RESUMEN
Diet-induced metabolic acidosis is associated with the impairment of bone metabolism and an increased risk of a number of chronic noncommunicable diseases, such as type 2 diabetes mellitus and hypertension. Low serum bicarbonate is associated with high mortality in healthy older individuals. Recently, we demonstrated that both coupling factor 6 (CF6)-overexpressing transgenic (TG) and high salt-fed mice which had sustained intracellular acidosis, due to enhanced proton import through ecto-F1Fo complex and/or reduced proton export through Na+-K+ ATPase inhibition, displayed shortened lifespan and early senescence-associated phenotypes such as signs of hair greying and alopecia, weight loss, and/or reduced organ mass. In this study, we searched causative genes of proton-induced aging in CF6-overexpressing TG and high salt-fed mice. We discovered NM_026333 as a novel anti-aging gene which was downregulated in the heart and kidney in both types of mice. NM_026333 protein consists of 269 amino acids with transmembrane region (90-193aa). Induction of NM_026333 or recombinant protein rescued TG cells and CF6-treated human cells from aging hallmarks of impaired autophagy, genomic instability, and epigenetic alteration. NM_026333 protein directly bound plasma membrane Na+-Ca2+ exchanger 1 (NCX1) to suppress its reverse mode, and cancelled proton-induced epigenetic regression of Atg7 that was caused by H3K4 and H4K20 tri-methylation via suppression of demethylase and H4K5 acetylation via suppression of nuclear HDAC3-HDAC4-emerin system. NM_026333 also attenuated proton-induced impaired formation of autolysosome, an increase in nuclear acetylated LC3 II, and acetylation of Atg7. These effects reappeared by NCX1 inhibitor. Furthermore, NCX1 inhibitor extended lifespan compared with vehicle-treatment in TG mice. This study will shed light on novel aging mechanism and provide implications in a target for anti-aging therapy.
Asunto(s)
ATPasas de Translocación de Protón Mitocondriales/metabolismo , Factores de Acoplamiento de la Fosforilación Oxidativa/metabolismo , Intercambiador de Sodio-Calcio/metabolismo , Envejecimiento/efectos de los fármacos , Animales , Autofagia/genética , Autofagia/fisiología , Membrana Celular/metabolismo , Células Cultivadas , Inmunoprecipitación de Cromatina , Epigenómica , Inestabilidad Genómica/efectos de los fármacos , Inestabilidad Genómica/genética , Células HEK293 , Humanos , Ratones , ATPasas de Translocación de Protón Mitocondriales/genética , Factores de Acoplamiento de la Fosforilación Oxidativa/genética , Protones , Transducción de Señal/efectos de los fármacosRESUMEN
Coupling factor 6 (CF6) forces a counter-clockwise rotation of plasma membrane F1 Fo complex, resulting in proton import and accelerated aging. Inhibitory factor peptide 1 (IF1) suppresses a unidirectional counter-clockwise rotation of F1 Fo complex without affecting ATP synthesis. We tested the hypothesis that IF1 may attenuate CF6-induced aging signaling in CF6-overexpressing transgenic (TG) cells. In IF1-GFP overexpressing wild type (WT) cells, the diffuse peripheral staining of tubular mitochondria was observed with a dense widely distributed network around the nucleus. In TG cells, however, the only peri-nuclear network of fragmented mitochondria was observed at 24 h, but it was developed to a widely distributed mitochondrial network of tubular mitochondria at 72 h. TG cells displayed aging hallmarks of telomere attrition, epigenetic alterations, defective proteostasis, and genomic instability with a decrease in emerin and lamin and loss of heterochromatin. IF1 induction rescued TG cells from telomere attrition, expression of genomic instability with the increase in emerin and lamin, and that of epigenetic alterations with recovery of heterochromatin. In defective proteostasis, IF1 induction restored a potent peri-nuclear staining of autolysosomes compared with the baseline weak staining. The decrease in Atg7 was restored, whereas the increase in P62 was abolished. We conclude that genetic disruption of proton signals by IF1 induction suppressed CF6-induced expression of aging hallmarks such as telomere attrition, epigenetic alterations, defective proteostasis, and genomic instability. Given the widespread biological actions of CF6, the physiological and pathological actions of IF1 may be complex.
Asunto(s)
Envejecimiento , Mitocondrias/metabolismo , ATPasas de Translocación de Protón Mitocondriales/metabolismo , Factores de Acoplamiento de la Fosforilación Oxidativa/metabolismo , Proteínas/metabolismo , Animales , Inestabilidad Genómica , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , ATPasas de Translocación de Protón Mitocondriales/genética , Factores de Acoplamiento de la Fosforilación Oxidativa/genética , Proteínas/genética , Proteína Inhibidora ATPasaRESUMEN
Diet-induced metabolic acidosis is associated with the impairment of bone metabolism and an increased risk of a number of chronic noncommunicable diseases, such as type 2 diabetes mellitus and hypertension. The serum bicarbonate level is an independent predictor of chronic kidney disease progression. We investigated whether proton accelerates aging by analyzing both coupling factor 6-overexpressing transgenic (TG) and high salt-fed mice which display sustained intracellular acidosis, due to enhanced proton import through ecto-F1 Fo complex and/or reduced proton export through Na+ -K+ ATPase inhibition. Both types of mice displayed shortened lifespan and early senescence-associated phenotypes such as signs of hair greying and alopecia, weight loss, and/or reduced organ mass. In chronic intracellular acidosis mice, autophagy was impaired by regression of Atg7, an increase in nuclear acetylated LC3 II, and acetylation of Atg7. The increase in histone 3 trimethylation at lysine 4 (H3K4me3) and H4K20me3 and the decrease in H3K9me3 and H3K27me3 were observed in the heart and kidney obtained from both TG and high salt-fed mice. The decrease in lamin A/C, emerin, and heterochromatin protein 1α without changes in barrier-to-autointegration factor and high-mobility group box 1 was confirmed in TG and high salt-fed mice. Suppression of nuclear histone deacetylase 3-emerin system is attributable to epigenetic regression of Atg7 and H4K5 acetylation. These findings will shed light on novel aging and impaired autophagy mechanism, and provide implications in a target for antiaging therapy.
Asunto(s)
Envejecimiento/fisiología , Epigénesis Genética , Inestabilidad Genómica , Acidosis/etiología , Animales , Autofagia/fisiología , Proteína 7 Relacionada con la Autofagia/genética , Proteína 7 Relacionada con la Autofagia/metabolismo , Presión Sanguínea , Ensamble y Desensamble de Cromatina , Femenino , Masculino , Ratones Endogámicos C57BL , Ratones Transgénicos , ATPasas de Translocación de Protón Mitocondriales/genética , Factores de Acoplamiento de la Fosforilación Oxidativa/genética , Protones , Cloruro de Sodio Dietético/farmacologíaRESUMEN
Coupling factor 6 (CF6) forces a counter-clockwise rotation of plasma membrane F1 Fo complex unlike a proton-mediated clockwise rotation in the mitochondria, resulting in ATP hydrolysis, proton import, and apoptosis. Inhibitory peptide 1 (IF1) inhibits a unidirectional counter-clockwise rotation of F1 Fo complex without affecting ATP synthesis by a clockwise rotation. We tested the hypothesis that IF1 may antagonize the biological action of CF6 in human embryonic kidney 293 cells. We generated mature and immature IF1 expression vectors and those labeled with GFP at the C-terminus. In the immature IF1-GFP overexpressing cells, the mitochondrial network of IF1-GFP was newly found at the plasma membrane after peripheral translocation, whereas in mature IF1-GFP transfected cells, a less punctuate rather homogenous pattern was found in the cytoplasm. IF1 protein was detected in the exosome fraction of culture media, and it was enhanced by mature or immature IF1 transfection. Extracellular ATP hydrolysis was enhanced by CF6, whereas immature or mature IF1 transfection suppressed ATP hydrolysis in response to CF6. Intracellular pH was decreased by CF6 but was unchanged after immature IF1 transfection. CF6-induced increase in apoptotic cells was blocked by immature or mature IF1, being accompanied by protein kinase B (PKB) phosphorylation. IF1 antagonizes the pro-apoptotic action of CF6 by relief of intracellular acidification and resultant phosphorylation of PKB. Given the widespread biological actions of CF6, the physiological and pathological functions of IF1 may be expected to be complex. J. Cell. Biochem. 117: 1680-1687, 2016. © 2015 Wiley Periodicals, Inc.
Asunto(s)
Apoptosis , Exosomas/metabolismo , ATPasas de Translocación de Protón Mitocondriales/antagonistas & inhibidores , ATPasas de Translocación de Protón Mitocondriales/metabolismo , Factores de Acoplamiento de la Fosforilación Oxidativa/antagonistas & inhibidores , Factores de Acoplamiento de la Fosforilación Oxidativa/metabolismo , Proteínas/metabolismo , Exosomas/genética , Células HEK293 , Humanos , ATPasas de Translocación de Protón Mitocondriales/genética , Factores de Acoplamiento de la Fosforilación Oxidativa/genética , Fosforilación/genética , Transporte de Proteínas/genética , Proteínas/genética , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , ATPasas de Translocación de Protón/genética , ATPasas de Translocación de Protón/metabolismo , Transfección , Proteína Inhibidora ATPasaRESUMEN
High sodium, high glucose, and obesity are important risk factors for age-related diseases such as cardiovascular disease (CVDs), stroke, and cancer. Coupling factor 6 (CF6) is released from vascular endothelial cells and functions as a circulating peptide that inhibits prostacyclin and nitric oxide generation by intracellular acidosis. High glucose elevates CF6 by activation of protein kinase C and p38 mitogen-activated protein kinase, whereas CF6 causes type 2 diabetes mellitus, resulting in a high glucose vicious cycle. Low glucose increases inhibitory factor peptide 1, an endogenous inhibitor of CF6. High salt intake increases CF6 through nuclear factor κB signaling, whereas CF6 induces salt-sensitive hypertension and salt-induced congestive heart failure. Oral administration of vitamin C cancels salt-induced increase in CF6, and estrogen replacement leads to the delayed onset of CF6-induced salt-sensitive hypertension and the rescue from cardiac systolic dysfunction. Because CF6 contributes to the onset of CVDs, nutritional regulation of CF6 will shed light on the understanding of preventive strategy and mechanisms for CVDs and a target for therapy.
Asunto(s)
Células Endoteliales/metabolismo , ATPasas de Translocación de Protón Mitocondriales/metabolismo , Factores de Acoplamiento de la Fosforilación Oxidativa/metabolismo , Administración Oral , Ácido Ascórbico/farmacología , Diabetes Mellitus Tipo 2/diagnóstico , Epoprostenol/antagonistas & inhibidores , Epoprostenol/metabolismo , Insuficiencia Cardíaca/diagnóstico , Insuficiencia Cardíaca/etiología , Humanos , Hipertensión/diagnóstico , FN-kappa B/metabolismo , Óxido Nítrico/antagonistas & inhibidores , Óxido Nítrico/metabolismo , Proteína Quinasa C/metabolismo , Transducción de Señal , Cloruro de Sodio Dietético/administración & dosificación , Cloruro de Sodio Dietético/efectos adversos , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
BACKGROUND: Coupling factor 6 (CF6), a component of ATP synthase, inhibits phospholipase A2 and induces vasoconstriction. However, because arachidonic acid acts in the widespread fields of vascular biology, CF6 might exert profound effects in addition to vasoconstriction. We investigated the effect of CF6 on the gene expression profile in human umbilical vein endothelial cells. METHODS AND RESULTS: The increased gene expression after 24-h exposure to CF6 at 10 mol/l, assessed by cDNA microarray (n = 3), included neuregulin-1 (1.84 +/- 0.07 fold compared with control, P < 0.05) and relaxin-1 (1.74 +/- 0.20, P < 0.05), both relating to congestive heart failure, urokinase type plasminogen activator receptor (1.77 +/- 0.24, P = 0.06) and estrogen receptor beta (1.74 +/- 0.36, P = 0.08), both relating to vascular inflammation and cell infiltration, and protein arginine methyltransferase (PRMT-1; 1.73 +/- 0.20, P < 0.05). Out of these genes, the enzyme relating to the synthesis (PRMT-1) of asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase (NOS), was further examined concomitantly with the degradation enzyme, dimethylarginine dimethylaminohydrolase 2 (DDAH-2). The ratio of PRMT-1 to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA, measured by real-time quantitative reverse transcription-polymerase chain reaction, was increased by 9 +/- 2% (n = 10, P < 0.01) at 48 h after CF6 at 10 mol/l, whereas the ratio of DDAH-2 to GAPDH was decreased by 12 +/- 2% (n = 8, P < 0.01). DDAH-2 protein and activity were decreased by 28 +/- 5% (n = 5, P < 0.01) and 19 +/- 2% (n = 6, P < 0.01) by CF6, respectively. ADMA release was enhanced by 20 +/- 8% and NOS activity was decreased by 13 +/- 1% (both n = 8, P < 0.05) by CF6. CONCLUSIONS: CF6 changes the gene expression profile to be proatherogenic and functions as a novel stimulator for ADMA release by enhancing its synthesis and suppressing its degradation.
Asunto(s)
Arginina/análogos & derivados , Células Endoteliales/efectos de los fármacos , ATPasas de Translocación de Protón Mitocondriales/farmacología , Factores de Acoplamiento de la Fosforilación Oxidativa/farmacología , Amidohidrolasas/metabolismo , Arginina/metabolismo , Western Blotting , Células Cultivadas , Perfilación de la Expresión Génica , Humanos , ATPasas de Translocación de Protón Mitocondriales/metabolismo , Óxido Nítrico Sintasa/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Factores de Acoplamiento de la Fosforilación Oxidativa/metabolismo , Proteína-Arginina N-Metiltransferasas/metabolismo , Venas UmbilicalesRESUMEN
OBJECTIVE: We previously showed that mitochondrial coupling factor 6 (CF6), an endogenous inhibitor of prostacyclin synthesis and a vasoconstrictor, is present on the surface of human umbilical vein endothelial cells (HUVEC) and is released outside of the cells by shear stress. We investigated the intracellular signaling mechanism for shear-induced release of CF6 in HUVEC and the effects of troglitazone and 15-deoxy-delta(12,14)-prostaglandin J2 (15d-PGJ2), both peroxisome proliferator-activated receptor (PPAR)-gamma ligands, on it. METHODS AND RESULTS: The release and gene expression of CF6 in HUVEC were enhanced by shear stress at 25 dyn/cm2, measured by radioimmunoassay and real-time RT-PCR, respectively. The intracellular content of CF6 was decreased after exposure to shear stress at 25 dyn/cm2. Transfection experiments with deletional and mutational CF6 promoter constructs, and with dominant negative mutant IkappaB kinase alpha (K44M) demonstrated that shear-induced CF6 transcription was dependent on nuclear factor-kappa B (NF-kappaB) activation. Pretreatment with troglitazone or 15d-PGJ2 inhibited the shear-induced release and gene expression of CF6, whereas fenofibric acid, a PPAR-alpha ligand, had no influence on them. Western blot and immunostaining showed that troglitazone and 15d-PGJ2 inhibited the shear-induced, reactive oxygen species (ROS)-mediated activation of NF-kappaB at the level of IkappaB protein. CONCLUSIONS: The shear-induced gene expression and release of CF6 in HUVEC are mediated by the ROS-related activation of NF-kappaB signaling pathway. Troglitazone and 15d-PGJ2 inhibit them at the IkappaB protein level.
Asunto(s)
Cromanos/farmacología , Células Endoteliales/metabolismo , Endotelio Vascular/metabolismo , ATPasas de Translocación de Protón Mitocondriales/metabolismo , Factores de Acoplamiento de la Fosforilación Oxidativa/metabolismo , PPAR gamma/metabolismo , Prostaglandina D2/análogos & derivados , Tiazolidinedionas/farmacología , Western Blotting/métodos , Células Cultivadas , Células Endoteliales/efectos de los fármacos , Endotelio Vascular/efectos de los fármacos , Expresión Génica , Humanos , Proteínas I-kappa B/metabolismo , Ligandos , Microscopía Fluorescente , ATPasas de Translocación de Protón Mitocondriales/genética , FN-kappa B/metabolismo , Factores de Acoplamiento de la Fosforilación Oxidativa/genética , Prostaglandina D2/farmacología , Radioinmunoensayo , Especies Reactivas de Oxígeno/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Estrés Mecánico , TroglitazonaRESUMEN
Extracellular ATP synthesis on human umbilical vein endothelial cells (HUVECs) was examined, and it was found that HUVECs possess high ATP synthesis activity on the cell surface. Extracellular ATP generation was detected within 5 s after addition of ADP and inorganic phosphate and reached a maximal level at 15 s. This type of ATP synthesis was almost completely inhibited by mitochondrial H(+)-ATP synthase inhibitors (e.g., efrapeptins, resveratrol, and piceatannol), which target the F(1) catalytic domain. Oligomycin and carbonyl cyanide m-chlorophenylhydrazone, but not potassium cyanide, also inhibited extracellular ATP synthesis on HUVECs, suggesting that cell surface ATP synthase employs the transmembrane electrochemical potential difference of protons to synthesize ATP as well as mitochondrial H(+)-ATP synthase. The F(1)-targeting H(+)-ATP synthase inhibitors markedly inhibited the proliferation of HUVECs, but intracellular ATP levels in HUVECs treated with these inhibitors were only slightly affected, as shown by comparison with the control cells. Interestingly, piceatannol inhibited only partially the activation of Syk (a nonreceptor tyrosine kinase), which has been shown to play a role in a number of endothelial cell functions, including cell growth and migration. These findings suggest that H(+)-ATP synthase-like molecules on the surface of HUVECs play an important role not only in extracellular ATP synthesis but also in the proliferation of HUVECs. The present results demonstrate that the use of small molecular H(+)-ATP synthase inhibitors targeting the F(1) catalytic domain may lead to significant advances in potential antiangiogenic cancer therapies.
Asunto(s)
Adenosina Trifosfato/biosíntesis , Células Endoteliales/metabolismo , ATPasas de Translocación de Protón/metabolismo , Carbonil Cianuro m-Clorofenil Hidrazona/farmacología , División Celular/efectos de los fármacos , División Celular/fisiología , Línea Celular , Membrana Celular/enzimología , Relación Dosis-Respuesta a Droga , Células Endoteliales/efectos de los fármacos , Células Endoteliales/enzimología , Precursores Enzimáticos/biosíntesis , Espacio Extracelular/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular , Oligomicinas/farmacología , Péptidos/farmacología , Cianuro de Potasio/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/biosíntesis , ATPasas de Translocación de Protón/antagonistas & inhibidores , ATPasas de Translocación de Protón/fisiología , Resveratrol , Ribonucleótido Reductasas/antagonistas & inhibidores , Estilbenos/farmacología , Quinasa Syk , Venas Umbilicales/citología , Venas Umbilicales/efectos de los fármacos , Venas Umbilicales/metabolismoRESUMEN
OBJECTIVE: We recently showed that mitochondrial coupling factor 6 (CF6) is present as a pressor substance and a prostacyclin inhibitor in systemic circulation. However, the regulation mechanism for circulating CF6 is unknown. We investigated the role of tumor necrosis factor-alpha (TNF-alpha) in the generation and release of CF6. METHODS AND RESULTS: We used two kinds of cells, human umbilical vein endothelial cells (HUVEC) and ECV-304. The concentration of CF6 in the medium increased with time in both ECV-304 and HUVEC. Treatment of ECV-304 and HUVEC with TNF-alpha enhanced the release of CF6 in a dose-dependent manner concomitantly with the decrease in CF6 content in the mitochondria at 24 h. The released CF6 was characterized to be an active full-length peptide by Western blot. The ratio of CF6 to GAPDH mRNA, measured by real-time polymerase chain reaction, was 1.7 fold increased at 1 h after exposure to TNF-alpha in ECV-304 and HUVEC. This enhanced gene expression and release was blocked or suppressed by 70% by stable transfection of dominant negative mutant I kappa B kinase alpha whose efficacy was confirmed by blockade of translocation of NF-kappa B p65 protein and of degradation of I kappa B alpha protein. Flow cytometry analysis revealed that the cell surface-associated CF6 was significantly increased at 24 h after TNF-alpha in a dose-dependent manner. CONCLUSIONS: TNF-alpha stimulates the gene expression of CF6 via activation of NF-kappa B signaling pathway, and promotes the release of CF6 from ECV-304 and HUVEC.
Asunto(s)
Células Endoteliales/metabolismo , ATPasas de Translocación de Protón Mitocondriales/genética , FN-kappa B/metabolismo , Factores de Acoplamiento de la Fosforilación Oxidativa/genética , Factor de Necrosis Tumoral alfa/metabolismo , Western Blotting/métodos , Línea Celular , Células Cultivadas , Dinoprost/análisis , Relación Dosis-Respuesta a Droga , Expresión Génica/efectos de los fármacos , Humanos , Proteínas I-kappa B/análisis , Proteínas I-kappa B/metabolismo , Microscopía Fluorescente , ATPasas de Translocación de Protón Mitocondriales/sangre , Factores de Acoplamiento de la Fosforilación Oxidativa/sangre , Radioinmunoensayo/métodos , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
OBJECTIVE: Coupling factor 6 is an endogenous inhibitor of prostacyclin synthesis and might function as an endogenous vasoconstrictor in the fashion of a circulating hormone in rats. We investigated the role of coupling factor 6 in human hypertension. METHODS AND RESULTS: The patients with essential hypertension (EH) (n = 30) received a series of normal salt diet (12 g salt/day) for 3 days, low salt diet (2 g salt/day) for 7 days, and high salt diet (20-23 g salt/day) for 7 days. Normotensive control subjects (n = 27) received normal and low salt diets. The plasma level of coupling factor 6, measured by radioimmunoassay, during normal salt diet was higher in patients with EH than in normotensive subjects (17.6 +/- 1.7 versus 12.8 +/- 0.5 ng/ml, P < 0.01). Whereas the plasma level of coupling factor 6 was unchanged after salt restriction in normotensive subjects, it was decreased after salt restriction (from 12 g/day to 2 g/day) and was increased after salt loading (from 2 g/day to 20-23 g/day) in patients with EH. This increase in plasma level of coupling factor 6 was abolished by oral administration of ascorbic acid, but the level of blood pressure was unaffected. The percentage changes in plasma coupling factor 6 level after salt restriction and loading were positively correlated with those in mean blood pressure (r = 0.57, P < 0.01), and negatively correlated with those in plasma nitric oxide level (r = -0.51, P < 0.05). CONCLUSION: These indicate that circulating coupling factor 6 is elevated in human hypertension and modulated by salt intake presumably via reactive oxygen species.
Asunto(s)
Hipertensión/sangre , ATPasas de Translocación de Protón Mitocondriales/sangre , Factores de Acoplamiento de la Fosforilación Oxidativa/sangre , Antioxidantes/administración & dosificación , Ácido Ascórbico/administración & dosificación , Biomarcadores/sangre , Presión Sanguínea/efectos de los fármacos , Dieta Hiposódica , Dinoprost/análogos & derivados , Dinoprost/sangre , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Femenino , Humanos , Hipertensión/fisiopatología , Masculino , Persona de Mediana Edad , ATPasas de Translocación de Protón Mitocondriales/efectos de los fármacos , Nitratos/sangre , Nitritos/sangre , Norepinefrina/sangre , Factores de Acoplamiento de la Fosforilación Oxidativa/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Renina/metabolismo , Sodio en la Dieta/administración & dosificación , Estadística como AsuntoRESUMEN
Vascular endothelial cells are exposed to an acidic pH, and CXC chemokine receptor type 4 (CXCR4) is a key protective molecule against acidosis. We investigated the effect of coupling factor 6 (CF6), a novel proton import activator, on CXCR4 signaling and its molecular mechanism. CF6 decreased CXCR4 expression in human umbilical vein endothelial cells (HUVECs) in a time- and dose-dependent manner. Pretreatment with small interfering RNA (siRNA) for hypoxia-inducible factor (HIF)-1α or PP1, a specific c-Src inhibitor, attenuated the CF6-induced decrease in CXCR4 without affecting CF6-induced intracellular acidosis. Chromatin immunoprecipitation revealed that CF6 enhanced the interaction between HIF-1α and the CXCR4 promoter at the hypoxia response element. CF6 also enhanced protein-protein interactions between phospho-c-Src and histone deacetylase 3 (HDAC3), but did not affect the binding of HDAC3 to the CXCR4 promoter at the hypoxia response element. Apoptotic cells, as measured by an Annexin-V-FITC Propidium Iodide Kit, were increased by CF6 in normoxia and hypoxia at 24 h; however, this increase was abolished by pretreatment with either siRNA for HIF-1α or the CXCR4 ligand. The coronary arteries and perivascular tissues obtained from CF6-overexpressing transgenic mice showed a lower expression of CXCR4 in the heart, increased wall thickness and infiltration of CD16-positive, CD206-positive or apoptotic cells. CF6 decreases CXCR4 expression through both HIF-1α- and c-Src-mediated mechanisms in vascular endothelial cells. Because CXCR4 has an important role in survival function, CF6 may have a role in the progression of arteriosclerosis via these complex mechanisms.
Asunto(s)
Apoptosis/efectos de los fármacos , Endotelio Vascular/efectos de los fármacos , Genes src/fisiología , Subunidad alfa del Factor 1 Inducible por Hipoxia/farmacología , Inflamación/patología , ATPasas de Translocación de Protón Mitocondriales/farmacología , Factores de Acoplamiento de la Fosforilación Oxidativa/farmacología , Receptores CXCR4/biosíntesis , Transducción de Señal/efectos de los fármacos , Animales , Células Endoteliales/efectos de los fármacos , Genes src/efectos de los fármacos , Humanos , Hipoxia/patología , Inflamación/inducido químicamente , Ratones , Ratones TransgénicosRESUMEN
The spontaneous microaggregation of platelets (SMAPs) is a marker for the prognosis of patients with cardiovascular diseases. Coupling factor 6 (CF6) binds to the plasma membrane ATP synthase and functions as a pro-atherogenic molecule in the cardiovascular system. However, the role of CF6 in SMAPs and stroke remains unknown. In 650 consecutive patients, including those with acute-onset stroke, and 20 control subjects, platelet-rich plasma (PRP) was obtained, and SMAP was measured using a laser light-scattering aggregometer. The cytosolic cyclic adenosine monophosphate (cAMP) concentration in platelets was measured using an enzyme-linked immunosorbent assay. CF6 increased SMAPs in patients and control subjects to a similar degree by binding to the α- and ß-subunits of ATP synthase and inducing intracellular acidosis. It was abolished by PRP pretreatment with antibodies against CF6, and the α- or ß-subunit of the plasma membrane ATP synthase, and the ATP synthase inhibitor efrapeptin. CF6 increased SMAPs in patient groups with and without antiplatelet therapy to a similar degree, and no difference was found among the subgroups taking aspirin, thienopyridine or cilostazol. The cytosolic cAMP concentration in platelets was decreased by CF6 in the presence of the direct adenylate cyclase activator forskolin. Pretreatment of PRP with the Gs activator cholera toxin blocked the decrease, whereas the Gi inactivator pertussis toxin and cilostazol had no influence. The CF6-induced acceleration of SMAPs was suppressed by cholera toxin but not by cilostazol or pertussis toxin. CF6 enhanced SMAPs by decreasing cytosolic cAMP. Because it was observed irrespective of antiplatelet agents, CF6 appears to be a novel target for antiplatelet therapy.
Asunto(s)
AMP Cíclico/metabolismo , Citosol/metabolismo , ATPasas de Translocación de Protón Mitocondriales/farmacología , Factores de Acoplamiento de la Fosforilación Oxidativa/farmacología , Inhibidores de Agregación Plaquetaria/farmacología , Agregación Plaquetaria/efectos de los fármacos , Complejos de ATP Sintetasa/metabolismo , Anciano , Área Bajo la Curva , Western Blotting , Toxina del Cólera/farmacología , Cilostazol , Citosol/efectos de los fármacos , Ensayo de Inmunoadsorción Enzimática , Femenino , Citometría de Flujo , Humanos , Concentración de Iones de Hidrógeno , Masculino , Selectina-P/metabolismo , Toxina del Pertussis/farmacología , Radioinmunoensayo , Factores de Riesgo , Estimulación Química , Accidente Cerebrovascular/sangre , Tetrazoles/farmacologíaRESUMEN
In male coupling factor 6 (CF6)-overexpressing transgenic (TG) mice, a high-salt diet induces hypertension and cardiac systolic dysfunction with excessive reactive oxygen species generation. However, the role of gender in CF6-mediated pathophysiology is unknown. We investigated the effects of ovariectomy and estrogen replacement on hypertension, cardiac dysfunction and Rac1 activity, which activates radical generation and the mineralocorticoid receptor, in female TG mice. Fifteen-week-old male and female TG and wild-type (WT) mice were fed a normal- or high-salt diet for 60 weeks. Systolic and diastolic blood pressures were higher in the TG mice fed a high-salt diet than in those fed a normal-salt diet at 20-60 weeks in males but only at 60 weeks in females. The blood pressure elevation under high-salt diet conditions was concomitant with a decrease in left ventricular fractional shortening. In the WT mice, neither blood pressure nor cardiac systolic function was influenced by a high-salt diet. In the female TG mice, bilateral ovariectomy induced hypertension with cardiac systolic dysfunction 8 weeks after the initiation of a high-salt diet. The ratios of Rac1 bound to guanosine triphosphate (Rac1-GTP) to total Rac1 in the heart and kidneys were increased in the ovariectomized TG mice, and estrogen replacement abolished the CF6-mediated pathophysiology induced under the high-salt diet conditions. The overexpression of CF6 induced salt-sensitive hypertension, complicated by systolic cardiac dysfunction, but its onset was delayed in females. Estrogen has an important role in the regulation of CF6-mediated pathophysiology, presumably via the downregulation of Rac1.
Asunto(s)
Estrógenos/farmacología , Hipertensión/inducido químicamente , Hipertensión/fisiopatología , ATPasas de Translocación de Protón Mitocondriales/fisiología , Factores de Acoplamiento de la Fosforilación Oxidativa/fisiología , Cloruro de Sodio Dietético/efectos adversos , Animales , Femenino , Guanosina Trifosfato/metabolismo , Riñón/metabolismo , Masculino , Ratones , Ratones Transgénicos , Miocardio/metabolismo , Neuropéptidos/metabolismo , Ovariectomía , Factores Sexuales , Cloruro de Sodio Dietético/farmacología , Disfunción Ventricular Izquierda/tratamiento farmacológico , Disfunción Ventricular Izquierda/fisiopatología , Proteínas de Unión al GTP rac/metabolismo , Proteína de Unión al GTP rac1RESUMEN
BACKGROUND: Regular exercise improves systolic cardiac dysfunction through Akt cascade-mediated physiological hypertrophy in congestive heart failure. Tissue acidosis impairs Akt cascade, and coupling factor 6 induces tissue acidosis via activation of ecto-F(1)F(o) complex. We tested the hypothesis that coupling factor 6 attenuates physiological cardiac hypertrophy induced by exercise and its benefit in mice. METHODS AND RESULTS: Adult wild-type mice (nâ=â20) and coupling factor 6-overexpressing transgenic mice (nâ=â20) were divided into two groups with or without 4-week exercise consisting of 90-min swimming twice daily. Left ventricular posterior wall and interventricular septum thicknesses were increased by 0.12â±â0.1 and 0.16â±â0.1 mm, respectively, after 4-week swimming in wild-type mice (both Pâ<â0.01), but unchanged in transgenic mice. Fractional shortening was increased from 37â±â1 to 41â±â1% after 4-week swimming in wild-type mice (Pâ<â0.05), whereas it was unchanged in transgenic. The insulin-like growth factor 1 (IGF-1) receptor protein and its phosphorylated form in the heart were both increased by 1.83â±â0.23 and 1.83â±â0.09 times, respectively, after 4-week swimming in wild-type mice (both Pâ<â0.05), but were unchanged in transgenic. Downstream phosphoinsulin receptor substrate 1, phosphoinositide 3-kinase, and phospho-Akt were increased by 2.22â±â0.22, 1.78â±â0.31, and 2.24â±â0.49 times, respectively, in wild-type mice (all Pâ<â0.05), but were unchanged in transgenic. Restoration of phospho-Akt by IGF-1 injection recovered left ventricular hypertrophy and systolic function after 4-week swimming in transgenic. CONCLUSION: Overexpression of coupling factor 6 attenuates exercise-induced physiological cardiac hypertrophy by downregulating Akt signaling, thereby cancelling its benefit for cardiac function in mice. Reduction in coupling factor 6 level seems to be useful for drawing the exercising effects on cardiac function.
Asunto(s)
Hipertrofia Ventricular Izquierda/metabolismo , ATPasas de Translocación de Protón Mitocondriales/metabolismo , Factores de Acoplamiento de la Fosforilación Oxidativa/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Esfuerzo Físico , Proteínas Proto-Oncogénicas c-akt/metabolismo , Adaptación Fisiológica , Animales , Presión Sanguínea/efectos de los fármacos , Presión Sanguínea/fisiología , Modelos Animales de Enfermedad , Expresión Génica , Ventrículos Cardíacos/efectos de los fármacos , Ventrículos Cardíacos/patología , Hipertrofia Ventricular Izquierda/etiología , Hipertrofia Ventricular Izquierda/genética , Factor I del Crecimiento Similar a la Insulina/genética , Factor I del Crecimiento Similar a la Insulina/metabolismo , Factor I del Crecimiento Similar a la Insulina/farmacología , Ratones , Ratones Transgénicos , ATPasas de Translocación de Protón Mitocondriales/genética , Factores de Acoplamiento de la Fosforilación Oxidativa/genética , Fosfoproteínas , ARN Mensajero/metabolismo , Receptor IGF Tipo 1/metabolismo , Transducción de Señal , Natación , Tabique Interventricular/efectos de los fármacos , Tabique Interventricular/patologíaRESUMEN
BACKGROUND AND PURPOSE: Homocysteine (Hcy) is an independent predictor of stroke. Coupling factor 6 (CF6) is regulated by nuclear factor kappa B (NF-kappaB) signaling which is activated by Hcy. We tested the hypothesis that CF6 is elevated with Hcy in stroke. We also examined the effect of vitamin treatment on CF6 and Hcy levels. METHODS AND RESULTS: The 59 Japanese patients with a recent history of stroke were randomly assigned to a group without vitamin treatment (Group 1, n = 29) and to a group with treatment with both folic acid and vitamin B(12) for 2 months (Group 2, n = 30). The CF6 level was elevated in the patients with stroke compared with that in controls (n = 64) at admission. In a multiple regression model, the plasma CF6 level was weakly correlated to the total Hcy (tHcy) level. In Group 1, the plasma tHcy and CF6 levels were unchanged. In Group 2, however, they were both decreased, and there was a weak positive correlation between the decreases in plasma levels of CF6 and tHcy. CONCLUSION: CF6 is elevated in patients with stroke independently of risk factors. Since Hcy and vitamin treatment affect CF6 levels in stroke, CF6 appears to be a novel molecule for the pathogenesis and treatment of stroke.
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Ácido Fólico/uso terapéutico , Homocisteína/sangre , ATPasas de Translocación de Protón Mitocondriales/sangre , Factores de Acoplamiento de la Fosforilación Oxidativa/sangre , Accidente Cerebrovascular/tratamiento farmacológico , Vitamina B 12/uso terapéutico , Complejo Vitamínico B/uso terapéutico , Anciano , Femenino , Humanos , Japón , Masculino , Persona de Mediana Edad , Accidente Cerebrovascular/sangreRESUMEN
OBJECTIVE: Reactive oxygen species are involved in the pathogenesis of congestive heart failure. We recently showed that coupling factor 6, a component of adenosine trisphosphate (ATP) synthase, induces hypertension by intracellular acidosis, which is related to reactive oxygen species generation. We investigated the effect of high-salt diet on the cardiac performance and reactive oxygen species generation in coupling factor 6-overexpressing transgenic mice. METHODS AND RESULTS: Baseline echocardiographic findings, reactive oxygen species generation, protein expression of sarcoplasmic/endoplasmic reticulum of Ca-ATPase 2 and phospholamban, and ATP content in the heart were similar between 7-week-old transgenic and wild-type mice. When the mice were fed with 8% salt diet for 20-24 weeks, fractional shortening of the left ventricle was decreased in transgenic mice compared with wild-type mice and was recovered by intraperitoneal administration of anticoupling factor 6 antibody. Nicotinamide adenine dinucleotide phosphate oxidase activity in the heart was increased in transgenic mice after the high-salt diet concomitantly with c-Src activation. The level of 8-iso-prostaglandin F2α was increased in transgenic heart compared with wild-type heart. The protein expression of sarcoplasmic/endoplasmic reticulum of Ca-ATPase 2 was decreased and that of phospholamban was increased in transgenic heart. In cDNA microarray analysis, the genes related to ATP synthesis and glycolysis were decreased in transgenic heart, concomitantly with the decrease in ATP content and the increase in ß-myosin heavy chain. CONCLUSION: These suggest that coupling factor 6 induces the development of systolic dysfunction and upregulation of nicotinamide adenine dinucleotide phosphate oxidase in the heart under the high-salt diet.
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ATPasas de Translocación de Protón Mitocondriales/genética , ATPasas de Translocación de Protón Mitocondriales/fisiología , Factores de Acoplamiento de la Fosforilación Oxidativa/genética , Factores de Acoplamiento de la Fosforilación Oxidativa/fisiología , Animales , Calcitonina/química , Proteínas de Unión al Calcio/genética , ATPasas Transportadoras de Calcio/genética , Dinoprost/análogos & derivados , Dinoprost/genética , Corazón/fisiología , Humanos , Ratones , Ratones Transgénicos , NADPH Oxidasas/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Regiones Promotoras Genéticas , Especies Reactivas de Oxígeno , Sales (Química)/farmacología , SístoleRESUMEN
Coupling factor 6 (CF6) is composed of 76 amino acids and is present in the peripheral stalk of mitochondrial ATP synthase. The generation of CF6 is positively regulated by tumor necrosis factor alpha and shear stress via nuclear factor kappaB, and by high glucose via protein kinase C and p38 mitogen-activated protein kinase. CF6 is released outside of the cells from vascular endothelial cells, and binds to the beta-subunit of the plasma membrane-bound ATP synthase in vascular endothelial cells and leads to intracellular acidosis. CF6 produces vasoconstriction, and the biological active site resides at the C-terminal portion. CF6 suppresses prostacyclin generation via inhibition of cytosolic phospholipase A(2). CF6 also suppresses nitric oxide synthase activity via an increase in asymmetric dimethylarginine and a decrease in platelet/endothelial cell adhesion molecule-1. CF6 induces the gene and protein expression of proatherogenic molecules such as endothelin 2, urokinase type plasminogen activator receptor, estrogen receptor beta, a soluble short form of vascular endothelial growth factor receptor-1, and lectin-like oxidized low-density lipoprotein receptor-1. The plasma level of CF6 is elevated in patients with essential hypertension, diabetes mellitus, end-stage renal disease, acute myocardial infarction, and coronary heart disease. It is likely that CF6 contributes to the pathogenesis of cardiovascular diseases, but further intensive investigation is needed.
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Aterosclerosis/metabolismo , Células Endoteliales/metabolismo , Endotelio Vascular/metabolismo , ATPasas de Translocación de Protón Mitocondriales/metabolismo , Factores de Acoplamiento de la Fosforilación Oxidativa/metabolismo , Animales , Endotelio Vascular/citología , Humanos , Transducción de Señal , Vasoconstricción/fisiologíaRESUMEN
Vascular endothelial growth factor (VEGF) is a well-known promoter of angiogenesis, but its receptor VEGFR-1 and a soluble short form of VEGFR-1 (sFlt-1) play a negative role in the VEGF signal pathway by trapping VEGF. We recently showed that endogenous prostacyclin inhibitor coupling factor 6 (CF6) forces the clockwise rotation of F(1) motor of plasma membrane adenosine triphosphate synthase and induces intracellular acidosis and c-Src activation. We investigated the role of CF6 in regulation of sFlt-1, and its mechanism in human umbilical vein endothelial cells. The ratio of sFlt-1 to glyceraldehyde 3-phosphate dehydrogenase mRNA was increased at 24 h by 1.59+/-0.29-fold by 10(-7) M CF6 (P<0.05), concomitantly with the increases in intercellular adhesion molecule-1 and lectin-like oxidized low-density lipoprotein receptor-1 and no change in VEGF-A. When the dose of CF6 was increased to 10(-6) M, no further increase in sFlt-1 mRNA was observed. The release of sFlt-1 protein was increased by 1.72+/-0.24-fold (P<0.05) at 48 h after exposure to CF6 at 10(-7) M, and it was blocked by pretreatment with anti-CF6 antibody. The immunoreactive bands for sFlt-1 and VEGFR-1 were both increased by CF6 to similar degrees. Pretreatment with PP1, an inhibitor of c-Src, and 10(-5) Mefrapeptin, an inhibitor of F(1) motor, inhibited CF6-induced increases in expression and release of sFlt-1 (P<0.05). In mice overexpressing CF6, the plasma level of sFlt-1 was increased by 1.36+/-0.29-fold compared with that in wild-type mice (P<0.05). These indicate that CF6 might increase the expression and release of sFlt-1 in the vessels through acidosis-induced c-Src activation.
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Acidosis/fisiopatología , Células Endoteliales/efectos de los fármacos , Endotelio Vascular/efectos de los fármacos , ATPasas de Translocación de Protón Mitocondriales/farmacología , Factores de Acoplamiento de la Fosforilación Oxidativa/farmacología , Proteínas Proto-Oncogénicas pp60(c-src)/fisiología , Receptor 1 de Factores de Crecimiento Endotelial Vascular/biosíntesis , Animales , Biotransformación/efectos de los fármacos , Western Blotting , Línea Celular , Endotelio Vascular/citología , Expresión Génica/efectos de los fármacos , Vectores Genéticos , Humanos , Hipoxia/metabolismo , Ratones , Ratones Transgénicos , ARN Mensajero/análisis , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Regulación hacia Arriba/fisiología , Receptor 1 de Factores de Crecimiento Endotelial Vascular/efectos de los fármacos , Receptor 1 de Factores de Crecimiento Endotelial Vascular/genéticaRESUMEN
OBJECTIVES: Coupling factor 6 (CF6) attenuates the endothelial generation of prostacyclin. However, the role of CF6 in the resistance arteriole that is directly related to vascular tone is not determined yet. We investigated the effect of endogenous and exogenous CF6 on prostacyclin generation in cultured vascular smooth muscle cells (VSMCs). METHODS AND RESULTS: We cultured resistance arteriole VSMCs from the mesenteric artery network of spontaneously hypertensive rats (SHRs, n = 8) and Wistar-Kyoto rats (WKY, n = 8) by enzymatic method. The gene expression of CF6 was higher by 76 +/- 24% in SHR-derived VSMCs compared with WKY rat-derived VSMCs (P < 0.05) concomitant with the reduced degradation rate of CF6 mRNA. The release of CF6 in SHRs was higher than that in WKY rats (11.0 +/- 0.8 vs. 3.8 +/- 0.4 pg/microg protein, P < 0.05). Prostacyclin generation was attenuated in mesenteric arteriolar VSMCs from SHRs compared with those from WKY rats, but it was restored by neutralization of CF6 with its antibody. Exogenous administration of CF6 suppressed arachidonic acid release in a dose-dependent manner, and it was greater in SHRs than in WKY rats. Pretreatment with PP1, an inhibitor of tyrosine kinase c-Src, or receptor blockers such as ADP, efrapeptin, and an antibody to beta-subunit of ATP synthase blocked CF6-induced decrease in prostacyclin generation. CONCLUSION: These data suggest that CF6 suppresses prostacyclin generation in resistance arteriole VSMCs in an autocrine or paracrine fashion, and it is enhanced in SHRs by the overproduction of CF6 and the hyperresponsiveness to CF6.
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Epoprostenol/biosíntesis , Hipertensión/metabolismo , ATPasas de Translocación de Protón Mitocondriales/metabolismo , Miocitos del Músculo Liso/metabolismo , Factores de Acoplamiento de la Fosforilación Oxidativa/metabolismo , Animales , Comunicación Autocrina , Células Cultivadas , Masculino , Arterias Mesentéricas/citología , Arterias Mesentéricas/metabolismo , Músculo Liso Vascular/metabolismo , Comunicación Paracrina , Ratas , Ratas Endogámicas SHR , Ratas Endogámicas WKYRESUMEN
AIMS: Coupling factor 6 (CF6) induces hypertension by attenuating the endothelial generation of prostacyclin. However, intracellular signalling of CF6 in the resistance arteriole vascular smooth muscle cells (VSMCs) that are directly related to vasoconstriction has not been determined. Here we investigated the direct effect of exogenous CF6 on Ca2+ signalling in cultured VSMCs and the in vivo role of endogenous CF6 in the genesis of hypertension using CF6 transgenic (TG) mice. METHODS AND RESULTS: CF6 induced a monophasic increase in the intracellular free Ca2+ concentration ([Ca2+]i) through nifedipine-sensitive Ca2+ channels in A7r5 cells, a cell line of VSMCs, and enhanced the angiotensin II-induced spike phase of [Ca2+]i to a greater degree in VSMCs derived from spontaneously hypertensive rats (SHRs). In the mesenteric arterioles obtained from CF6-TG mice that manifested hypertension, angiotensin II-induced vasoconstriction was enhanced, compared with wild-type mice, and its enhancement was abolished by an anti-CF6 antibody. Pre-treatment with PP1, a tyrosine kinase c-Src inhibitor, blocked CF6-induced increase in Ca2+ signalling in VSMCs and vasoconstriction in TG mice. The receptor of CF6 was F1 motor of adenosine triphosphate (ATP) synthase with a higher affinity in SHRs. CF6 decreased intracellular pH via activation of ATPase activity and led to c-Src activation to a greater degree in SHR-derived VSMCs. CONCLUSION: CF6 causes hypertension by directly enhancing Ca2+ signalling in VSMCs and vasoconstriction in the mesenteric arteriolar network via c-Src activation.