RESUMO
OBJECTIVE: Sister-of-Mammalian Grainyhead (SOM) is a member of the Grainyhead family of transcription factors. In humans, 3 isoforms are derived from differential first exon usage and alternative splicing and differ only in their N terminal domain. SOM2, the only variant also present in mouse, induces endothelial cell migration and protects against apoptosis. The functions of the human specific isoforms SOM1 and SOM3 have not yet been investigated. Therefore we wanted to elucidate their functions in endothelial cells. APPROACH AND RESULTS: Overexpression of SOM1 in primary human endothelial cells induced migration, phosphorylation of Akt1 and endothelial nitric oxide synthase, and protected against apoptosis, whereas SOM3 had opposite effects; isoform-specific knockdowns confirmed the disparate effects on apoptosis. After reporter assays demonstrated that both are active transcription factors, microarray analyses revealed that they induce different target genes, which could explain the different cellular effects. Overexpression of SOM3 in zebrafish embryos resulted in increased lethality and severe deformations, whereas SOM1 had no deleterious effect. CONCLUSIONS: Our data demonstrate that the splice variant-derived isoforms SOM1 and SOM3 induce opposing effects in primary human endothelial cells and in a whole animal model, most likely through the induction of different target genes.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Fatores de Transcrição/metabolismo , Animais , Apoptose , Movimento Celular , Proteínas de Ligação a DNA/genética , Ativação Enzimática , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica , Genes Reporter , Células HEK293 , Humanos , Células MCF-7 , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Fosforilação , Isoformas de Proteínas , Proteínas Proto-Oncogênicas c-akt/metabolismo , Interferência de RNA , Fatores de Transcrição/genética , Transcrição Gênica , Transfecção , Peixe-Zebra/genética , Peixe-Zebra/metabolismoRESUMO
BACKGROUND: The underlying molecular mechanisms of the vasculoprotective effects of physical exercise are incompletely understood. Telomere erosion is a central component of aging, and telomere-associated proteins regulate cellular senescence and survival. This study examines the effects of exercising on vascular telomere biology and endothelial apoptosis in mice and the effects of long-term endurance training on telomere biology in humans. METHODS AND RESULTS: C57/Bl6 mice were randomized to voluntary running or no running wheel conditions for 3 weeks. Exercise upregulated telomerase activity in the thoracic aorta and in circulating mononuclear cells compared with sedentary controls, increased vascular expression of telomere repeat-binding factor 2 and Ku70, and reduced the expression of vascular apoptosis regulators such as cell-cycle-checkpoint kinase 2, p16, and p53. Mice preconditioned by voluntary running exhibited a marked reduction in lipopolysaccharide-induced aortic endothelial apoptosis. Transgenic mouse studies showed that endothelial nitric oxide synthase and telomerase reverse transcriptase synergize to confer endothelial stress resistance after physical activity. To test the significance of these data in humans, telomere biology in circulating leukocytes of young and middle-aged track and field athletes was analyzed. Peripheral blood leukocytes isolated from endurance athletes showed increased telomerase activity, expression of telomere-stabilizing proteins, and downregulation of cell-cycle inhibitors compared with untrained individuals. Long-term endurance training was associated with reduced leukocyte telomere erosion compared with untrained controls. CONCLUSIONS: Physical activity regulates telomere-stabilizing proteins in mice and in humans and thereby protects from stress-induced vascular apoptosis.
Assuntos
Vasos Sanguíneos/citologia , Vasos Sanguíneos/fisiologia , Senescência Celular/fisiologia , Exercício Físico/fisiologia , Leucócitos/citologia , Leucócitos/fisiologia , Condicionamento Físico Animal , Esforço Físico/fisiologia , Adolescente , Adulto , Animais , Endotélio Vascular/citologia , Endotélio Vascular/fisiologia , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Pessoa de Meia-Idade , Condicionamento Físico Animal/métodos , Corrida/fisiologia , Telômero/enzimologia , Telômero/fisiologia , Adulto JovemRESUMO
Owing to their specific physico/chemical properties, engineered as well as environmental nanoparticles can induce pathogenic endpoints in humans. Earlier studies demonstrated that pure carbon nanoparticles induce cell signaling events at the level of membrane receptor activation in lung epithelial cells. As a possible link between receptor activation and subsequent MAP-kinase signaling, the involvement of Src family kinases was investigated in cell lines of organs potentially exposed to environmental nanoparticles. Human cells from bronchus, intestine, and skin (keratinocytes) as well as rat lung epithelial cells showed similar time patterns for the activation of mitogen-activated protein kinases Erk1/2 as well as Src family kinases (SFK) when treated with carbon nanoparticles. Moreover, c-Src was identified as an integral part of the signaling mediating the transfer of information from membrane receptors to members of the proliferative signaling cascade in lung epithelial cells. Pretreatment of cells with the compatible solute ectoine, which is known to stabilize macromolecules, reduced the nanoparticle specific phosphorylation of SFK. Together with earlier in vivo and in vitro data, this demonstrates that compatible solutes prevent nanoparticle-induced signaling steps at the level of membrane-coupled signaling.
Assuntos
Carbono , Células Epiteliais/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteína Quinase 1 Ativada por Mitógeno , Proteína Quinase 3 Ativada por Mitógeno , Nanopartículas , Diamino Aminoácidos/antagonistas & inibidores , Animais , Brônquios/metabolismo , Células CACO-2 , Carbono/toxicidade , Colo/metabolismo , Receptores ErbB/metabolismo , Humanos , Pulmão/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Nanopartículas/química , Nanopartículas/toxicidade , Fosforilação , Ratos , Pele/metabolismo , Quinases da Família src/metabolismoRESUMO
OBJECTIVE: The enzyme telomerase and its catalytic subunit the telomerase reverse transcriptase (TERT) are important for maintenance of telomere length in the nucleus. Recent studies provided evidence for a mitochondrial localization of TERT. Therefore, we investigated the exact localization of TERT within the mitochondria and its function. METHODS AND RESULTS: Here, we demonstrate that TERT is localized in the matrix of the mitochondria. TERT binds to mitochondrial DNA at the coding regions for ND1 and ND2. Binding of TERT to mitochondrial DNA protects against ethidium bromide-induced damage. TERT increases overall respiratory chain activity, which is most pronounced at complex I and dependent on the reverse transcriptase activity of the enzyme. Moreover, mitochondrial reactive oxygen species are increased after genetic ablation of TERT by shRNA. Mitochondrially targeted TERT and not wild-type TERT revealed the most prominent protective effect on H(2)O(2)-induced apoptosis. Lung fibroblasts from 6-month-old TERT(-/-) mice (F2 generation) showed increased sensitivity toward UVB radiation and heart mitochondria exhibited significantly reduced respiratory chain activity already under basal conditions, demonstrating the protective function of TERT in vivo. CONCLUSIONS: Mitochondrial TERT exerts a novel protective function by binding to mitochondrial DNA, increasing respiratory chain activity and protecting against oxidative stress-induced damage.
Assuntos
Dano ao DNA , DNA Mitocondrial/metabolismo , Mitocôndrias/enzimologia , Telomerase/metabolismo , Animais , Apoptose/efeitos dos fármacos , Células Cultivadas , Transporte de Elétrons , Células Endoteliais/enzimologia , Etídio/farmacologia , Fibroblastos/enzimologia , Humanos , Peróxido de Hidrogênio/toxicidade , Pulmão/enzimologia , Camundongos , Camundongos Knockout , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/patologia , Mitocôndrias/efeitos da radiação , Mitocôndrias Cardíacas/enzimologia , Estresse Oxidativo , Ligação Proteica , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Telomerase/deficiência , Telomerase/genética , Transfecção , Raios UltravioletaRESUMO
Diet and pollution are environmental factors known to compromise "healthy aging" of the cardiovascular and respiratory systems. The molecular consequences of this permanent burden in these cells are still unknown. Therefore, this study investigates the impact of unhealthy diet on aging-related signaling pathways of human, primary cardiovascular cells and of airborne particles on lung epithelial and human endothelial cells. Nutrition health reports have shown that the diet in industrialized countries contains more than 100mg/dl low density lipoprotein (LDL) and a high fraction of added sugars, especially fructose. Several studies demonstrated that ultrafine particles can enter the circulation and thus may interact with endothelial cells directly. Both, dietary compounds and pollution derived particles, have been shown to increase the risk for cardiovascular diseases. To simulate an unhealthy diet, we supplemented cell culture media of human primary endothelial cells, smooth muscle cells and cardiomyocytes with LDL and replaced 1/3 of glucose with fructose. We observed hypertrophy in cardiomyocytes, enhanced proliferation in smooth muscle cells and increased senescence, loss of endothelial nitric oxide synthase and increased nuclear FoxO3A in endothelial cells. With respect to pollution we have used ultrafine carbon black particles (ufCB), one of the major constituents of industrial and exhaust emissions, in concentrations our lungs and vessels are constantly exposed to. These concentrations of ufCB increased reactive oxygen species in lung epithelial and vascular endothelial cells and reduced the S-NO content, a marker for NO-bioavailability, in endothelial cells. NO increases activation of Telomerase Reverse Transcriptase (TERT), an enzyme essential for telomere maintenance. TERT is required for proper endothelial cell function and is inactivated by Src kinase under conditions of oxidative stress. ufCB significantly increased Src kinase activation and reduced Telomerase activity in endothelial and lung epithelial cells. As a consequence, ufCB increased senescence of endothelial cells. To investigate whether ufCB show also effects in vivo, we instilled ufCB in concentrations not inducing inflammation into mice. Indeed, eNOS expression was reduced in the abdominal aorta of animals treated with ufCB. Thus, a combination of fructose and LDL in the diet and ufCB, as a major constituent of air pollution, seem to accelerate respiratory and cardiovascular cellular changes, which may compromise "healthy aging" and can lead to cardiovascular and pulmonary diseases.
Assuntos
Senescência Celular/efeitos dos fármacos , Dieta , Fuligem/farmacologia , Poluentes Atmosféricos/farmacologia , Poluentes Atmosféricos/toxicidade , Animais , Aorta Abdominal/enzimologia , Proliferação de Células/efeitos dos fármacos , Senescência Celular/fisiologia , LDL-Colesterol/farmacologia , Meios de Cultura/farmacologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Feminino , Frutose/farmacologia , Glucose/farmacologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Óxido Nítrico/biossíntese , Óxido Nítrico Sintase/biossíntese , Óxido Nítrico Sintase Tipo III/biossíntese , Tamanho da Partícula , Fenótipo , Espécies Reativas de Oxigênio/metabolismo , Fuligem/toxicidade , Telomerase/biossíntese , Quinases da Família src/biossínteseRESUMO
One distinguishing feature of eukaryotic cells is their compartmentalization into organelles, which all have a unique structural and functional identity. Some proteins are exclusively localized in a single organelle, whereas others are found in more than one. A few proteins, whose function was thought to be completely understood, were only recently found to be present in the mitochondria. Although these proteins come from diverse functional classes, their common new denominator is the regulation of respiratory chain activity. Therefore, this review focuses on new functions of the Signal Transducer and Activator of Transcription 3, originally described as a transcription factor, the most prominent Src kinase family members, Src, Fyn, and Yes, which were so far known as plasma membrane-associated molecular effectors of a variety of extracellular stimuli, the tyrosine phosphatase Shp-2 previously characterized as a modulator of cytosolic signal transduction involved in cell growth, development, inflammation, and chemotaxis, and Telomerase Reverse Transcriptase, the key enzyme preventing telomere erosion in the nucleus. Their unexpected localization in other organelles and regulation of mitochondrial and/or nuclear functions by them adds a new layer of regulatory complexity. This extends the flexibility to cope with changing environmental demands using a limited number of genes and proteins.
Assuntos
Núcleo Celular/metabolismo , Mitocôndrias/metabolismo , Animais , Núcleo Celular/genética , Humanos , Mitocôndrias/genética , Modelos Biológicos , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismoRESUMO
Telomerase with its catalytic subunit telomerase reverse transcriptase (TERT) prevents telomere erosion in the nucleus. In addition, telomerase has also telomere-independent functions in protection from apoptosis. Unexpectedly, TERT was found in the mitochondria. However, its regulation in this organelle is completely unknown. Here, we demonstrate that mitochondrial TERT is downregulated by exposure to H(2)O(2) in primary human endothelial cells. This depletion is dependent on the Src phosphorylation site within TERT, tyrosine 707. In accordance with this finding, we also detected Src in the mitochondria and demonstrated that Src is activated upon H(2)O(2) treatment. This regulation of mitochondrial TERT is reminiscent of the situation in the nucleus from where TERT is exported under conditions of oxidative stress in a Src kinase dependent manner. In addition, Akt1 was also found in the mitochondria and H(2)O(2) treatment led to reduced active Akt1 in these organelles, suggesting that similar regulatory mechanisms operate in mitochondria and the nucleus.
Assuntos
Peróxido de Hidrogênio/farmacologia , Telomerase/metabolismo , Quinases da Família src/metabolismo , Sequência de Bases , Sítios de Ligação , Núcleo Celular/enzimologia , Células Cultivadas , Primers do DNA/genética , Regulação para Baixo/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/enzimologia , Ativação Enzimática/efeitos dos fármacos , Humanos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/enzimologia , Estresse Oxidativo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Telomerase/química , Telomerase/genética , Tirosina/químicaRESUMO
Reactive oxygen species have been described to modulate proteins within the cell, a process called redox regulation. However, the importance of compartment-specific redox regulation has been neglected for a long time. In the early 1980s and 1990s, many in vitro studies introduced the possibility that nuclear redox signaling exists. However, the functional relevance for that has been greatly disregarded. Recently, it has become evident that nuclear redox signaling is indeed one important signaling mechanism regulating a variety of cellular functions. Transcription factors, and even kinases and phosphatases, have been described to be redox regulated in the nucleus. This review describes several of these proteins in closer detail and explains their functions resulting from nuclear localization and redox regulation. Moreover, the redox state of the nucleus and several important nuclear redox regulators [Thioredoxin-1 (Trx-1), Glutaredoxins (Grxs), Peroxiredoxins (Prxs), and APEX nuclease (multifunctional DNA-repair enzyme) 1 (APEX1)] are introduced more precisely, and their necessity for regulation of transcription factors is emphasized.
Assuntos
Núcleo Celular/metabolismo , Transdução de Sinais , Animais , Núcleo Celular/enzimologia , Humanos , Oxirredução , Espécies Reativas de Oxigênio/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Aging is one major risk factor for numerous diseases. The enzyme telomerase reverse transcriptase (TERT) plays an important role for aging and apoptosis. Previously, we demonstrated that inhibition of oxidative stress-induced Src kinase family-dependent nuclear export of TERT results in delayed replicative senescence and reduced apoptosis sensitivity. Therefore, the aim of this study was to investigate mechanisms inhibiting nuclear export of TERT. First, we demonstrated that H2O2-induced nuclear export of TERT was abolished in Src, Fyn, and Yes-deficient embryonic fibroblasts. Next, we wanted to identify one potential negative regulator of this export process. One candidate is the protein tyrosine phosphatase Shp-2 (Shp-2), which can counteract activities of the Src kinase family. Indeed, Shp-2 was evenly distributed between the nucleus and cytosol. Nuclear Shp-2 associates with TERT in endothelial cells and dissociates from TERT prior to its nuclear export. Overexpression of Shp-2 wt inhibited H2O2-induced export of TERT. Overexpression of the catalytically inactive, dominant negative Shp-2 mutant (Shp-2(C459S)) reduced endogenous as well as overexpressed nuclear TERT protein and telomerase activity, whereas it had no influence on TERT(Y707F). Binding of TERT(Y707F) to Shp-2 is reduced compared with TERTwt. Ablation of Shp-2 expression led only to an increased tyrosine phosphorylation of TERTwt, but not of TERT(Y707F). Moreover, reduced Shp-2 expression decreased nuclear telomerase activity, whereas nuclear telomerase activity was increased in Shp-2-overexpressing endothelial cells. In conclusion, Shp-2 retains TERT in the nucleus by regulating tyrosine 707 phosphorylation.