RESUMEN
The N-terminal caveolin-binding motif (CBM) in Na/K-ATPase (NKA) α1 subunit is essential for cell signaling and somitogenesis in animals. To further investigate the molecular mechanism, we have generated CBM mutant human-induced pluripotent stem cells (iPSCs) through CRISPR/Cas9 genome editing and examined their ability to differentiate into skeletal muscle (Skm) cells. Compared with the parental wild-type human iPSCs, the CBM mutant cells lost their ability of Skm differentiation, which was evidenced by the absence of spontaneous cell contraction, marker gene expression, and subcellular myofiber banding structures in the final differentiated induced Skm cells. Another NKA functional mutant, A420P, which lacks NKA/Src signaling function, did not produce a similar defect. Indeed, A420P mutant iPSCs retained intact pluripotency and ability of Skm differentiation. Mechanistically, the myogenic transcription factor MYOD was greatly suppressed by the CBM mutation. Overexpression of a mouse Myod cDNA through lentiviral delivery restored the CBM mutant cells' ability to differentiate into Skm. Upstream of MYOD, Wnt signaling was demonstrated from the TOPFlash assay to have a similar inhibition. This effect on Wnt activity was further confirmed functionally by defective induction of the presomitic mesoderm marker genes BRACHYURY (T) and MESOGENIN1 (MSGN1) by Wnt3a ligand or the GSK3 inhibitor/Wnt pathway activator CHIR. Further investigation through immunofluorescence imaging and cell fractionation revealed a shifted membrane localization of ß-catenin in CBM mutant iPSCs, revealing a novel molecular component of NKA-Wnt regulation. This study sheds light on a genetic regulation of myogenesis through the CBM of NKA and control of Wnt/ß-catenin signaling.
Asunto(s)
Glucógeno Sintasa Quinasa 3 , beta Catenina , Animales , Caveolina 1/genética , Caveolina 1/metabolismo , Caveolina 1/farmacología , Diferenciación Celular , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3/farmacología , Ratones , Desarrollo de Músculos/genética , ATPasa Intercambiadora de Sodio-Potasio/genética , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Vía de Señalización Wnt , beta Catenina/metabolismoRESUMEN
This review summarizes data from several laboratories that have demonstrated a role of the Na/K-ATPase, specifically its α1 subunit, in the generation of reactive oxygen species (ROS) via the negative regulator of Src. Together with Src and other signaling proteins, the Na/K-ATPase forms an oxidant amplification loop (NKAL), amplifies ROS, and participates in cytokines storm in obesity. The development of a peptide fragment of the α1 subunit, NaKtide, has been shown to negatively regulate Src. Several groups showed that the systemic administration of the cell permeable modification of NaKtide (pNaKtide) or its selective delivery to fat tissue-adipocyte specific expression of NaKtide-ameliorate the systemic elevation of inflammatory cytokines seen in chronic obesity. Severe acute respiratory syndrome - coronavirus 2 (SARS-CoV-2), the RNA Coronavirus responsible for the COVID-19 global pandemic, invades cells via the angiotensin converting enzyme 2 (ACE-2) receptor (ACE2R) that is appended in inflamed fat tissue and exacerbates the formation of the cytokines storm. Both obesity and heart and renal failure are well known risks for adverse outcomes in patients infected with COVID-19. White adipocytes express ACE-2 receptors in high concentration, especially in obese patients. Once the virus invades the white adipocyte cell, it creates a COVID19-porphyrin complex which degrades and produces free porphyrin and iron and increases ROS. The increased formation of ROS and activation of the NKAL results in a further potentiated formation of ROS production, and ultimately, adipocyte generation of more inflammatory mediators, leading to systemic cytokines storm and heart failure. Moreover, chronic obesity also results in the reduction of antioxidant genes such as heme oxygenase-1 (HO-1), increasing adipocyte susceptibility to ROS and cytokines. It is the systemic inflammation and cytokine storm which is responsible for many of the adverse outcomes seen with COVID-19 infections in obese subjects, leading to heart failure and death. This review will also describe the potential antioxidant drugs and role of NaKtide and their demonstrated antioxidant effect used as a major strategy for improving obesity and epicardial fat mediated heart failure in the context of the COVID pandemic.
RESUMEN
We have demonstrated that Na/K-ATPase acts as a receptor for reactive oxygen species (ROS), regulating renal Na+ handling and blood pressure. TALLYHO/JngJ (TH) mice are believed to mimic the state of obesity in humans with a polygenic background of type 2 diabetes. This present work is to investigate the role of Na/K-ATPase signaling in TH mice, focusing on susceptibility to hypertension due to chronic excess salt ingestion. Age-matched male TH and the control C57BL/6J (B6) mice were fed either normal diet or high salt diet (HS: 2, 4, and 8% NaCl) to construct the renal function curve. Na/K-ATPase signaling including c-Src and ERK1/2 phosphorylation, as well as protein carbonylation (a commonly used marker for enhanced ROS production), were assessed in the kidney cortex tissues by Western blot. Urinary and plasma Na+ levels were measured by flame photometry. When compared to B6 mice, TH mice developed salt-sensitive hypertension and responded to a high salt diet with a significant rise in systolic blood pressure indicative of a blunted pressure-natriuresis relationship. These findings were evidenced by a decrease in total and fractional Na+ excretion and a right-shifted renal function curve with a reduced slope. This salt-sensitive hypertension correlated with changes in the Na/K-ATPase signaling. Specifically, Na/K-ATPase signaling was not able to be stimulated by HS due to the activated baseline protein carbonylation, phosphorylation of c-Src and ERK1/2. These findings support the emerging view that Na/K-ATPase signaling contributes to metabolic disease and suggest that malfunction of the Na/K-ATPase signaling may promote the development of salt-sensitive hypertension in obesity. The increased basal level of renal Na/K-ATPase-dependent redox signaling may be responsible for the development of salt-sensitive hypertension in polygenic obese TH mice.
Asunto(s)
Hipertensión/metabolismo , Sistema de Señalización de MAP Quinasas , Síndrome Metabólico/metabolismo , Obesidad/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Animales , Hipertensión/etiología , Hipertensión/genética , Riñón/metabolismo , Masculino , Síndrome Metabólico/genética , Ratones , Ratones Endogámicos C57BL , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Obesidad/genética , Carbonilación Proteica , Especies Reactivas de Oxígeno/metabolismo , Sodio/sangre , Sodio/orina , Cloruro de Sodio Dietético/efectos adversos , Familia-src Quinasas/metabolismoRESUMEN
BACKGROUND: We have demonstrated that cardiotonic steroids, such as ouabain, signaling through the Na/K-ATPase, regulate sodium reabsorption in the renal proximal tubule. By direct carbonylation modification of the Pro222 residue in the actuator (A) domain of pig Na/K-ATPase α1 subunit, reactive oxygen species are required for ouabain-stimulated Na/K-ATPase/c-Src signaling and subsequent regulation of active transepithelial (22)Na(+) transport. In the present study we sought to determine the functional role of Pro222 carbonylation in Na/K-ATPase signaling and sodium handling. METHODS AND RESULTS: Stable pig α1 knockdown LLC-PK1-originated PY-17 cells were rescued by expressing wild-type rat α1 and rat α1 with a single mutation of Pro224 (corresponding to pig Pro222) to Ala. This mutation does not affect ouabain-induced inhibition of Na/K-ATPase activity, but abolishes the effects of ouabain on Na/K-ATPase/c-Src signaling, protein carbonylation, Na/K-ATPase endocytosis, and active transepithelial (22)Na(+) transport. CONCLUSIONS: Direct carbonylation modification of Pro224 in the rat α1 subunit determines ouabain-mediated Na/K-ATPase signal transduction and subsequent regulation of renal proximal tubule sodium transport.
Asunto(s)
Túbulos Renales Proximales/metabolismo , Carbonilación Proteica , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Sodio/metabolismo , Animales , Animales Modificados Genéticamente , Proteína Tirosina Quinasa CSK , Células Cultivadas , Técnicas de Silenciamiento del Gen , Túbulos Renales Proximales/citología , Mutación , Ouabaína/farmacología , Ratas , Transducción de Señal , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidores , ATPasa Intercambiadora de Sodio-Potasio/genética , Porcinos , Familia-src Quinasas/metabolismoRESUMEN
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
Cardiotonic steroids (such as ouabain) signaling through Na/K-ATPase regulate sodium reabsorption in the renal proximal tubule. We report here that reactive oxygen species are required to initiate ouabain-stimulated Na/K-ATPase·c-Src signaling. Pretreatment with the antioxidant N-acetyl-L-cysteine prevented ouabain-stimulated Na/K-ATPase·c-Src signaling, protein carbonylation, redistribution of Na/K-ATPase and sodium/proton exchanger isoform 3, and inhibition of active transepithelial (22)Na(+) transport. Disruption of the Na/K-ATPase·c-Src signaling complex attenuated ouabain-stimulated protein carbonylation. Ouabain-stimulated protein carbonylation is reversed after removal of ouabain, and this reversibility is largely independent of de novo protein synthesis and degradation by either the lysosome or the proteasome pathways. Furthermore, ouabain stimulated direct carbonylation of two amino acid residues in the actuator domain of the Na/K-ATPase α1 subunit. Taken together, the data indicate that carbonylation modification of the Na/K-ATPase α1 subunit is involved in a feed-forward mechanism of regulation of ouabain-mediated renal proximal tubule Na/K-ATPase signal transduction and subsequent sodium transport.
Asunto(s)
Complejos Multienzimáticos/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/fisiología , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Acetilcisteína/farmacología , Animales , Proteína Tirosina Quinasa CSK , Línea Celular , Inhibidores Enzimáticos/farmacología , Depuradores de Radicales Libres/farmacocinética , Complejos Multienzimáticos/genética , Ouabaína/farmacología , Carbonilación Proteica/efectos de los fármacos , Carbonilación Proteica/genética , Proteolisis/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , ATPasa Intercambiadora de Sodio-Potasio/genética , Porcinos , Familia-src QuinasasRESUMEN
Decreases in cardiac Na/K-ATPase have been documented in patients with heart failure. Reduction of Na/K-ATPase α1 also contributes to the deficiency in cardiac contractility in animal models. Our previous studies demonstrate that reduction of cellular Na/K-ATPase causes cell growth inhibition and cell death in renal proximal tubule cells. To test whether reduction of Na/K-ATPase in combination with increased cardiotonic steroids causes cardiac myocyte death and cardiac dysfunction, we examined heart function in Na/K-ATPase α1 heterozygote knock-out mice (α1(+/-)) in comparison to wild type (WT) littermates after infusion of marinobufagenin (MBG). Adult cardiac myocytes were also isolated from both WT and α1(+/-) mice for in vitro experiments. The results demonstrated that MBG infusion increased myocyte apoptosis and induced significant left ventricle dilation in α1(+/-) mice but not in their WT littermates. Mechanistically, it was found that in WT myocytes MBG activated the Src/Akt/mTOR signaling pathway, which further increased phosphorylation of ribosome S6 kinase (S6K) and BAD (Bcl-2-associated death promoter) and protected cells from apoptosis. In α1(+/-) myocytes, the basal level of phospho-BAD is higher compared with WT myocytes, but MBG failed to induce further activation of the mTOR pathway. Reduction of Na/K-ATPase also caused the activation of caspase 9 but not caspase 8 in these cells. Using cultures of neonatal cardiac myocytes, we demonstrated that inhibition of the mTOR pathway by rapamycin also enabled MBG to activate caspase 9 and induce myocyte apoptosis.
Asunto(s)
Apoptosis/efectos de los fármacos , Bufanólidos/efectos adversos , Inhibidores Enzimáticos/efectos adversos , Cardiopatías/enzimología , Proteínas Musculares/metabolismo , Miocitos Cardíacos/enzimología , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Animales , Antibacterianos/farmacología , Apoptosis/genética , Bufanólidos/farmacología , Caspasa 8/genética , Caspasa 8/metabolismo , Caspasa 9/genética , Caspasa 9/metabolismo , Células Cultivadas , Activación Enzimática/efectos de los fármacos , Activación Enzimática/genética , Inhibidores Enzimáticos/farmacología , Cardiopatías/inducido químicamente , Cardiopatías/genética , Ratones , Ratones Mutantes , Proteínas Musculares/antagonistas & inhibidores , Proteínas Musculares/genética , Miocitos Cardíacos/patología , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Sirolimus/farmacología , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidores , ATPasa Intercambiadora de Sodio-Potasio/genética , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismo , Familia-src Quinasas/genética , Familia-src Quinasas/metabolismoRESUMEN
Cardiotonic steroids, including marinobufagenin, are a group of new steroid hormones found in plasma and urine of patients with congestive heart failure, myocardial infarction, and chronic renal failure. In animal studies, partial nephrectomy induces marinobufagenin elevation, cardiac hypertrophy, and fibrosis. The objective of this study is to test the effect of renal ischemia on marinobufagenin levels in humans with renal artery stenosis (RAS). To test this, plasma marinobufagenin levels were measured in patients with RAS of the Prospective Randomized Study Comparing Renal Artery Stenting With or Without Distal Protection, non-RAS patient controls who were scheduled for coronary angiography, and normal healthy individuals. Marinobufagenin levels were significantly higher in patients with RAS compared with those of the other 2 groups. Multivariate analysis shows that occurrence of RAS is independently related to marinobufagenin levels. In addition, renal artery revascularization by stenting partially reversed marinobufagenin levels in the patients with RAS (0.77±0.06 nmol/L at baseline; 0.66±0.06 nmol/L at 24 hours; and 0.61±0.05 nmol/L at 1 month). In conclusion, we have found that marinobufagenin levels are increased in patients with RAS, whereas reversal of renal ischemia by stenting treatment reduces marinobufagenin levels. These results suggest that RAS-induced renal ischemia may be a major cause of marinobufagenin release.
Asunto(s)
Bufanólidos/sangre , Hipertensión/sangre , Isquemia/sangre , Riñón/irrigación sanguínea , Obstrucción de la Arteria Renal/sangre , Anciano , Análisis de Varianza , Ensayo de Inmunoadsorción Enzimática , Femenino , Tasa de Filtración Glomerular , Humanos , Hipertensión/complicaciones , Modelos Logísticos , Masculino , Persona de Mediana Edad , Selección de Paciente , Estudios Prospectivos , Obstrucción de la Arteria Renal/terapia , StentsRESUMEN
Recent studies have demonstrated that the Na(+)/K(+)-ATPase is not only an ion pump, but also a membrane receptor that confers the ligand-like effects of cardiotonic steroids (CTS) such as ouabain on protein kinases and cell growth. Because CTS have been implicated in cardiac fibrosis, this study examined the role of caveolae in the regulation of Na(+)/K(+)-ATPase function and CTS signaling in cardiac fibroblasts. In cardiac fibroblasts prepared from wild-type and caveolin-1 knockout [Cav-1(-/-)] mice, we found that the absence of caveolin-1 did not affect total cellular amount or surface expression of Na(+)/K(+)-ATPase alpha1 subunit. However, it did increase ouabain-sensitive (86)Rb(+) uptake. While knockout of caveolin-1 increased basal activities of Src and ERK1/2, it abolished the activation of these kinases induced by ouabain but not angiotensin II. Finally, ouabain stimulated collagen synthesis and cell proliferation in wild type but not Cav-1(-/-) cardiac fibroblasts. Thus, we conclude that caveolae are important for regulating both pumping and signal transducing functions of Na(+)/K(+)-ATPase. While depletion of caveolae increases the pumping function of Na(+)/K(+)-ATPase, it suppresses CTS-induced signal transduction, growth, and collagen production in cardiac fibroblasts.
Asunto(s)
Caveolina 1/fisiología , Fibroblastos/metabolismo , Corazón/fisiología , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Animales , Western Blotting , Cardiotónicos/farmacología , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Proliferación Celular , Colágeno/metabolismo , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Corazón/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ouabaína/farmacología , Transducción de SeñalRESUMEN
The Na/K-ATPase was discovered as an energy transducing ion pump. A major difference between the Na/K-ATPase and other P-type ATPases is its ability to bind a group of chemicals called cardiotonic steroids (CTS). The plant-derived CTS such as digoxin are valuable drugs for the management of cardiac diseases, whereas ouabain and marinobufagenin (MBG) have been identified as a new class of endogenous hormones. Recent studies have demonstrated that the endogenous CTS are important regulators of renal Na(+) excretion and blood pressure. The Na/K-ATPase is not only an ion pump, but also an important receptor that can transduce the ligand-like effect of CTS on intracellular protein kinases and Ca(2+) signaling. Significantly, these CTS-provoked signaling events are capable of reducing the surface expression of apical NHE3 (Na/H exchanger isoform 3) and basolateral Na/K-ATPase in renal proximal tubular cells. These findings suggest that endogenous CTS may play an important role in regulation of tubular Na(+) excretion under physiological conditions; conversely, a defect at either the receptor level (Na/K-ATPase) or receptor-effector coupling would reduce the ability of renal proximal tubular cells to excrete Na(+), thus culminating/resulting in salt-sensitive hypertension.
Asunto(s)
Bufanólidos/metabolismo , Señalización del Calcio , Túbulos Renales Proximales/metabolismo , Ouabaína/metabolismo , Proteínas Quinasas/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Animales , Humanos , Hipertensión/metabolismo , Hipertensión/patología , Hipertensión/fisiopatología , Túbulos Renales Proximales/patología , Túbulos Renales Proximales/fisiopatología , Transporte de Proteínas , Intercambiador 3 de Sodio-Hidrógeno , Intercambiadores de Sodio-Hidrógeno/metabolismoRESUMEN
Here, we show that the Na/K-ATPase interacts with caveolin-1 (Cav1) and regulates Cav1 trafficking. Graded knockdown of Na/K-ATPase decreases the plasma membrane pool of Cav1, which results in a significant reduction in the number of caveolae on the cell surface. These effects are independent of the pumping function of Na/K-ATPase, and instead depend on interaction between Na/K-ATPase and Cav1 mediated by an N-terminal caveolin-binding motif within the ATPase alpha1 subunit. Moreover, knockdown of the Na/K-ATPase increases basal levels of active Src and stimulates endocytosis of Cav1 from the plasma membrane. Microtubule-dependent long-range directional trafficking in Na/K-ATPase-depleted cells results in perinuclear accumulation of Cav1-positive vesicles. Finally, Na/K-ATPase knockdown has no effect on processing or exit of Cav1 from the Golgi. Thus, the Na/K-ATPase regulates Cav1 endocytic trafficking and stabilizes the Cav1 plasma membrane pool.
Asunto(s)
Transporte Biológico/fisiología , Caveolina 1/metabolismo , Membrana Celular/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Animales , Caveolas/metabolismo , Caveolas/ultraestructura , Caveolina 1/genética , Membrana Celular/ultraestructura , Vesículas Citoplasmáticas/metabolismo , Endocitosis/fisiología , Transferencia Resonante de Energía de Fluorescencia , Aparato de Golgi/metabolismo , Aparato de Golgi/ultraestructura , Microtúbulos/metabolismo , Mutación Puntual , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Interferencia de ARN , Ratas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal/fisiología , ATPasa Intercambiadora de Sodio-Potasio/genética , Proteínas de Unión al GTP rab/genética , Proteínas de Unión al GTP rab/metabolismo , Proteínas de Unión al GTP rab5/genética , Proteínas de Unión al GTP rab5/metabolismo , Proteínas de Unión a GTP rab7 , Familia-src Quinasas/antagonistas & inhibidores , Familia-src Quinasas/genética , Familia-src Quinasas/metabolismoRESUMEN
The Na-K-ATPase is an energy-transducing ion pump that converts the free energy of ATP into transmembrane ion gradients. It also serves as a functional receptor for cardiotonic steroids such as ouabain and digoxin. Binding of ouabain to the Na-K-ATPase can activate calcium signaling in a cell-specific manner. The exquisite calcium modulation via the Na-K-ATPase is achieved by the ability of the pump to integrate signals from numerous protein and non-protein molecules, including ion transporters, channels, protein kinases/phosphatases, as well as cellular Na+. This review focuses on the unique properties of the Na-K-ATPase and its role in the formation of different calcium-signaling microdomains.
Asunto(s)
Señalización del Calcio/fisiología , Compartimento Celular/fisiología , Células Epiteliales/enzimología , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Animales , HumanosRESUMEN
Mitochondrial injury, characterized by outer membrane permeabilization and consequent release of apoptogenic factors, is a key to apoptosis of mammalian cells. Bax and Bak, two multidomain Bcl-2 family proteins, provide a requisite gateway to mitochondrial injury. However it is unclear how Bax and Bak cooperate to provoke mitochondrial injury and whether their roles are redundant. Here, we have identified a unique role of Bak in mitochondrial fragmentation, a seemingly morphological event that contributes to mitochondrial injury during apoptosis. We show that mitochondrial fragmentation is attenuated in Bak-deficient mouse embryonic fibroblasts, baby mouse kidney cells, and, importantly, also in primary neurons isolated from brain cortex of Bak-deficient mice. In sharp contrast, Bax deficiency does not prevent mitochondrial fragmentation during apoptosis. Bcl-2 and Bcl-XL inhibit mitochondrial fragmentation, and their inhibitory effects depend on the presence of Bak. Reconstitution of Bak into Bax/Bak double-knockout cells restores mitochondrial fragmentation, whereas reconstitution of Bax is much less effective. Bak interacts with Mfn1 and Mfn2, two mitochondrial fusion proteins. During apoptosis, Bak dissociates from Mfn2 and enhances the association with Mfn1. Mutation of Bak in the BH3 domain prevents its dissociation from Mfn2 and diminishes its mitochondrial fragmentation activity. This study has uncovered a previously unrecognized function of Bak in the regulation of mitochondrial morphological dynamics during apoptosis. By this function, Bak may collaborate with Bax to permeabilize the outer membrane of mitochondria, unleashing the apoptotic cascade.
Asunto(s)
Apoptosis/fisiología , GTP Fosfohidrolasas/fisiología , Mitocondrias/metabolismo , Mitocondrias/patología , Proteína Destructora del Antagonista Homólogo bcl-2/fisiología , Animales , Animales Recién Nacidos , Apoptosis/genética , Células Cultivadas , Células HeLa , Humanos , Membranas Intracelulares/metabolismo , Membranas Intracelulares/patología , Ratones , Mitocondrias/genética , Permeabilidad , Proteína Destructora del Antagonista Homólogo bcl-2/deficiencia , Proteína Destructora del Antagonista Homólogo bcl-2/genética , Proteína X Asociada a bcl-2/deficiencia , Proteína X Asociada a bcl-2/genética , Proteína X Asociada a bcl-2/fisiologíaRESUMEN
Recent studies have ascribed many non-pumping functions to the Na/K-ATPase. Here, we present experimental evidence demonstrating that over half of the plasma membrane Na/K-ATPase in LLC-PK1 cells is performing cellular functions other than ion pumping. This "non-pumping" pool of Na/K-ATPase, like the pumping pump, binds ouabain. Depletion of either cholesterol or caveolin-1 moves some of the "non-pumping" Na/K-ATPase into the pumping pool. Graded knock-down of the alpha1 subunit of the Na/K-ATPase eventually results in loss of this "non-pumping" pool while preserving the pumping pool. Our prior studies indicate that a loss of the non-pumping pool is associated with a loss of receptor function as evidenced by the failure of ouabain administration to induce the activation of Src and/or ERK. Therefore, our new findings suggest that a substantial amount of surface-expressed Na/K-ATPase, at least in some types of cells, may function as non-canonical ouabain-binding receptors.
Asunto(s)
Caveolina 1/metabolismo , Membrana Celular/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Animales , Caveolina 1/genética , Línea Celular , Membrana Celular/genética , Activación Enzimática/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Ouabaína/farmacología , Subunidades de Proteína/deficiencia , Subunidades de Proteína/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/genética , Familia-src Quinasas/metabolismoRESUMEN
We have shown that the Na/K-ATPase and Src form a signaling receptor complex. Here we determined how alterations in the amount and properties of the Na/K-ATPase affect basal Src activity and ouabain-induced signal transduction. Several alpha1 subunit knockdown cell lines were generated by transfecting LLC-PK1 cells with a vector expressing alpha1-specific small interference RNA. Although the alpha1 knockdown resulted in significant decreases in Na/K-ATPase activity, it increased the basal Src activity and tyrosine phosphorylation of focal adhesion kinase, a Src effector. Concomitantly it also abolished ouabain-induced activation of Src and ERK1/2. When the knockdown cells were rescued by a rat alpha1, both Na/K-ATPase activity and the basal Src activity were restored. In addition, ouabain was able to stimulate Src and ERK1/2 in the rescued cells at a much higher concentration, consistent with the established differences in ouabain sensitivity between pig and rat alpha1. Finally both fluorescence resonance energy transfer analysis and co-immunoprecipitation assay indicated that the pumping-null rat alpha1 (D371E) mutant could also bind Src. Expression of this mutant restored the basal Src activity and focal adhesion kinase tyrosine phosphorylation. Taken together, the new findings suggest that LLC-PK1 cells contain a pool of Src-interacting Na/K-ATPase that not only regulates Src activity but also serves as a receptor for ouabain to activate protein kinases.
Asunto(s)
Interferencia de ARN , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Familia-src Quinasas/metabolismo , Animales , Secuencia de Bases , Línea Celular , Activación Enzimática , Humanos , Células LLC-PK1 , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Datos de Secuencia Molecular , Ratas , Transducción de Señal , PorcinosRESUMEN
We have shown that ouabain activates Src, resulting in subsequent tyrosine phosphorylation of multiple effectors. Here, we tested if the Na+/K+-ATPase and Src can form a functional signaling complex. In LLC-PK1 cells the Na+/K+-ATPase and Src colocalized in the plasma membrane. Fluorescence resonance energy transfer analysis indicated that both proteins were in close proximity, suggesting a direct interaction. GST pulldown assay showed a direct, ouabain-regulated, and multifocal interaction between the 1 subunit of Na+/K+-ATPase and Src. Although the interaction between the Src kinase domain and the third cytosolic domain (CD3) of 1 is regulated by ouabain, the Src SH3SH2 domain binds to the second cytosolic domain constitutively. Functionally, binding of Src to either the Na+/K+-ATPase or GST-CD3 inhibited Src activity. Addition of ouabain, but not vanadate, to the purified Na+/K+-ATPase/Src complex freed the kinase domain and restored the Src activity. Consistently, exposure of intact cells to ouabain apparently increased the distance between the Na+/K+-ATPase and Src. Concomitantly, it also stimulated tyrosine phosphorylation of the proteins that are associated with the Na+/K+-ATPase. These new findings illustrate a novel molecular mechanism of signal transduction involving the interaction of a P-type ATPase and a nonreceptor tyrosine kinase.
Asunto(s)
Transducción de Señal , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Familia-src Quinasas/metabolismo , Animales , Línea Celular , Pollos , Activación Enzimática/efectos de los fármacos , Humanos , Ratones , Complejos Multiproteicos/química , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Ouabaína/farmacología , Fosfotirosina/metabolismo , Unión Proteica , Ratas , ATPasa Intercambiadora de Sodio-Potasio/genética , ATPasa Intercambiadora de Sodio-Potasio/aislamiento & purificación , Porcinos , Familia-src Quinasas/química , Familia-src Quinasas/genéticaRESUMEN
Coronary artery disease and its sequelae-ischemia, myocardial infarction, and heart failure-are leading causes of morbidity and mortality in man. Considerable effort has been devoted toward improving functional recovery and reducing the extent of infarction after ischemic episodes. As a step in this direction, it was found that the heart was significantly protected against ischemia-reperfusion injury if it was first preconditioned by brief ischemia or by administering a potassium channel opener. Both of these preconditioning strategies were found to require opening of a K(ATP) channel, and in 1997 we showed that this pivotal role was mediated by the mitochondrial ATP-sensitive K(+) channel (mitoK(ATP)). This paper will review the evidence showing that opening mitoK(ATP) is cardioprotective against ischemia-reperfusion injury and, moreover, that mitoK(ATP) plays this role during all three phases of the natural history of ischemia-reperfusion injury preconditioning, ischemia, and reperfusion. We discuss two distinct mechanisms by which mitoK(ATP) opening protects the heart-increased mitochondrial production of reactive oxygen species (ROS) during the preconditioning phase and regulation of intermembrane space (IMS) volume during the ischemic and reperfusion phases. It is likely that cardioprotection by ischemic preconditioning (IPC) and K(ATP) channel openers (KCOs) arises from utilization of normal physiological processes. Accordingly, we summarize the results of new studies that focus on the role of mitoK(ATP) in normal cardiomyocyte physiology. Here, we observe the same two mechanisms at work. In low-energy states, mitoK(ATP) opening triggers increased mitochondrial ROS production, thereby amplifying a cell signaling pathway leading to gene transcription and cell growth. In high-energy states, mitoK(ATP) opening prevents the matrix contraction that would otherwise occur during high rates of electron transport. MitoK(ATP)-mediated volume regulation, in turn, prevents disruption of the structure-function of the IMS and facilitates efficient energy transfers between mitochondria and myofibrillar ATPases.
Asunto(s)
Proteínas de la Membrana/fisiología , Mitocondrias Cardíacas/fisiología , Potasio/metabolismo , Animales , Transporte Biológico , Cardiotónicos , Humanos , Daño por Reperfusión Miocárdica/prevención & control , Canales de PotasioRESUMEN
Tea pigments are oxidized products of polyphenols derived from tea leaves (Camellia sinensis). Theaflavins are constituents of tea pigments with antioxidant, antineoplastic and antiinflammatory properties similar to their parent compounds. The biological properties of polyphenols and theaflavins have been linked to their capacity to inhibit the activation of nuclear factor-kappaB (NF-kappaB), a transcription factor, which is critically involved in the molecular regulation of a number of proinflammatory cytokines. The current study examines the requirement for NF-kappaB in the immunosuppressive effects mediated by tea antioxidants. Specifically, we tested the hypothesis that cytokines produced by type 1 (T(H1)) CD4(+) T cells which require NF-kappaB for gene expression, such as interleukin-2 (IL-2) and interferon gamma (IFN gamma), are selectively inhibited by tea pigments. We found that tea pigments potently suppress IL-2 secretion, IL-2 gene expression and the activation of NF-kappaB in murine spleens enriched for CD4(+) T cells, as expected. Consistent with our hypothesis, tea pigments also inhibited the induction of IFNgamma mRNA. However, the expression of the T(H2) cytokines IL-4 and IL-5, which lack functional NF-kappaB sites within their promoters was unexpectedly suppressed by tea pigments, as well. The results indicate that NF-kappaB may be only one of multiple transcription factors inhibited by tea pigments.