RESUMO
The mucosa of the small intestine is renewed completely every 3-5 d throughout the entire lifetime by small populations of adult stem cells that are believed to reside in the bottom of the crypts and to migrate and differentiate into all the different populations of intestinal cells. When the cells reach the apex of the villi and are fully differentiated, they undergo cell death and are shed into the lumen. Reactive oxygen species (ROS) production is proportional to the electron transfer activity of the mitochondrial respiration chain. ROS homeostasis is maintained to control cell death and is finely tuned by an inducible antioxidant program. Here we show that peroxisome proliferator-activated receptor-γ coactivator-1ß (PGC-1ß) is highly expressed in the intestinal epithelium and possesses dual activity, stimulating mitochondrial biogenesis and oxygen consumption while inducing antioxidant enzymes. To study the role of PGC-1ß gain and loss of function in the gut, we generated both intestinal-specific PGC-1ß transgenic and PGC-1ß knockout mice. Mice overexpressing PGC-1ß present a peculiar intestinal morphology with very long villi resulting from increased enterocyte lifespan and also demonstrate greater tumor susceptibility, with increased tumor number and size when exposed to carcinogens. PGC-1ß knockout mice are protected from carcinogenesis. We show that PGC-1ß triggers mitochondrial respiration while protecting enterocytes from ROS-driven macromolecule damage and consequent apoptosis in both normal and dysplastic mucosa. Therefore, PGC-1ß in the gut acts as an adaptive self-point regulator, capable of providing a balance between enhanced mitochondrial activity and protection from increased ROS production.
Assuntos
Neoplasias do Colo/patologia , Enterócitos/citologia , Mucosa Intestinal/patologia , Intestino Delgado/patologia , Fatores de Transcrição/metabolismo , Animais , Antioxidantes/metabolismo , Apoptose , Carcinogênese , Transporte de Elétrons , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Homeostase , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/metabolismo , Oxigênio/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Espécies Reativas de Oxigênio/metabolismo , Fatores de Transcrição/genéticaRESUMO
Development of hepatic steatosis and its progression to steatohepatitis may be the consequence of dysfunction of several metabolic pathways, such as triglyceride synthesis, very low-density lipoprotein (VLDL) secretion, and fatty acid ß-oxidation. Peroxisome proliferator-activated receptor γ coactivator-1ß (PGC-1ß) is a master regulator of mitochondrial biogenesis and oxidative metabolism, lipogenesis, and triglyceride (TG) secretion. Here we generated a novel mouse model with constitutive hepatic activation of PGC-1ß and studied the role of this transcriptional coactivator in dietary-induced steatosis and steatohepatitis. Selective activation of PGC-1ß within hepatocytes is able to protect the liver from lipid overload and from progression to fibrosis. The protective function exerted by PGC-1ß is due to its ability to induce mitochondrial oxidative phosphorylation, fatty acid ß-oxidation, and citrate cycle, as well as to decrease oxidative stress and promote TG secretion in the blood stream. These findings bolster the concept that a combined hepatic specific action of PGC-1ß on lipid synthesis and secretion, as well as on mitochondrial biogenesis and function, could protect against steatohepatitis.
Assuntos
Fígado Gorduroso/metabolismo , Fígado Gorduroso/prevenção & controle , Fígado/metabolismo , Transativadores/metabolismo , Animais , Apoptose/fisiologia , Deficiência de Colina/complicações , Dieta Hiperlipídica/efeitos adversos , Modelos Animais de Doenças , Fígado Gorduroso/etiologia , Fibrose , Metabolismo dos Lipídeos/fisiologia , Fígado/patologia , Camundongos , Camundongos Transgênicos , Renovação Mitocondrial/fisiologia , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Transativadores/genética , Fatores de Transcrição , Triglicerídeos/sangueRESUMO
Reduction of nutrient intake without malnutrition positively influences lifespan and healthspan from yeast to mice and exerts some beneficial effects also in humans. The AMPK-FoxO axis is one of the evolutionarily conserved nutrient-sensing pathways, and the FOXO3A locus is associated with human longevity. Interestingly, FoxO3A has been reported to be also a mitochondrial protein in mammalian cells and tissues. Here we report that glucose restriction triggers FoxO3A accumulation into mitochondria of fibroblasts and skeletal myotubes in an AMPK-dependent manner. A low-glucose regimen induces the formation of a protein complex containing FoxO3A, SIRT3, and mitochondrial RNA polymerase (mtRNAPol) at mitochondrial DNA-regulatory regions causing activation of the mitochondrial genome and a subsequent increase in mitochondrial respiration. Consistently, mitochondrial transcription increases in skeletal muscle of fasted mice, with a mitochondrial DNA-bound FoxO3A/SIRT3/mtRNAPol complex detectable also in vivo. Our results unveil a mitochondrial arm of the AMPK-FoxO3A axis acting as a recovery mechanism to sustain energy metabolism upon nutrient restriction.
Assuntos
Adenilato Quinase/fisiologia , Fatores de Transcrição Forkhead/fisiologia , Glucose/metabolismo , Mitocôndrias/metabolismo , Sirtuína 3/fisiologia , Adenilato Quinase/genética , Adenilato Quinase/metabolismo , Animais , Células Cultivadas , DNA Mitocondrial/metabolismo , Transporte de Elétrons , Metabolismo Energético , Privação de Alimentos , Proteína Forkhead Box O3 , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Regulação da Expressão Gênica , Genoma Mitocondrial , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Biológicos , Células NIH 3T3 , Sirtuína 3/genética , Sirtuína 3/metabolismoRESUMO
Peroxisome proliferator-activated receptor-γ coactivator 1-α (PGC1α) is a transcriptional coactivator able to up-regulate mitochondrial biogenesis, respiratory capacity, oxidative phosphorylation, and fatty acid ß-oxidation with the final aim of providing a more efficient pathway for aerobic energy production. In the continuously renewed intestinal epithelium, proliferative cells in the crypts migrate along the villus axis and differentiate into mature enterocytes, increasing their respiratory capacity and finally undergoing apoptosis. Here we show that in the intestinal epithelial surface, PGC1α drives mitochondrial biogenesis and respiration in the presence of reduced antioxidant enzyme activities, thus determining the accumulation of reactive oxygen species and fostering the fate of enterocytes toward apoptosis. Combining gain- and loss-of-function genetic approaches in human cells and mouse models of intestinal cancer, we present an intriguing scenario whereby PGC1α regulates enterocyte cell fate and protects against tumorigenesis.
Assuntos
Antioxidantes/metabolismo , Enterócitos/metabolismo , Proteínas de Choque Térmico/metabolismo , Neoplasias Intestinais/metabolismo , Proteínas de Neoplasias/metabolismo , Fatores de Transcrição/metabolismo , Animais , Transformação Celular Neoplásica , Enterócitos/patologia , Proteínas de Choque Térmico/genética , Humanos , Neoplasias Intestinais/genética , Neoplasias Intestinais/patologia , Camundongos , Camundongos Nus , Proteínas de Neoplasias/genética , Neoplasias Experimentais/genética , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Consumo de Oxigênio/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Fatores de Transcrição/genéticaRESUMO
Ever increasing evidence has been provided on the accumulation of mutations in the mitochondrial DNA (mtDNA) during the aging process. However, the lack of direct functional consequences of the mutant mtDNA load on the mitochondria-dependent cell metabolism has raised many questions on the physiological importance of the age-related mtDNA variations. In the present work, we have analyzed the bioenergetic properties associated with the age-related T414G mutation of the mtDNA control region in transmitochondrial cybrids. The results show that the T414G mutation does not cause per se any detectable bioenergetic change. Moreover, three mtDNA mutations clustered in the 16S ribosomal RNA gene cosegregated together with the T414G in the same cybrid cell line. Two of them, namely T1843C and A1940G, are novel and associate with a negative bioenergetic phenotype. The results are discussed in the more general context of the complex heterogeneity and the dramatic instability of the mitochondrial genome during cell culture of transmitochondrial cybrids.
Assuntos
Envelhecimento/genética , Genes de RNAr , Mutação Puntual , RNA Ribossômico 16S/genética , RNA/genética , Sequência de Bases , Linhagem Celular , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Metabolismo Energético , Humanos , Células Híbridas , Mitocôndrias/enzimologia , Mitocôndrias/metabolismo , Dados de Sequência Molecular , RNA/química , RNA Mitocondrial , RNA Ribossômico 16S/químicaRESUMO
BACKGROUND: Chronic renal disease (CKD) is characterized by complex changes in cell metabolism leading to an increased production of oxygen radicals, that, in turn has been suggested to play a key role in numerous clinical complications of this pathological condition. Several reports have focused on the identification of biological elements involved in the development of systemic biochemical alterations in CKD, but this abundant literature results fragmented and not exhaustive. RESULTS: To better define the cellular machinery associated to this condition, we employed a high-throughput genomic approach based on a whole transcriptomic analysis associated with classical molecular methodologies. The genomic screening of peripheral blood mononuclear cells revealed that 44 genes were up-regulated in both CKD patients in conservative treatment (CKD, n = 9) and hemodialysis (HD, n = 17) compared to healthy subjects (HS, n = 8) (p < 0.001, FDR = 1%). Functional analysis demonstrated that 11/44 genes were involved in the oxidative phosphorylation system. Western blotting for COXI and COXIV, key constituents of the complex IV of oxidative phosphorylation system, performed on an independent testing-group (12 healthy subjects, 10 CKD and 14 HD) confirmed an higher synthesis of these subunits in CKD/HD patients compared to the control group. Only for COXI, the comparison between CKD and healthy subjects reached the statistical significance. However, complex IV activity was significantly reduced in CKD/HD patients compared to healthy subjects (p < 0.01). Finally, CKD/HD patients presented higher reactive oxygen species and 8-hydroxydeoxyguanosine levels compared to controls. CONCLUSION: Taken together these results suggest, for the first time, that CKD/HD patients may have an impaired mitochondrial respiratory system and this condition may be both the consequence and the cause of an enhanced oxidative stress.
Assuntos
Perfilação da Expressão Gênica , Mitocôndrias/patologia , Estresse Oxidativo , Insuficiência Renal Crônica/fisiopatologia , Adulto , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Feminino , Expressão Gênica , Humanos , Leucócitos Mononucleares/metabolismo , Masculino , Pessoa de Meia-Idade , Mitocôndrias/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Oxirredução , Fosforilação , Espécies Reativas de Oxigênio/metabolismo , Diálise Renal , Insuficiência Renal Crônica/metabolismoRESUMO
E-cadherin is the core protein of the epithelial adherens junction. Through its cytoplasmic domain, E-cadherin interacts with several signaling proteins; among them, α- and ß-catenins mediate the link of E-cadherin to the actin cytoskeleton. Loss of E-cadherin expression is a crucial step of epithelial-mesenchymal transition (EMT) and is involved in cancer invasion and metastatization. In human tumors, down-regulation of E-cadherin is frequently associated with poor prognosis. Despite the critical role of E-cadherin in cancer progression, little is known about proteome alterations linked with its down-regulation. To address this point, we investigated proteomics, biophysical and functional changes of epithelial breast cancer cell lines upon shRNA-mediated stable knockdown of E-cadherin expression (shEcad). shEcad cells showed a distinct proteomic signature including altered expression of enzymes and proteins involved in cytoskeletal dynamic and migration. Moreover, these results suggest that, besides their role in mechanical adhesion, loss of E-cadherin expression may contribute to cancer progression by modifying a complex network of pathways that tightly regulate fundamental processes as oxidative stress, immune evasion and cell metabolism. Altogether, these results extend our knowledge on the cellular modifications associated with E-cadherin down-regulation in breast cancer cells.
Assuntos
Neoplasias da Mama/patologia , Caderinas/antagonistas & inibidores , Regulação Neoplásica da Expressão Gênica , Neoplasias Epiteliais e Glandulares/patologia , Proteômica/métodos , Antígenos CD , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Caderinas/genética , Linhagem Celular Tumoral , Movimento Celular , Transição Epitelial-Mesenquimal , Feminino , Técnicas de Silenciamento de Genes , Células Hep G2 , Humanos , Células MCF-7 , Invasividade Neoplásica , Neoplasias Epiteliais e Glandulares/genética , Neoplasias Epiteliais e Glandulares/metabolismo , RNA Interferente Pequeno/metabolismoRESUMO
PURPOSE: The aim of this study was to investigate the angiogenic role of the hepatocyte growth factor (HGF)/cMET pathway and its inhibition in bone marrow endothelial cells (EC) from patients with multiple myeloma versus from patients with monoclonal gammopathy of undetermined significance (MGUS) or benign anemia (control group). EXPERIMENTAL DESIGN: The HGF/cMET pathway was evaluated in ECs from patients with multiple myeloma (multiple myeloma ECs) at diagnosis, at relapse after bortezomib- or lenalidomide-based therapies, or on refractory phase to these drugs; in ECs from patients with MGUS (MGECs); and in those patients from the control group. The effects of a selective cMET tyrosine kinase inhibitor (SU11274) on multiple myeloma ECs' angiogenic activities were studied in vitro and in vivo. RESULTS: Multiple myeloma ECs express more HGF, cMET, and activated cMET (phospho (p)-cMET) at both RNA and protein levels versus MGECs and control ECs. Multiple myeloma ECs are able to maintain the HGF/cMET pathway activation in absence of external stimulation, whereas treatment with anti-HGF and anti-cMET neutralizing antibodies (Ab) is able to inhibit cMET activation. The cMET pathway regulates several multiple myeloma EC activities, including chemotaxis, motility, adhesion, spreading, and whole angiogenesis. Its inhibition by SU11274 impairs these activities in a statistically significant fashion when combined with bortezomib or lenalidomide, both in vitro and in vivo. CONCLUSIONS: An autocrine HGF/cMET loop sustains multiple myeloma angiogenesis and represents an appealing new target to potentiate the antiangiogenic management of patients with multiple myeloma.
Assuntos
Comunicação Autócrina , Células da Medula Óssea/metabolismo , Células Endoteliais/metabolismo , Fator de Crescimento de Hepatócito/metabolismo , Mieloma Múltiplo/metabolismo , Proteínas Proto-Oncogênicas c-met/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Inibidores da Angiogênese/farmacologia , Movimento Celular , Citocinas/biossíntese , Feminino , Expressão Gênica , Fator de Crescimento de Hepatócito/genética , Humanos , Indóis/farmacologia , Masculino , Pessoa de Meia-Idade , Terapia de Alvo Molecular , Gamopatia Monoclonal de Significância Indeterminada/genética , Gamopatia Monoclonal de Significância Indeterminada/metabolismo , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/genética , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/metabolismo , Piperazinas/farmacologia , Proteoma , Proteômica , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-met/genética , Sulfonamidas/farmacologiaRESUMO
Mitochondria are involved in a variety of cellular biochemical pathways among which the ATP production by oxidative phosphorylation (OXPHOS) represents the most important function of the organelle. Since mitochondria contain their own genome encoding subunits of the OXPHOS apparatus, mtDNA mutations can cause different mitochondrial diseases. The impact of these mutations can be characterized by the trans-mitochondrial cybrid technique based on mtDNA-depleted cells (ρ(0)) as acceptors of exogenous mitochondria. The aim of the present work was to compare ρ(0) cells obtained by long term ethidium bromide treatment and by a mitochondrial targeted restriction endonuclease, respectively, as mitochondrial acceptors for trans-mitochondrial cybrid generation. Fusion cells have mitochondrial respiratory functions comparable to their parental wild type cells, regardless the strategy utilized to obtain the ρ(0) acceptor cells. Therefore, the newly developed enzymatic strategy for mtDNA depletion is a more convenient and suitable tool for a broader range of applications.
Assuntos
Trifosfato de Adenosina/biossíntese , Mitocôndrias/metabolismo , Sequência de Bases , Fusão Celular , Linhagem Celular Tumoral , Primers do DNA , DNA Mitocondrial/metabolismo , Transporte de Elétrons , Humanos , Microscopia Confocal , Fosforilação Oxidativa , Reação em Cadeia da Polimerase em Tempo RealRESUMO
In the present work we have critically examined the use of the KCN-titration technique in the study of the control of the cellular respiration by cytochrome c oxidase (COX) in the presence of the mitochondrial membrane potential (Δψ(mito)) in HepG2 cells. We clearly show that the apparent high inhibition threshold of COX in the presence of maximal Δψ(mito) is due to the KCN-induced decrease of Δψ(mito) and not to a low control of COX on the mitochondrial respiration. The tight control exerted by COX on the Δψ(mito) provides further insights for understanding the pathogenetic mechanisms associated with mitochondrial defects in human neuromuscular degenerative disorders.
Assuntos
Complexo IV da Cadeia de Transporte de Elétrons/fisiologia , Potenciais da Membrana , Mitocôndrias/fisiologia , Linhagem Celular , Humanos , Consumo de OxigênioRESUMO
We have recently shown that the transcriptional coactivator PGC1α, a master regulator of mitochondrial biogenesis and function, is involved in the control of the intestinal epithelium cell fate. Furthermore, PGC1α protects against colon cancer formation by promoting ROS accumulation and, consequently, mitochondria-mediated apoptosis. Here we provide an additional mechanistic insight into the tumor suppressor activity of PGC1α showing that its pro-apoptotic effect is mediated by Bax. In fact, PGC1α overexpression in HCT116 Bax (-/-) colorectal cancer cells stimulates mitochondrial production and activity, but it fails to induce cell death as well as to oppose tumor growth in the xenograft model. The lack of ROS accumulation in the Bax (-/-) cells strengthens our view that the PGC1α-induced oxidative burst represents one of the main apoptosis-driving factors in colorectal cancer cells.
Assuntos
Apoptose , Neoplasias do Colo/patologia , Proteínas de Choque Térmico/metabolismo , Fatores de Transcrição/metabolismo , Proteína X Associada a bcl-2/metabolismo , Adenoviridae/genética , Adenoviridae/metabolismo , Animais , Neoplasias do Colo/metabolismo , DNA Mitocondrial/análise , Células HCT116 , Humanos , Camundongos , Camundongos Nus , Mitocôndrias/genética , Mitocôndrias/metabolismo , PPAR gama/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Espécies Reativas de Oxigênio/metabolismo , Explosão Respiratória , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
SCO proteins are copper-donor chaperones involved in the assembly of mitochondrial cytochrome c oxidase (COX). Mutations in the two human SCO-encoding genes, SCO1 and SCO2, produce tissue-specific COX deficiencies associated with distinct clinical phenotypes. Here, we report the identification and characterization of scox, the single Drosophila melanogaster SCO-encoding gene. Null mutations of the scox gene are associated with larval lethality, while mutations in its 5'UTR are associated with motor dysfunction and female sterile phenotypes. All mutant phenotypes may be rescued by a transgene encompassing wild-type scox. The analysis of the phenotypes associated with the D. melanogaster scox mutations shows that unimpaired COX assembly and activity is required for biological processes that specifically depend on an adequate energy supply. Finally, we identified the SCO1 orthologs in 39 eukaryotic species informative for a tentative reconstruction of the evolutionary history of the SCO function. Comparison of the exon/intron structure and other key features suggest that eukaryotic SCO genes descend from an intron-rich ancestral gene already present in the last common ancestor of lineages that diverged as early as metazoans and flowering plants.