RESUMEN
Inflammation, oxidative stress, and copper (Cu) play an important role in cardiovascular disease, including atherosclerosis. We previously reported that cytosolic Cu chaperone antioxidant-1 (Atox1) translocates to the nucleus in response to inflammatory cytokines or exogenous Cu and that Atox1 is localized at the nucleus in the endothelium of inflamed atherosclerotic aorta. However, the roles of nuclear Atox1 and their function are poorly understood. Here we showed that Atox1 deficiency in ApoE-/- mice with a Western diet exhibited a significant reduction of atherosclerotic lesion formation. In vitro, adenovirus-mediated overexpression of nuclear-targeted Atox1 (Ad-Atox1-NLS) in cultured human endothelial cells (ECs) increased monocyte adhesion and reactive oxygen species (ROS) production compared to control cells (Ad-null). To address the underlying mechanisms, we performed genome-wide mapping of Atox1-regulated targets in ECs, using an unbiased systemic approach integrating sequencing data. Combination of ChIP-Seq and RNA-Seq analyses in ECs transfected with Ad-Atox1-NLS or Ad-null identified 1387 differentially expressed genes (DEG). Motif enrichment assay and KEGG pathway enrichment analysis revealed that 248 differentially expressed genes, including inflammatory and angiogenic genes, were regulated by Atox1-NLS, which was then confirmed by real-time qPCR. Among these genes, functional analysis of inflammatory responses identified CD137, CSF1, and IL5RA as new nuclear Atox1-targeted inflammatory genes, while CD137 is also a key regulator of Atox1-NLS-induced ROS production. These findings uncover new nuclear Atox1 downstream targets involved in inflammation and ROS production and provide insights into the nuclear Atox1 as a potential therapeutic target for the treatment of inflammatory diseases such as atherosclerosis.
Asunto(s)
Aterosclerosis , Cobre , Animales , Aterosclerosis/genética , Cobre/metabolismo , Proteínas Transportadoras de Cobre , Citocinas/metabolismo , Células Endoteliales/metabolismo , Humanos , Inflamación/genética , Ratones , Ratones Noqueados para ApoE , Chaperonas Moleculares/metabolismo , Especies Reactivas de Oxígeno/metabolismo , TranscriptomaRESUMEN
Vascular endothelial growth factor receptor type 2 (VEGFR2, also known as KDR and FLK1) signalling in endothelial cells (ECs) is essential for developmental and reparative angiogenesis. Reactive oxygen species and copper (Cu) are also involved in these processes. However, their inter-relationship is poorly understood. Evidence of the role of the endothelial Cu importer CTR1 (also known as SLC31A1) in VEGFR2 signalling and angiogenesis in vivo is lacking. Here, we show that CTR1 functions as a redox sensor to promote angiogenesis in ECs. CTR1-depleted ECs showed reduced VEGF-induced VEGFR2 signalling and angiogenic responses. Mechanistically, CTR1 was rapidly sulfenylated at Cys189 at its cytosolic C terminus after stimulation with VEGF, which induced CTR1-VEGFR2 disulfide bond formation and their co-internalization to early endosomes, driving sustained VEGFR2 signalling. In vivo, EC-specific Ctr1-deficient mice or CRISPR-Cas9-generated redox-dead Ctr1(C187A)-knockin mutant mice had impaired developmental and reparative angiogenesis. Thus, oxidation of CTR1 at Cys189 promotes VEGFR2 internalization and signalling to enhance angiogenesis. Our study uncovers an important mechanism for sensing reactive oxygen species through CTR1 to drive neovascularization.
Asunto(s)
Transportador de Cobre 1/metabolismo , Cobre/metabolismo , Neovascularización Fisiológica/fisiología , Especies Reactivas de Oxígeno/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Animales , Bovinos , Línea Celular , Transportador de Cobre 1/genética , Cisteína/metabolismo , Femenino , Células HEK293 , Células Endoteliales de la Vena Umbilical Humana , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Oxidación-Reducción , Transducción de Señal/fisiologíaRESUMEN
VEGFR2 (KDR/Flk1) signaling in endothelial cells (ECs) plays a central role in angiogenesis. The P-type ATPase transporter ATP7A regulates copper homeostasis, and its role in VEGFR2 signaling and angiogenesis is entirely unknown. Here, we describe the unexpected crosstalk between the Copper transporter ATP7A, autophagy, and VEGFR2 degradation. The functional significance of this Copper transporter was demonstrated by the finding that inducible EC-specific ATP7A deficient mice or ATP7A-dysfunctional ATP7Amut mice showed impaired post-ischemic neovascularization. In ECs, loss of ATP7A inhibited VEGF-induced VEGFR2 signaling and angiogenic responses, in part by promoting ligand-induced VEGFR2 protein degradation. Mechanistically, VEGF stimulated ATP7A translocation from the trans-Golgi network to the plasma membrane where it bound to VEGFR2, which prevented autophagy-mediated lysosomal VEGFR2 degradation by inhibiting autophagic cargo/adapter p62/SQSTM1 binding to ubiquitinated VEGFR2. Enhanced autophagy flux due to ATP7A dysfunction in vivo was confirmed by autophagy reporter CAG-ATP7Amut -RFP-EGFP-LC3 transgenic mice. In summary, our study uncovers a novel function of ATP7A to limit autophagy-mediated degradation of VEGFR2, thereby promoting VEGFR2 signaling and angiogenesis, which restores perfusion recovery and neovascularization. Thus, endothelial ATP7A is identified as a potential therapeutic target for treatment of ischemic cardiovascular diseases.
Asunto(s)
Autofagia/genética , Vasos Sanguíneos/metabolismo , ATPasas Transportadoras de Cobre/genética , ATPasas Tipo P/genética , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genética , Animales , Vasos Sanguíneos/efectos de los fármacos , Vasos Sanguíneos/fisiología , Células COS , Células Cultivadas , Chlorocebus aethiops , ATPasas Transportadoras de Cobre/metabolismo , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Células Endoteliales/fisiología , Humanos , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Proteínas Asociadas a Microtúbulos/metabolismo , ATPasas Tipo P/metabolismo , Interferencia de ARN , Transducción de Señal/genética , Factor A de Crecimiento Endotelial Vascular/farmacología , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
OBJECTIVE: Copper (Cu) is essential micronutrient, and its dysregulation is implicated in aortic aneurysm (AA) development. The Cu exporter ATP7A (copper-transporting P-type ATPase/Menkes ATPase) delivers Cu via the Cu chaperone Atox1 (antioxidant 1) to secretory Cu enzymes, such as lysyl oxidase, and excludes excess Cu. Lysyl oxidase is shown to protect against AA formation. However, the role and mechanism of ATP7A in AA pathogenesis remain unknown. Approach and Results: Here, we show that Cu chelator markedly inhibited Ang II (angiotensin II)-induced abdominal AA (AAA) in which ATP7A expression was markedly downregulated. Transgenic ATP7A overexpression prevented Ang II-induced AAA formation. Conversely, Cu transport dysfunctional ATP7Amut/+/ApoE-/- mice exhibited robust AAA formation and dissection, excess aortic Cu accumulation as assessed by X-ray fluorescence microscopy, and reduced lysyl oxidase activity. In contrast, AAA formation was not observed in Atox1-/-/ApoE-/- mice, suggesting that decreased lysyl oxidase activity, which depends on both ATP7A and Atox1, was not sufficient to develop AAA. Bone marrow transplantation suggested importance of ATP7A in vascular cells, not bone marrow cells, in AAA development. MicroRNA (miR) array identified miR-125b as a highly upregulated miR in AAA from ATP7Amut/+/ApoE-/- mice. Furthermore, miR-125b target genes (histone methyltransferase Suv39h1 and the NF-κB negative regulator TNFAIP3 [tumor necrosis factor alpha induced protein 3]) were downregulated, which resulted in increased proinflammatory cytokine expression, aortic macrophage recruitment, MMP (matrix metalloproteinase)-2/9 activity, elastin fragmentation, and vascular smooth muscle cell loss in ATP7Amut/+/ApoE-/- mice and reversed by locked nucleic acid-anti-miR-125b infusion. CONCLUSIONS: ATP7A downregulation/dysfunction promotes AAA formation via upregulating miR-125b, which augments proinflammatory signaling in a Cu-dependent manner. Thus, ATP7A is a potential therapeutic target for inflammatory vascular disease.
Asunto(s)
Aneurisma de la Aorta Abdominal/genética , Aneurisma de la Aorta Abdominal/fisiopatología , ATPasas Transportadoras de Cobre/fisiología , MicroARNs/fisiología , Angiotensina II/efectos de los fármacos , Animales , Apoptosis , Células Cultivadas , Quelantes/farmacología , Cobre/metabolismo , Proteínas Transportadoras de Cobre/metabolismo , ATPasas Transportadoras de Cobre/genética , Modelos Animales de Enfermedad , Regulación hacia Abajo , Femenino , Humanos , Inflamación/genética , Inflamación/fisiopatología , Masculino , Ratones Endogámicos C57BL , Ratones Transgénicos , Chaperonas Moleculares/metabolismo , Molibdeno/farmacología , Músculo Liso Vascular/citología , Regulación hacia ArribaRESUMEN
Copper (Cu) has emerged as an important modifier of body lipid metabolism. However, how Cu contributes to the physiology of fat cells remains largely unknown. We found that adipocytes require Cu to establish a balance between main metabolic fuels. Differentiating adipocytes increase their Cu uptake along with the ATP7A-dependent transport of Cu into the secretory pathway to activate a highly up-regulated amino-oxidase copper-containing 3 (AOC3)/semicarbazide-sensitive amine oxidase (SSAO); in vivo, the activity of SSAO depends on the organism's Cu status. Activated SSAO oppositely regulates uptake of glucose and long-chain fatty acids and remodels the cellular proteome to coordinate changes in fuel availability and related downstream processes, such as glycolysis, de novo lipogenesis, and sphingomyelin/ceramide synthesis. The loss of SSAO-dependent regulation due to Cu deficiency, limited Cu transport to the secretory pathway, or SSAO inactivation shifts metabolism towards lipid-dependent pathways and results in adipocyte hypertrophy and fat accumulation. The results establish a role for Cu homeostasis in adipocyte metabolism and identify SSAO as a regulator of energy utilization processes in adipocytes.
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Adipocitos/enzimología , Adipocitos/metabolismo , Amina Oxidasa (conteniendo Cobre)/metabolismo , Cobre/metabolismo , Células 3T3-L1 , Animales , Secuencia de Bases , Transporte Biológico , Diferenciación Celular , Forma de la Célula , Tamaño de la Célula , Cobre/deficiencia , ATPasas Transportadoras de Cobre/metabolismo , Metabolismo Energético , Activación Enzimática , Ácidos Grasos/biosíntesis , Glucosa/metabolismo , Homeostasis , Hipertrofia , Masculino , Ratones , Proteómica , Ratas Wistar , Vías Secretoras , Triglicéridos/metabolismoRESUMEN
Copper (Cu) is an essential micronutrient but excess Cu is potentially toxic. Its important propensity to cycle between two oxidation states accounts for its frequent presence as a cofactor in many physiological processes through Cu-containing enzymes, including mitochondrial energy production (via cytochrome c-oxidase), protection against oxidative stress (via superoxide dismutase), and extracellular matrix stability (via lysyl oxidase). Since free Cu is potentially toxic, the bioavailability of intracellular Cu is tightly controlled by Cu transporters and Cu chaperones. Recent evidence reveals that these Cu transport systems play an essential role in the physiological responses of cardiovascular cells, including cell growth, migration, angiogenesis and wound repair. In response to growth factors, cytokines, and hypoxia, their expression, subcellular localization, and function are tightly regulated. Cu transport systems and their regulators have also been linked to various cardiovascular pathophysiologies such as hypertension, inflammation, atherosclerosis, diabetes, cardiac hypertrophy, and cardiomyopathy. A greater appreciation of the central importance of Cu transporters and Cu chaperones in cell signaling and gene expression in cardiovascular biology offers the possibility of identifying new therapeutic targets for cardiovascular disease.
Asunto(s)
Enfermedades Cardiovasculares/metabolismo , Sistema Cardiovascular/metabolismo , Proteínas de Transporte de Catión/metabolismo , Cobre/metabolismo , Chaperonas Moleculares/metabolismo , Animales , Enfermedades Cardiovasculares/patología , Sistema Cardiovascular/fisiopatología , Expresión Génica/fisiología , Humanos , Transducción de Señal/fisiologíaRESUMEN
The Na,K-ATPase α2 subunit plays a key role in cardiac muscle contraction by regulating intracellular Ca2+, whereas α1 has a more conventional role of maintaining ion homeostasis. The ß subunit differentially regulates maturation, trafficking, and activity of α-ß heterodimers. It is not known whether the distinct role of α2 in the heart is related to selective assembly with a particular one of the three ß isoforms. We show here by immunofluorescence and co-immunoprecipitation that α2 is preferentially expressed with ß2 in T-tubules of cardiac myocytes, forming α2ß2 heterodimers. We have expressed human α1ß1, α2ß1, α2ß2, and α2ß3 in Pichia pastoris, purified the complexes, and compared their functional properties. α2ß2 and α2ß3 differ significantly from both α2ß1 and α1ß1 in having a higher K0.5K+ and lower K0.5Na+ for activating Na,K-ATPase. These features are the result of a large reduction in binding affinity for extracellular K+ and shift of the E1P-E2P conformational equilibrium toward E1P. A screen of perhydro-1,4-oxazepine derivatives of digoxin identified several derivatives (e.g. cyclobutyl) with strongly increased selectivity for inhibition of α2ß2 and α2ß3 over α1ß1 (range 22-33-fold). Molecular modeling suggests a possible basis for isoform selectivity. The preferential assembly, specific T-tubular localization, and low K+ affinity of α2ß2 could allow an acute response to raised ambient K+ concentrations in physiological conditions and explain the importance of α2ß2 for cardiac muscle contractility. The high sensitivity of α2ß2 to digoxin derivatives explains beneficial effects of cardiac glycosides for treatment of heart failure and potential of α2ß2-selective digoxin derivatives for reducing cardiotoxicity.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Proteínas de Transporte de Catión/metabolismo , Moléculas de Adhesión Celular Neuronal/metabolismo , Inhibidores Enzimáticos/química , Miocardio/enzimología , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Adenosina Trifosfatasas/antagonistas & inhibidores , Adenosina Trifosfatasas/química , Animales , Proteínas de Transporte de Catión/antagonistas & inhibidores , Proteínas de Transporte de Catión/química , Moléculas de Adhesión Celular Neuronal/antagonistas & inhibidores , Moléculas de Adhesión Celular Neuronal/química , Dimerización , Inhibidores Enzimáticos/metabolismo , Humanos , Isoenzimas/antagonistas & inhibidores , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Ratones , Miocardio/química , Potasio/química , Potasio/metabolismo , Sodio/química , Sodio/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidores , ATPasa Intercambiadora de Sodio-Potasio/química , ATPasa Intercambiadora de Sodio-Potasio/genéticaRESUMEN
The photolysis quantum yield, Qp, of 1-(2-nitrophenyl)ethyl phosphate (caged Pi) measured in the near-UV (342 nm peak with 60 nm half-bandwidth) is 0.53 and is based on results reported in 1978 (Biochemistry, 17, 1929-1935). This article amplifies methodology for determining that Qp in view of different recent estimates. Some general principles together with other examples relating to measurement of Qp values are discussed together with their relevance to biological research.
Asunto(s)
Organofosfatos/química , Fotólisis , Espectrofotometría Ultravioleta , Rayos UltravioletaRESUMEN
Cu ion (Cu) entry into human cells is mediated by CTR1 (also known as SLC31A1), the high-affinity Cu transporter. When extracellular Cu is raised, the cell is protected against excess accumulation by rapid internalization of the transporter. When Cu is lowered, the transporter returns to the membrane. We show in HEK293 cells overexpressing CTR1 that expression of either the C-terminal domain of AP180 (also known as SNAP91), a clathrin-coat assembly protein that sequesters clathrin, or a dominant-negative mutant of dynamin, decreases Cu-induced endocytosis of CTR1, as does a dynamin inhibitor and clathrin knockdown using siRNA. Utilizing imaging, siRNA techniques and a new high-throughput assay for endocytosis employing CLIP-tag methodology, we show that internalized CTR1 accumulates in early sorting endosomes and recycling compartments (containing Rab5 and EEA1), but not in late endosomes or lysosomal pathways. Using live cell fluorescence, we find that upon extracellular Cu removal CTR1 recycles to the cell surface through the slower-recycling Rab11-mediated pathway. These processes enable cells to dynamically alter transporter levels at the plasma membrane and acutely modulate entry as a safeguard against excess cellular Cu.
Asunto(s)
Proteínas de Transporte de Catión/metabolismo , Clatrina/metabolismo , Cobre/metabolismo , Dinaminas/metabolismo , Endosomas/metabolismo , Clatrina/genética , Transportador de Cobre 1 , Dinaminas/genética , Endocitosis , Células HEK293 , Homeostasis , Humanos , Transporte Iónico , Proteínas de Ensamble de Clatrina Monoméricas/genética , Proteínas de Ensamble de Clatrina Monoméricas/metabolismo , Mutación/genética , Transporte de Proteínas , ARN Interferente Pequeño , Proteínas de Transporte Vesicular/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Proteínas de Unión al GTP rab5/metabolismoRESUMEN
Cu is an essential micronutrient, and its role in an array of critical physiological processes is receiving increasing attention. Among these are wound healing, angiogenesis, protection against reactive oxygen species, neurotransmitter synthesis, modulation of normal cell and tumor growth, and many others. Free Cu is absent inside cells, and a network of proteins has evolved to deliver this essential, but potentially toxic, metal ion to its intracellular target sites following uptake. Although the total body content is low (â¼100 mg), dysfunction of proteins involved in Cu homeostasis results in several well-characterized human disease states. The initial step in cellular Cu handling is its transport across the plasma membrane, a subject of study for only about the last 25 years. This review focuses on the initial step in Cu homeostasis, the properties of the major protein, hCTR1, that mediates Cu uptake, and the status of our understanding of this highly specialized transport system. Although a high-resolution structure of the protein is still lacking, an array of biochemical and biophysical studies have provided a picture of how hCTR1 mediates Cu(I) transport and how Cu is delivered to the proteins in the intracellular milieu. Recent studies provide evidence that the transporter also plays a key protective role in the regulation of cellular Cu via regulatory endocytosis, lowering its surface expression, in response to elevated Cu loads.
Asunto(s)
Células/metabolismo , Cobre/metabolismo , Animales , Transporte Biológico , Proteínas de Transporte de Catión/química , Proteínas de Transporte de Catión/metabolismo , Cobre/toxicidad , Regulación de la Expresión Génica , Homeostasis , HumanosRESUMEN
This paper is the third in a series of reviews published in this issue resulting from the University of California Davis Cardiovascular Symposium 2014: Systems approach to understanding cardiac excitation-contraction coupling and arrhythmias: Na(+) channel and Na(+) transport. The goal of the symposium was to bring together experts in the field to discuss points of consensus and controversy on the topic of sodium in the heart. The present review focuses on cardiac Na(+)/Ca(2+) exchange (NCX) and Na(+)/K(+)-ATPase (NKA). While the relevance of Ca(2+) homeostasis in cardiac function has been extensively investigated, the role of Na(+) regulation in shaping heart function is often overlooked. Small changes in the cytoplasmic Na(+) content have multiple effects on the heart by influencing intracellular Ca(2+) and pH levels thereby modulating heart contractility. Therefore it is essential for heart cells to maintain Na(+) homeostasis. Among the proteins that accomplish this task are the Na(+)/Ca(2+) exchanger (NCX) and the Na(+)/K(+) pump (NKA). By transporting three Na(+) ions into the cytoplasm in exchange for one Ca(2+) moved out, NCX is one of the main Na(+) influx mechanisms in cardiomyocytes. Acting in the opposite direction, NKA moves Na(+) ions from the cytoplasm to the extracellular space against their gradient by utilizing the energy released from ATP hydrolysis. A fine balance between these two processes controls the net amount of intracellular Na(+) and aberrations in either of these two systems can have a large impact on cardiac contractility. Due to the relevant role of these two proteins in Na(+) homeostasis, the emphasis of this review is on recent developments regarding the cardiac Na(+)/Ca(2+) exchanger (NCX1) and Na(+)/K(+) pump and the controversies that still persist in the field.
Asunto(s)
Potenciales de Acción , Arritmias Cardíacas/metabolismo , Miocitos Cardíacos/metabolismo , Intercambiador de Sodio-Calcio/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Animales , Congresos como Asunto , Humanos , Miocitos Cardíacos/fisiologíaRESUMEN
Pulmonary vascular remodeling and increased arterial wall stiffness are two major causes for the elevated pulmonary vascular resistance and pulmonary arterial pressure in patients and animals with pulmonary hypertension. Cellular copper (Cu) plays an important role in angiogenesis and extracellular matrix remodeling; increased Cu in vascular smooth muscle cells has been demonstrated to be associated with atherosclerosis and hypertension in animal experiments. In this study, we show that the Cu-uptake transporter 1, CTR1, and the Cu-efflux pump, ATP7A, were both upregulated in the lung tissues and pulmonary arteries of mice with hypoxia-induced pulmonary hypertension. Hypoxia also significantly increased expression and activity of lysyl oxidase (LOX), a Cu-dependent enzyme that causes crosslinks of collagen and elastin in the extracellular matrix. In vitro experiments show that exposure to hypoxia or treatment with cobalt (CoCl2) also increased protein expression of CTR1, ATP7A, and LOX in pulmonary arterial smooth muscle cells (PASMC). In PASMC exposed to hypoxia or treated with CoCl2, we also confirmed that the Cu transport is increased using 64Cu uptake assays. Furthermore, hypoxia increased both cell migration and proliferation in a Cu-dependent manner. Downregulation of hypoxia-inducible factor 1α (HIF-1α) with siRNA significantly attenuated hypoxia-mediated upregulation of CTR1 mRNA. In summary, the data from this study indicate that increased Cu transportation due to upregulated CTR1 and ATP7A in pulmonary arteries and PASMC contributes to the development of hypoxia-induced pulmonary hypertension. The increased Cu uptake and elevated ATP7A also facilitate the increase in LOX activity and thus the increase in crosslink of extracellular matrix, and eventually leading to the increase in pulmonary arterial stiffness.
Asunto(s)
Proteínas de Transporte de Catión/genética , Cobre/metabolismo , Hipertensión Pulmonar/etiología , Hipertensión Pulmonar/genética , Hipoxia/complicaciones , Regulación hacia Arriba/genética , Animales , Apoptosis/efectos de los fármacos , Proteínas de Transporte de Catión/metabolismo , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Quelantes/farmacología , Cobalto/farmacología , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/genética , Técnicas de Silenciamiento del Gen , Humanos , Hipertensión Pulmonar/patología , Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Pulmón/patología , Masculino , Ratones Endogámicos C57BL , Miocitos del Músculo Liso/metabolismo , Antígeno Nuclear de Célula en Proliferación/metabolismo , Proteína-Lisina 6-Oxidasa/antagonistas & inhibidores , Proteína-Lisina 6-Oxidasa/genética , Proteína-Lisina 6-Oxidasa/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Arteria Pulmonar/efectos de los fármacos , Arteria Pulmonar/metabolismo , Arteria Pulmonar/patología , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Cardiotonic steroids (CTS), specific inhibitors of Na,K-ATPase activity, have been widely used for treating cardiac insufficiency. Recent studies suggest that low levels of endogenous CTS do not inhibit Na,K-ATPase activity but play a role in regulating blood pressure, inducing cellular kinase activity, and promoting cell viability. Higher CTS concentrations inhibit Na,K-ATPase activity and can induce reactive oxygen species, growth arrest, and cell death. CTS are being considered as potential novel therapies in cancer treatment, as they have been shown to limit tumor cell growth. However, there is a lack of information on the relative toxicity of tumor cells and comparable non-tumor cells. We have investigated the effects of CTS compounds, ouabain, digitoxin, and bufalin, on cell growth and survival in cell lines exhibiting the full spectrum of non-cancerous to malignant phenotypes. We show that CTS inhibit membrane Na,K-ATPase activity equally well in all cell lines tested regardless of metastatic potential. In contrast, the cellular responses to the drugs are different in non-tumor and tumor cells. Ouabain causes greater inhibition of proliferation and more extensive apoptosis in non-tumor breast cells compared to malignant or oncogene-transfected cells. In tumor cells, the effects of ouabain are accompanied by activation of anti-apoptotic ERK1/2. However, ERK1/2 or Src inhibition does not sensitize tumor cells to CTS cytotoxicity, suggesting that other mechanisms provide protection to the tumor cells. Reduced CTS-sensitivity in breast tumor cells compared to non-tumor cells indicates that CTS are not good candidates as cancer therapies.
Asunto(s)
Apoptosis/efectos de los fármacos , Neoplasias de la Mama/tratamiento farmacológico , Glicósidos Cardíacos/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Glándulas Mamarias Humanas/metabolismo , Animales , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Femenino , Humanos , Glándulas Mamarias Humanas/patología , Ratones , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Proteínas de Neoplasias/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Familia-src Quinasas/metabolismoRESUMEN
Human copper transporter 1 (hCTR1) is a homotrimer of a 190-amino acid monomer having three transmembrane domains believed to form a pore for copper permeation through the plasma membrane. The hCTR1-mediated copper transport mechanism is not well understood, nor has any measurement been made of the rate at which copper ions are transported by hCTR1. In this study, we estimated the rate of copper transport by the hCTR1 trimer in cultured cells using (64)Cu uptake assays and quantification of plasma membrane hCTR1. For endogenous hCTR1, we estimated a turnover number of about 10 ions/trimer/s. When overexpressed in HEK293 cells, a second transmembrane domain mutant of hCTR1 (H139R) had a 3-fold higher Km value and a 4-fold higher turnover number than WT. Truncations of the intracellular C-terminal tail and an AAA substitution of the putative metal-binding HCH C-terminal tripeptide (thought to be required for transport) also exhibited elevated transport rates and Km values when compared with WT hCTR1. Unlike WT hCTR1, H139R and the C-terminal mutants did not undergo regulatory endocytosis in elevated copper. hCTR1 mutants combining methionine substitutions that block transport (M150L,M154L) on the extracellular side of the pore and the high transport H139R or AAA intracellular side mutations exhibited the blocked transport of M150L,M154L, confirming that Cu(+) first interacts with the methionines during permeation. Our results show that hCTR1 elements on the intracellular side of the hCTR1 pore, including the carboxyl tail, are not essential for permeation, but serve to regulate the rate of copper entry.
Asunto(s)
Proteínas de Transporte de Catión/metabolismo , Membrana Celular/metabolismo , Cobre/metabolismo , Endocitosis , Western Blotting , Células CACO-2 , Proteínas de Transporte de Catión/química , Proteínas de Transporte de Catión/genética , Cobre/farmacocinética , Radioisótopos de Cobre , Transportador de Cobre 1 , Células HEK293 , Humanos , Transporte Iónico , Cinética , Microscopía Confocal , Mutación , Multimerización de ProteínaRESUMEN
Cisplatin (cDDP) is an anticancer drug used in a number of malignancies, including testicular, ovarian, cervical, bladder, lung, head, and neck cancers. Its use is limited by the development of resistance, often rationalized via effects on cellular uptake. It has been claimed that human copper transporter 1 (hCTR1), the human high-affinity copper transporter, is the major entry pathway for cDDP and related drugs via a mechanism that mimics copper. This is an unexpected property of hCTR1, a highly selective copper (I) transporter. We compared the uptake rates of copper with cDDP (and several analogs) into human embryonic kidney 293 cells overexpressing wild-type or mutant hCTR1, mouse embryonic fibroblasts that do or do not express CTR1, and human ovarian tumor cells that are sensitive or resistant to cDDP. We have also compared the effects of extracellular copper, which causes regulatory endocytosis of hCTR1, to those of cDDP. We confirm the correlation between higher hCTR1 levels and higher platinum drug uptake in tumor cells sensitive to the drug. However, we show that hCTR1 is not the major entry route of platinum drugs, and that the copper transporter is not internalized in response to extracellular drug. Our data suggest the major entry pathway for platinum drugs is not saturable at relevant concentrations and not protein-mediated. Clinical trials have been initiated that depend upon regulating membrane levels of hCTR1. If reduced drug uptake is a major factor in resistance, hCTR1 is unlikely to be a productive target in attempts to enhance efficacy, although the proteins involved in copper homeostasis may play a role.
Asunto(s)
Antineoplásicos/metabolismo , Proteínas de Transporte de Catión/metabolismo , Cisplatino/metabolismo , Resistencia a Antineoplásicos , Animales , Antineoplásicos/farmacología , Proteínas de Transporte de Catión/genética , Línea Celular Tumoral , Cisplatino/farmacología , Transportador de Cobre 1 , Fibroblastos/metabolismo , Técnicas de Silenciamiento del Gen , Células HEK293 , Humanos , RatonesRESUMEN
Copper is an essential micronutrient. Following entry via the human copper transporter 1 (hCTR1), copper is delivered to several copper chaperones, which subsequently transfer the metal to specific targets via protein:protein interactions. It is has been assumed, but not demonstrated, that chaperones acquire copper directly from hCTR1. However, some reports have pointed to an intermediary role for glutathione (GSH), an abundant copper-binding tri-peptide. To address the issue of how transported copper is acquired by the copper chaperones in vivo, we measured the initial rate of (64)Cu uptake in cells in which the cellular levels of copper chaperones or GSH were substantially depleted or elevated. Knockdown or overexpression of copper chaperones ATOX1, CCS, or both had no effect on the initial rate of (64)Cu entry into HEK293 cells having endogenous or overexpressed hCTR1. In contrast, depleting cellular GSH using L-buthionine-sulfoximine (BSO) caused a 50% decrease in the initial rate of (64)Cu entry in HEK293 cells and other cell types. This decrease was reversed by washout of BSO or GSH replenishment with a permeable ester. BSO treatment under our experimental conditions had no significant effects on the viability, ATP levels, or metal content of the cells. Attenuated (64)Cu uptake in BSO was not due to oxidation of the cysteine in the putative metal-binding motif (HCH) at the intracellular hCTR1 COOH terminus, because a mutant lacking this motif was fully active, and (64)Cu uptake was still reduced by BSO treatment. Our data suggest that GSH plays an important role in copper handling at the entry step.
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Proteínas de Transporte de Catión/fisiología , Cobre/metabolismo , Glutatión/fisiología , Transporte Biológico/fisiología , Membrana Celular/metabolismo , Transportador de Cobre 1 , Células HEK293 , HumanosRESUMEN
Restoration of the functional potency of pancreatic islets either through enhanced proliferation (hyperplasia) or increase in size (hypertrophy) of beta cells is a major objective for intervention in diabetes. We have obtained experimental evidence that global knock-out of a small, single-span regulatory subunit of Na,K-ATPase, FXYD2, alters glucose control. Adult Fxyd2(-/-) mice showed significantly lower blood glucose levels, no signs of peripheral insulin resistance, and improved glucose tolerance compared with their littermate controls. Strikingly, there was a substantial hyperplasia in pancreatic beta cells from the Fxyd2(-/-) mice compared with the wild type littermates, compatible with an observed increase in the level of circulating insulin. No changes were seen in the exocrine compartment of the pancreas, and the mice had only a mild, well-adapted renal phenotype. Morphometric analysis revealed an increase in beta cell mass in KO compared with WT mice. This appears to explain a phenotype of hyperinsulinemia. By RT-PCR, Western blot, and immunocytochemistry we showed the FXYD2b splice variant in pancreatic beta cells from wild type mice. Phosphorylation of Akt kinase was significantly higher under basal conditions in freshly isolated islets from Fxyd2(-/-) mice compared with their WT littermates. Inducible expression of FXYD2 in INS 832/13 cells produced a reduction in the phosphorylation level of Akt, and phosphorylation was restored in parallel with degradation of FXYD2. Thus we suggest that in pancreatic beta cells FXYD2 plays a role in Akt signaling pathways associated with cell growth and proliferation.
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Glucemia/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/sangre , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Empalme Alternativo , Animales , Western Blotting , Línea Celular Tumoral , Femenino , Regulación Enzimológica de la Expresión Génica , Prueba de Tolerancia a la Glucosa , Hiperplasia , Inmunohistoquímica , Células Secretoras de Insulina/patología , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , ATPasa Intercambiadora de Sodio-Potasio/genéticaRESUMEN
Owing to their redox and coordination chemistry copper ions play essential roles in cellular function. Research over the past 20 years has shed much light on the biochemistry of copper homeostasis, and the emergence of high-resolution crystal structures for many of the proteins that partake in cellular copper biology have began to provide insight into the molecular mechanisms by which cells handle this important metal. A notable gap in our understanding is related to the process by which cells acquire copper ions. This chapter describes recent progress in the structure determination of cellular copper uptake transporters and how the emerging structural information aids understanding of the molecular mechanisms that govern cellular copper acquisition and distribution.
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Proteínas de Transporte de Catión/metabolismo , Cobre/metabolismo , Transporte Biológico , Proteínas de Transporte de Catión/química , Transportador de Cobre 1 , Humanos , Estructura Terciaria de Proteína , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
There has been a vigorous debate for decades concerning the heritability of intelligence. In recent years, the debate has been focused on whether the components of IQ variability change with age and on separating environmental effects into shared and unshared components. Citing evidence from adoption studies, studies comparing identical and fraternal twins, and studies of identical twins raised apart, some prominent psychologists have concluded that the shared environment has a significant effect on the intelligence of children but little or no effect on the intelligence of adults. In this article, the evidence from such studies is reviewed. The article reaches the conclusion that while there is some evidence from adoption studies supporting the claim that shared environment has little or no effect on adult intelligence, that evidence is inconclusive and is inconsistent with evidence from twin studies and from studies of identical twins reared apart.