RESUMO
Dyslipidemia is a well-established condition proved to accelerate the progression of chronic kidney disease leading to tubulo-interstitial injury. However, the molecular aspects of the dyslipidemia-induced renal damage have not been fully clarified and in particular the role played by low-density lipoproteins (LDLs). This study aimed to examine the effects of native non-oxidized LDL on cellular oxidative metabolism in cultured human proximal tubular cells. By means of confocal microscopy imaging combined to respirometric and enzymatic assays it is shown that purified native LDL caused a marked increase of cellular reactive oxygen species (ROS) production, which was mediated by activation of NADPH oxidase(s) and by mitochondrial dysfunction by means of a ROS-induced ROS release mechanism. The LDL-dependent mitochondrial alterations comprised inhibition of the respiratory chain activity, enhanced ROS production, uncoupling of the oxidative phosphorylation efficiency, collapse of the mtΔΨ, increased Ca(2+) uptake and loss of cytochrome c. All the above LDL-induced effects were completely abrogated by chelating extracellular Ca(2+) as well as by inhibition of the Ca(2+) -activated cytoplasmic phospholipase A2, NADPH oxidase and mitochondrial permeability transition. We propose a mechanicistic model whereby the LDL-induced intracellular redox unbalance is triggered by a Ca(2+) inward flux-dependent commencement of cPLA2 followed by activation of a lipid- and ROS-based cross-talking signalling pathway. This involves first oxidants production via the plasmamembrane NADPH oxidase and then propagates downstream to mitochondria eliciting redox- and Ca(2+) -dependent dysfunctions leading to cell-harming conditions. These findings may help to clarify the mechanism of dyslipidemia-induced renal damage and suggest new potential targets for specific therapeutic strategies to prevent oxidative stress implicated in kidney diseases.
Assuntos
Túbulos Renais Proximais/metabolismo , Lipoproteínas LDL/metabolismo , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Cálcio/metabolismo , Sinalização do Cálcio , Linhagem Celular , Citocromos c/metabolismo , Dislipidemias/metabolismo , Humanos , Mitocôndrias/patologia , NADPH Oxidases/antagonistas & inibidores , NADPH Oxidases/biossíntese , NADPH Oxidases/metabolismo , Inibidores de Fosfolipase A2 , Fosfolipases A2/metabolismo , Transdução de SinaisRESUMO
Hepatitis C virus (HCV) infection induces a state of oxidative stress by affecting mitochondrial-respiratory-chain activity. By using cell lines inducibly expressing different HCV constructs, we showed previously that viral-protein expression leads to severe impairment of mitochondrial oxidative phosphorylation and to major reliance on nonoxidative glucose metabolism. However, the bioenergetic competence of the induced cells was not compromised, indicating an efficient prosurvival adaptive response. Here, we show that HCV protein expression activates hypoxia-inducible factor 1 (HIF-1) by normoxic stabilization of its alpha subunit. In consequence, expression of HIF-controlled genes, including those coding for glycolytic enzymes, was significantly upregulated. Similar expression of HIF-controlled genes was observed in cell lines inducibly expressing subgenomic HCV constructs encoding either structural or nonstructural viral proteins. Stabilization and transcriptional activation of HIF-1alpha was confirmed in Huh-7.5 cells harboring cell culture-derived infectious HCV and in liver biopsy specimens from patients with chronic hepatitis C. The HCV-related HIF-1alpha stabilization was insensitive to antioxidant treatment. Mimicking an impairment of mitochondrial oxidative phosphorylation by treatment of inducible cell lines with oligomycin resulted in stabilization of HIF-1alpha. Similar results were obtained by treatment with pyruvate, indicating that accumulation of intermediate metabolites is sufficient to stabilize HIF-1alpha. These observations provide new insights into the pathogenesis of chronic hepatitis C and, possibly, the HCV-related development of hepatocellular carcinoma.
Assuntos
Adaptação Fisiológica , Glicólise , Hepacivirus/patogenicidade , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Mitocôndrias/virologia , Antioxidantes/farmacologia , Linhagem Celular Tumoral , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/análise , Fígado/metabolismo , Mitocôndrias/metabolismo , Fosforilação Oxidativa , Estabilidade Proteica , Proteínas ViraisRESUMO
Retroviral vectors are used in human gene therapy trials to stably introduce therapeutic genes in the genome of patients' cells. Their applicability, however, is frustrated by the limited viability of transformed cells and/or by risks linked to selection of oncogene-mutated clones. The reasons for these drawbacks are not yet completely understood. In this study, we show that LXSN-NeoR gene/interleukin-7-engineered mesenchymal stromal cells exhibited a marked enhancement of reactive oxygen species production compared with untransfected cells. This effect resulted to be independent on the product of the gene carried by the retroviral vehicle as it was reproducible in cells transfected with the empty vector alone. Stable transfection of mesenchymal stromal cells with the different retroviral vectors pBabe-puro and PINCO-puro and the lentiviral vector pSico PGK-puro caused similar redox imbalance, unveiling a phenomenon of more general impact. The enhanced production of reactive oxygen species over the basal level was attributable to mitochondrial dysfunction and brought back to altered activity of the NADH-CoQ oxidoreductase (complex I) of the respiratory chain. The oxidative stress in transfected mesenchymal stem cells was completely reversed by treatment with a cAMP analog, thus pointing to alteration in the protein kinase A-dependent signaling pathway of the host cell. Transfection of mesenchymal stromal cells with a PINCO-parental vector harboring the green fluorescent protein gene as selection marker in place of the puromycin-resistance gene resulted in no alteration of the redox phenotype. These novel findings provide insights and caveats to the applicability of cell- or gene-based therapies and indicate possible intervention to improve them. Disclosure of potential conflicts of interest is found at the end of this article.
Assuntos
Células da Medula Óssea/citologia , AMP Cíclico/metabolismo , AMP Cíclico/fisiologia , Mitocôndrias/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Linhagem Celular , Respiração Celular/fisiologia , Transformação Celular Viral , Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , Complexo I de Transporte de Elétrons/antagonistas & inibidores , Complexo I de Transporte de Elétrons/fisiologia , Humanos , Interleucina-7/metabolismo , Lentivirus/genética , Mesoderma/citologia , Vírus da Leucemia Murina de Moloney/genética , Oxirredução , Células Estromais/citologia , Transdução GenéticaRESUMO
In the present study mitochondrial respiratory function of fibroblasts from a patient affected by early-onset parkinsonism carrying the homozygous W437X nonsense mutation in the PINK1 gene has been thoroughly characterized. When compared with normal fibroblasts, the patient's fibroblast mitochondria exhibited a lower respiratory activity and a decreased respiratory control ratio with cellular ATP supply relying mainly on enhanced glycolytic production. The quantity, specific activity and subunit pattern of the oxidative phosphorylation complexes were normal. However, a significant decrease of the cellular cytochrome c content was observed and this correlated with a reduced cytochrome c oxidase in situ-activity. Measurement of ROS revealed in mitochondria of the patient's fibroblasts enhanced O(2)(*-) and H(2)O(2) production abrogated by inhibition of complex I. No change in the glutathione-based redox buffering was, however, observed.
Assuntos
Mitocôndrias/fisiologia , Mutação , Doença de Parkinson/genética , Proteínas Quinases/genética , Trifosfato de Adenosina/metabolismo , Adulto , Células Cultivadas , Citocromos c/metabolismo , Glutationa/metabolismo , Humanos , Imuno-Histoquímica , Microscopia Confocal , Mitocôndrias/enzimologia , Mitocôndrias/metabolismo , Fosforilação Oxidativa , Doença de Parkinson/metabolismo , Doença de Parkinson/fisiopatologia , Espécies Reativas de Oxigênio/metabolismo , Frações Subcelulares/metabolismoRESUMO
UNLABELLED: Hepatitis C virus (HCV) infection induces a state of oxidative stress that is more pronounced than that in many other inflammatory diseases. In this study we used well-characterized cell lines inducibly expressing the entire HCV open-reading frame to investigate the impact of viral protein expression on cell bioenergetics. It was shown that HCV protein expression has a profound effect on cell oxidative metabolism, with specific inhibition of complex I activity, depression of mitochondrial membrane potential and oxidative phosphorylation coupling efficiency, increased production of reactive oxygen and nitrogen species, as well as loss of the Pasteur effect. Importantly, all these effects were causally related to mitochondrial calcium overload, as inhibition of mitochondrial calcium uptake completely reversed the observed bioenergetic alterations. CONCLUSION: Expression of HCV proteins causes deregulation of mitochondrial calcium homeostasis. This event occurs upstream of further mitochondrial dysfunction, leading to alterations in the bioenergetic balance and nitro-oxidative stress. These observations provide new insights into the pathogenesis of hepatitis C and may offer new opportunities for therapeutic intervention.
Assuntos
Cálcio/fisiologia , Metabolismo Energético/fisiologia , Hepacivirus/genética , Mitocôndrias Hepáticas/metabolismo , Estresse Oxidativo/fisiologia , Proteínas Virais/genética , Trifosfato de Adenosina/metabolismo , Neoplasias Ósseas , Linhagem Celular Tumoral , Humanos , Fases de Leitura Aberta , Osteossarcoma , Fosforilação Oxidativa , Consumo de Oxigênio , Espécies Reativas de Oxigênio/metabolismoRESUMO
The hypoxia-inducible factor (HIF) transcriptional system enables cell adaptation to limited O(2) availability, transducing this signal into patho-physiological responses such as angiogenesis, erythropoiesis, vasomotor control, and altered energy metabolism, as well as cell survival decisions. However, other factors beyond hypoxia are known to activate this pleiotropic transcription factor. The aim of this study was to characterize HIF in human hematopoietic stem cells (HSCs) and evidence is provided that granulocyte colony stimulating factor-mobilized CD34+- and CD133+-HSCs express a stabilized cytoplasmic form of HIF-1alpha under normoxic conditions. It is shown that HIF-1alpha stabilization correlates with down-regulation of the tumour suppressor von Hippel-Lindau protein (pVHL) and is positively controlled by NADPH-oxidase-dependent production of reactive oxygen species, indicating a specific O(2)-independent post-transcriptional control of HIF in mobilized HSCs. This novel finding is discussed in the context of the proposed role of HIF as a mediator of progenitor cell recruitment to injured ischemic tissues and/or in the control of the maintenance of the undifferentiated state.
Assuntos
Células-Tronco Hematopoéticas/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Consumo de Oxigênio/fisiologia , Antígenos CD34/metabolismo , Circulação Sanguínea , Hipóxia Celular , Células Cultivadas , Células-Tronco Hematopoéticas/efeitos dos fármacos , Humanos , Modelos Biológicos , NADPH Oxidases/metabolismo , Processamento de Proteína Pós-Traducional , Espécies Reativas de Oxigênio/metabolismo , Espécies Reativas de Oxigênio/farmacologia , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismoRESUMO
Consolidated evidence highlights the importance of redox signalling in poising the balance between self-renewal and differentiation in adult stem cells. The present study shows that human hematopoietic stem/progenitor cells (HSCs) constitutively generate low levels of hydrogen peroxide whose production is inhibited by DPI, apocynin, catalase, and LY294002 and scarcely stimulated by PMA. Moreover, it is shown that HSCs express at the mRNA and protein levels the catalytic subunits of NOX1, NOX2, and NOX4 isoforms of the NADPH oxidase family along with the complete battery of the regulatory subunits p22, p40, p47, p67, rac1, rac2, NOXO1, and NOXA1 as well as the splicing variant NOX2s and that the three NOX isoforms are largely co-expressed in the same HSC. These findings are interpreted in terms of a positive feed-back mechanism of NOXs activation enabling a fine tuning of the ROS level to be possibly used in redox-mediated signalling for growth and differentiation of HSCs.
Assuntos
Células da Medula Óssea/metabolismo , Células-Tronco Hematopoéticas/metabolismo , NADPH Oxidases/genética , Espécies Reativas de Oxigênio/metabolismo , Acetofenonas/farmacologia , Antígenos CD34/análise , Western Blotting , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Catalase/genética , Catalase/metabolismo , Cromonas/farmacologia , Inibidores Enzimáticos/farmacologia , Citometria de Fluxo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Glutationa Peroxidase/genética , Glutationa Peroxidase/metabolismo , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Humanos , Peróxido de Hidrogênio/metabolismo , Imuno-Histoquímica , Isoenzimas/genética , Isoenzimas/metabolismo , Microscopia Confocal , Modelos Biológicos , Morfolinas/farmacologia , NADPH Oxidases/metabolismo , Oniocompostos/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Glutationa Peroxidase GPX1RESUMO
This mini-review summarizes evidence, provided by our group, relevant to the understanding of how redox signalling may control the fate of adult hematopoietic stem/progenitor cells (HSPCs). In particular it is shown that bone marrow-derived human HSPC are endowed with a composite panel of constitutively active NADPH-oxidases (NOXs) comprising the cell membrane-localized catalytic subunits of the NOX1, NOX2 and NOX4 isoforms. It is proposed that the coordinated activity of the NOX isoforms in HSPCs function as environmental oxygen sensor and generate low level of ROS, which likely serve as second messengers. The pro-oxidant setting, entering into play when HSPCs leave the hypoxic bone marrow niche, would enable them to be more responsive to proliferative/differentiative stimuli. Moreover it is suggested that enhanced ROS elicit mitochondrial "differentiation" in a pre-commitment phase needed to match the bioenergetic request in the oncoming proliferation/differentiation process.
Assuntos
Células-Tronco Adultas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Células-Tronco Adultas/citologia , Células-Tronco Adultas/enzimologia , Proliferação de Células , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/enzimologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Mitocôndrias/metabolismo , Modelos Biológicos , NADPH Oxidases/metabolismo , OxirreduçãoRESUMO
The impact of cAMP on ROS-balance in human and mammalian cell cultures was studied. cAMP reduced accumulation of ROS induced by serum-limitation, under conditions in which there was no significant change in the activity of scavenger systems. This effect was associated with cAMP-dependent activation of the NADH-ubiquinone oxidoreductase activity of complex I. In fibroblasts from a patient a genetic defect in the 75 kDa FeS-protein subunit of complex I resulted in inhibition of the activity of the complex and enhanced ROS production, which were reversed by cAMP. A missense genetic defect in the NDUFS4 subunit, putative substrate of PKA, suppressed, on the other hand, the activity of the complex and prevented ROS production.
Assuntos
AMP Cíclico/fisiologia , Mitocôndrias/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Animais , Linhagem Celular , Meios de Cultura Livres de Soro , Complexo I de Transporte de Elétrons/metabolismo , Sequestradores de Radicais Livres/metabolismo , Glutationa/metabolismo , Humanos , Camundongos , Superóxido Dismutase/metabolismoRESUMO
Recent measurements of the flux control exerted by cytochrome c oxidase on the respiratory activity in intact cells have led to a re-appraisal of its regulatory function. We have further extended this in vivo study in the framework of the Metabolic Control Analysis and evaluated the impact of the mitochondrial transmembrane electrochemical potential (Deltamu(H+)) on the control strength of the oxidase. The results indicate that, under conditions mimicking the mitochondrial State 4 of respiration, both the flux control coefficient and the threshold value of cytochrome oxidase are modified with respect to the uncoupled condition. The results obtained are consistent with a model based on changes in the assembly state of the oxidative phosphorylation enzyme complexes and possible implications in the understanding of exercise-intolerance of human neuromuscular degenerative diseases are discussed.
Assuntos
Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Metabolismo Energético/fisiologia , Mitocôndrias/fisiologia , Fosforilação Oxidativa/efeitos dos fármacos , Ácido Ascórbico/metabolismo , Linhagem Celular Tumoral , Humanos , Cinética , Potenciais da Membrana/fisiologia , Membranas Mitocondriais/fisiologia , Oligomicinas/farmacologia , Consumo de Oxigênio , Tetrametilfenilenodiamina/farmacologia , Valinomicina/farmacologiaRESUMO
This study was aimed to characterize the mitochondrial and extra-mitochondrial oxygen consuming reactions in human CD34+ hematopoietic stem cells. Cell samples were collected by apheresis following pre-conditioning by granulocyte colony-stimulating factor and isolated by anti-CD34 positive immunoselection. Polarographic analysis of the CN-sensitive endogenous cell respiration revealed a low mitochondrial oxygen consumption rate. Differential absorbance spectrometry on whole cell lysate and two-dimensional blue native-PAGE analysis of mitoplast proteins confirmed a low amount of mitochondrial respiratory chain complexes thus qualifying the hematopoietic stem cell as a poor oxidative phosphorylating cell type. Confocal microscopy imaging showed, however, that the intracellular content of mitochondria was not homogeneously distributed in the CD34+ hematopoietic stem cell sample displaying a clear inverse correlation of their density with the expression of the CD34 commitment marker. About half of the endogenous oxygen consumption was extra-mitochondrial and completely inhibitable by enzymatic scavengers of reactive oxygen species and by diphenylene iodinium. By spectral analysis, flow cytometry, reverse transcriptase-PCR, immunocytochemistry, and immunoprecipitation it was shown that the extra-mitochondrial oxygen consumption was contributed by the NOX2 and NOX4 isoforms of the O2-*. producer plasma membrane NAD(P)H oxidase with low constitutive activity. A model is proposed suggesting for the NAD(P)H oxidase a role of O2 sensor and/or ROS source serving as redox messengers in the activation of intracellular signaling pathways leading (or contributing) to mitochondriogenesis, cell survival, and differentiation in hematopoietic stem cells.
Assuntos
Células-Tronco Hematopoéticas/citologia , Mitocôndrias/metabolismo , Oxigênio/metabolismo , Antígenos CD34/biossíntese , Linhagem Celular , Células Cultivadas , Transporte de Elétrons , Eletroforese em Gel Bidimensional , Eletroforese em Gel de Poliacrilamida , Citometria de Fluxo , Fator Estimulador de Colônias de Granulócitos e Macrófagos , Humanos , Processamento de Imagem Assistida por Computador , Immunoblotting , Imuno-Histoquímica , Imunoprecipitação , Microscopia Confocal , Microscopia de Fluorescência , Modelos Biológicos , NADPH Oxidases/metabolismo , Oxirredução , Oxigênio/química , Consumo de Oxigênio , Fosforilação , Ligação Proteica , Estrutura Quaternária de Proteína , Espécies Reativas de Oxigênio , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , EspectrofotometriaRESUMO
A study is presented on proton transfer associated with the reaction of the fully reduced, purified bovine heart cytochrome c oxidase with molecular oxygen or ferricyanide. The proton consumption associated with aerobic oxidation of the four metal centers changed significantly with pH going from approximately 3.0 H(+)/COX at pH 6.2-6.3 to approximately 1.2 H(+)/COX at pH 8.0-8.5. Rereduction of the metal centers was associated with further proton uptake which increased with pH from approximately 1.0 H(+)/COX at pH 6.2-6.3 to approximately 2.8 H(+)/COX at pH 8.0-8.5. Anaerobic oxidation of the four metal centers by ferricyanide resulted in the net release of 1.3-1.6 H(+)/COX in the pH range 6.2-8.2, which were taken up by the enzyme on rereduction of the metal centers. The proton transfer elicited by ferricyanide represents the net result of deprotonation/protonation reactions linked to anaerobic oxidoreduction of the metal centers. Correction for the ferricyanide-induced pH changes of the proton uptake observed in the oxidation and rereduction phase of the reaction of the reduced oxidase with oxygen gave a measure of the proton consumption in the reduction of O(2) to 2H(2)O. The results show that the expected stoichiometric proton consumption of 4H(+) in the reduction of O(2) to 2H(2)O is differently associated, depending on the actual pH, with the oxidation and reduction phase of COX. Two H(+)/COX are initially taken up in the reduction of O(2) to two OH(-) groups bound to the binuclear Fe a(3)-Cu(B) center. At acidic pHs the third and fourth protons are also taken up in the oxidative phase with formation of 2H(2)O. At alkaline pHs the third and fourth protons are taken up with formation of 2H(2)O only upon rereduction of COX.