks within the cell, and that their perinuclear localization may secondarily result in the reduction of nuclear [O2]. We next tested whether mitochondrial respiratory activity can directly affect nuclear [O2] as previously shown for mitochondria (21Penjweini R. Andreoni A. Rosales T. Kim J. Brenner M.D. Sackett D.L. et al.Intracellular oxygen mapping using a myoglobin-mCherry probe with fluorescence lifetime imaging.J. Biomed. Opt. 2018; 23: 1-14PubMed Google Scholar). The pharmacologic inhibition of mitochondrial respiratory complex I by rotenone (Rot) or complex III by myxothiazol (Myxo) resulted in increased subcellular [O2] both in the mitochondrial and nuclear compartments of HCT116 and HEK293T cells (Figs. 3A and S2B). Notably, there was also a pattern of significantly increased [O2] even in the cytosol, consistent with a previous study utilizing a nontargeted phosphorescent O2 probe that showed intracellular [O2] dependence on respiration (Fig. 3A) (11Sung H.J. Ma W. Wang P.-y. Hynes J. O'Riordan T.C. Combs C.A. et al.Mitochondrial respiration protects against oxygen-associated DNA damage.Nat. Commun. 2010; 1: 1-8Crossref PubMed Scopus (95) Google Scholar). Because cytochrome c oxidase (COX) (complex IV) is the major site of O2 consumption, we predicted that reconstituting its activity even with inhibition of upstream electron transfer would prevent the rise in mitochondrial and nuclear O2 levels. Indeed, in the presence of Myxo, directly feeding electrons to COX by using the reductant ascorbate (Asc) and redox mediator N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) resulted in decreased mitochondrial and nuclear O2 levels (Fig. 3B). In contrast to the observation with respiratory inhibitors, stimulating mitochondrial O2 consumption with the uncoupling agent 2,4-dinitrophenol resulted in a further lowering of [O2] in all three subcellular compartments as expected in our proposed model (Fig. 3C). To further confirm the effects of these pharmacologic agents on mitochondrial and nuclear [O2], we utilized a nonrespiring HCT116 cell line that had been created previously by homozygous disruption of SCO2 (SCO2−/−), a metallochaperone gene involved in cellular copper homeostasis and essential for COX assembly (25Leary S.C. Cobine P.A. Kaufman B.A. Guercin G.H. Mattman A. Palaty J. et al.The human cytochrome c oxidase assembly factors SCO1 and SCO2 have regulatory roles in the maintenance of cellular copper homeostasis.Cell Metab. 2007; 5: 9-20Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar, 26Matsumoto T. Wang P.Y. Ma W. Sung H.J. Matoba S. Hwang P.M. Polo-like kinases mediate cell survival in mitochondrial dysfunction.Proc. Natl. Acad. Sci. U. S. A. 2009; 106: 14542-14546Crossref PubMed Scopus (53) Google Scholar, 27Pacheu-Grau D. Bareth B. Dudek J. Juris L. Vogtle F.N. Wissel M. et al.Cooperation between COA6 and SCO2 in COX2 maturation during cytochrome c oxidase assembly links two mitochondrial cardiomyopathies.Cell Metab. 2015; 21: 823-833Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Measurements of O2 consumption using mitochondrial inhibitors and uncoupler carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) revealed the complete absence of respiration in SCO2−/− cells in comparison with isogenic wildtype cells (Fig. 4A). Furthermore, the absence of O2 consumption upon treatment with TMPD and Asc was consistent with the essential role that SCO2 plays in the assembly and stabilization of the COX complex, whereas the reintroduction of SCO2 complementary DNA (cDNA) (transgene, Tg) into SCO2−/− cells (SCO2−/− Tg) rescued COX3 subunit protein levels, respiratory activity, and the effects of TMPD/Asc on O2 consumption (Fig. 4, A and B). Importantly, the genetic disruption of respiration in SCO2−/− cells increased [O2] levels in both the mitochondrial and nuclear compartments, whereas the respiration-rescued SCO2−/− Tg cells showed reversal of these changes (Fig. 4C). These observations confirmed the specificity of the pharmacologic inhibitors of mitochondrial respiration used in our current and previous FLIM O2 studies (21Penjweini R. Andreoni A. Rosales T. Kim J. Brenner M.D. Sackett D.L. et al.Intracellular oxygen mapping using a myoglobin-mCherry probe with fluorescence lifetime imaging.J. Biomed. Opt. 2018; 23: 1-14PubMed Google Scholar). The protein levels of nuclear transcription factor hypoxia-inducible factor 1-alpha (HIF-1α) are sensitive to intracellular [O2] as its hydroxylation by HIF prolyl hydroxylase for subsequent degradation requires O2 as substrate. Because inhibiting respiration increases intracellular O2 levels, we examined whether the levels of HIF-1α could serve as a sensitive biologic read out of changes in [O2]. Treating wildtype HCT116 cells with mitochondrial inhibitors targeting the different respiratory complexes all prevented the stabilization of HIF-1α under both 5% and 0.5% O2, which normally increase its levels, suggesting a state of increased intracellular O2 availability confirmed by our FLIM measurements (Fig. 5A). Bypassing the electron transfer blockade at complex III (Myxo or antimycin A) using TMPD/Asc to resume O2 consumption resulted in recovery of HIF-1α and HIF-2α stabilization, whereas stimulating O2 consumption with the uncoupling agent FCCP further increased their levels compared with controls (Fig. 5, B and C). Thus, these observations demonstrated that mitochondrial O2 consumption may contribute to the stabilization of HIF-1α by decreasing intracellular [O2]. Consistent with this mechanism, there was mild stabilization of HIF-1α only at the most hypoxic imposed [O2] (0.5% O2) in nonrespiring SCO2−/− cells, further supporting an important role for mitochondrial respiration in O2 homeostasis (Fig. 5, C and D). We also confirmed that the diminished stabilization of HIF-1α in SCO2−/− cells by hypoxia was not because of a defect in HIF or its intrinsic regulation because treatment with CoCl2, a direct inactivator of HIF prolyl hydroxylase, stabilized HIF-1α to levels identical to that in wildtype cells (Fig. 5D). Changes in intracellular O2 levels may impact gene expression regulated not only by HIF transcription factors but also by chromatin modifications such as histone methylations that induce or silence genes. Among the post-translational modifications of histones, lysine (K) methylation in particular has been shown to be sensitive to the cellular levels of O2 (28Shmakova A. Batie M. Druker J. Rocha S. Chromatin and oxygen sensing in the context of JmjC histone demethylases.Biochem. J. 2014; 462: 385-395Crossref PubMed Scopus (75) Google Scholar). Methylation of histone H3 at amino acid residues K4, K9, and K27 has been shown to be increased under hypoxia, possibly because of limited O2, a substrate required for demethylation by histone lysine demethylases (29Lee S. Lee J. Chae S. Moon Y. Lee H.Y. Park B. et al.Multi-dimensional histone methylations for coordinated regulation of gene expression under hypoxia.Nucl. Acids Res. 2017; 45: 11643-11657Crossref PubMed Scopus (18) Google Scholar, 30Batie M. Frost J. Frost M. Wilson J.W. Schofield P. Rocha S. Hypoxia induces rapid changes to histone methylation and reprograms chromatin.Science. 2019; 363: 1222-1226Crossref PubMed Scopus (187) Google Scholar, 31Chakraborty A.A. Laukka T. Myllykoski M. Ringel A.E. Booker M.A. Tolstorukov M.Y. et al.Histone demethylase KDM6A directly senses oxygen to control chromatin and cell fate.Science. 2019; 363: 1217-1222Crossref PubMed Scopus (211) Google Scholar). Therefore, as a biologic read out of intracellular O2 levels to complement the FLIM probe measurements and a simple demonstration of respiration-driven alterations in [O2], we examined the expression of genes known to be sensitive to O2 leve
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