RESUMEN
Acute lung injury (ALI) is a common respiratory syndrome accompanied with an inflammation response. Annexin A5 (AnxA5) has anti-thrombotic, anti-apoptotic, and anti-inflammatory properties. The current study aims to explore the potential effect of AnxA5 on lipopolysaccharide (LPS)-induced inflammatory response in alveolar macrophages (AMs). Rat AMs (NR8383) were used in this study, and the cell viabilities at 4, 8, and 16 h after LPS administration with gradient concentrations were determined using cell counting kit-8 assay. Cell apoptosis and expressions of messenger RNAs (mRNAs) and protein were determined by flow cytometry, quantitative real-time polymerase chain reaction (qRT-PCR), and western blot, respectively. We found that LPS suppressed the viability of AMs in a dose-dependent manner, and it elevated the expression of AnxA5 in AMs. Inhibition of AnxA5 improved the cell viability compared with the LPS group and could reduce the apoptosis rate in comparison with LPS treatment. The knockdown of AnxA5 suppressed the expressions of tumor necrosis factor-α (TNF-α), interleukin (IL-1ß), and IL-6 at both protein and mRNA levels and regulated the expressions of apoptosis-related molecules (Bax, Bcl-2, and caspase-3). Moreover, the knockdown of AnxA5 improved the expression levels of inhibitory κB (IκB) and nuclear factor E2-related factor 2 (Nrf2) but inhibited the expression of nuclear transcription factor κB (NF-κB), compared with the LPS group. SN50 and ML385 were used to validate this signaling, and the inhibition of AnxA5 suppressed the LPS-induced inflammation, indicating that AnxA5 may be a potential anti-inflammatory target. In addition, NF-κB/Nrf2 signaling pathway may also be involved in the LPS-induced inflammatory response of rat alveolar macrophages.
Asunto(s)
Anexina A5/antagonistas & inhibidores , Antiinflamatorios/farmacología , Apoptosis , Inflamación/prevención & control , Lipopolisacáridos/toxicidad , Macrófagos Alveolares/inmunología , Animales , Anexina A5/metabolismo , Supervivencia Celular , Células Cultivadas , Inflamación/inducido químicamente , Inflamación/metabolismo , Inflamación/patología , Macrófagos Alveolares/metabolismo , Macrófagos Alveolares/patología , FN-kappa B/metabolismo , Ratas , Transducción de SeñalRESUMEN
OBJECTIVE: To investigate the contributions and underlying molecular mechanisms of annexin A5 toward silica-induced pulmonary fibrosis. METHODS: Male C57BL/6 mice were randomly divided into three groups and instilled intratracheally with silica, saline, or air. Mice were euthanized at 3, 7, 14, or 28 days following treatment. Annexin A5 levels in serum and lung tissues were detected by enzyme-linked immunosorbant assay (ELISA) assays or Western blots. The association of annexin A5 levels with silica-induced lung fibrosis was further investigated in the macrophage cell line, RAW264.7. Following exposure of these cells to silica at a concentration of 200 µg/ml for 6 or 12 h, the expression levels of transforming growth factor ß1 (TGF-ß1), interleukin 1α (IL-1α), Fas ligand (FasL), and their downstream targets were evaluated by Western blots. Furthermore, annexin A5 and FasL were knocked down by small interfering ribonucleic acid (siRNA) and TGF-ß1 secretion into the cell culture medium was measured by ELISA assays or Western blots. RESULTS: Mice treated with silica demonstrated lung fibrosis at 28 days following exposure, whereas, in controls, only mild and transient inflammation was evident at day 3 and day 7 postinstillation and was not present at day 14. Furthermore, silica-exposed mice exhibited significantly (p < 0.05) elevated levels of annexin A5 in serum and lung tissues, relative to control groups. Consistent with these findings, silica exposure of RAW264.7 cells for 6 or 12 h, led to an annexin A5-dependent increase in the expression levels of TGF-ß1, IL-1α, FasL, and their downstream target molecules. These silica-induced changes were reversed by siRNA-mediated knockdown of annexin A5, but downregulation of FasL led to increased annexin A5 expression and reduced levels of TGF-ß1, IL-1α, and FasL downstream target molecules. CONCLUSIONS: These findings define a role of annexin A5 in promoting macrophage activation via Fas/FasL pathways in silica-induced lung fibrosis.
Asunto(s)
Anexina A5/metabolismo , Modelos Animales de Enfermedad , Activación de Macrófagos/efectos de los fármacos , Fibrosis Pulmonar/etiología , Mucosa Respiratoria/efectos de los fármacos , Dióxido de Silicio/toxicidad , Silicosis/fisiopatología , Contaminantes Atmosféricos/química , Contaminantes Atmosféricos/toxicidad , Animales , Anexina A5/antagonistas & inhibidores , Anexina A5/sangre , Anexina A5/genética , Citocinas/agonistas , Citocinas/metabolismo , Proteína Ligando Fas/antagonistas & inhibidores , Proteína Ligando Fas/genética , Proteína Ligando Fas/metabolismo , Pulmón/efectos de los fármacos , Pulmón/inmunología , Pulmón/metabolismo , Pulmón/patología , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Material Particulado/química , Material Particulado/toxicidad , Células RAW 264.7 , Interferencia de ARN , Distribución Aleatoria , Mucosa Respiratoria/inmunología , Mucosa Respiratoria/metabolismo , Mucosa Respiratoria/patología , Transducción de Señal/efectos de los fármacos , Dióxido de Silicio/química , Silicosis/inmunología , Silicosis/metabolismo , Silicosis/patología , Organismos Libres de Patógenos EspecíficosRESUMEN
Only a limited number of human cells can fuse to form a multinucleated syncytium. Cell fusion occurs as part of the differentiation of some cell types, including myotubes in muscle and osteoclasts in remodeling bone. In the differentiation of the human placenta, mononuclear cytotrophoblasts aggregate and fuse to form endocrinologically active, non-proliferative, multinucleated syncytia. These syncytia allow the exchange of nutrients and gases between the maternal and fetal circulation. Alteration of syncytial formation during pregnancy affects fetal growth and the outcome of the pregnancy. Here, we demonstrate the role of annexin A5 (AnxA5) in syncytial formation by cellular delivery of recombinant AnxA5 and RNA interference. By a variety of co-immunoprecipitation, immunolocalization and proximity experiments, we show that a pool of AnxA5 organizes at the inner-leaflet of the plasma membrane in the vicinity of a molecular complex that includes E-Cadherin, α-Catenin and ß-Catenin, three proteins previously shown to form adherens junctions implicated in cell fusion. A combination of knockdown and reconstitution experiments with AnxA5, with or without the ability to self-assemble in 2D-arrays, demonstrate that this AnxA5 2D-network mediates E-Cadherin mobility in the plasmalemma that triggers human trophoblasts aggregation and thereby cell fusion.
Asunto(s)
Anexina A5/genética , Cadherinas/genética , Membrana Celular/metabolismo , Células Gigantes/metabolismo , Trofoblastos/metabolismo , alfa Catenina/genética , beta Catenina/genética , Uniones Adherentes/metabolismo , Uniones Adherentes/ultraestructura , Adulto , Anexina A5/antagonistas & inhibidores , Anexina A5/metabolismo , Antígenos CD , Cadherinas/metabolismo , Comunicación Celular , Diferenciación Celular , Fusión Celular , Movimiento Celular , Femenino , Regulación de la Expresión Génica , Células Gigantes/citología , Humanos , Embarazo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Trofoblastos/citología , alfa Catenina/metabolismo , beta Catenina/metabolismoRESUMEN
Proteomic studies have suggested a biochemical interaction between α subunit of the large conductance, voltage- and Ca2+-activated potassium channel (BKCaα), and annexin A5 (ANXA5), which we verify here by coimmunoprecipitation and double labelling immunocytochemistry. The observation that annexin is flipped to the outer membrane leaflet of the plasma membrane during apoptosis, together with the knowledge that the intracellular C-terminal of BKCaα contains both Ca2+-binding and a putative annexin-binding motif, prompted us to investigate the functional consequences of this protein partnership to cell death. Membrane biotinylation demonstrated that ANXA5 was flipped to the outer membrane leaflet of HEK 293 cells early in serum deprivation-evoked apoptosis. As expected, serum deprivation caused caspase-3/7 activation and this was accentuated in BKCaα expressing HEK 293 cells. The functional consequences of ANXA5 partnership with BKCaα were striking, with ANXA5 knockdown causing an increase and ANXA5 overexpression causing a decrease, in single BKCa channel Ca2+-sensitivity, measured in inside-out membrane patches by patch-clamp. Taken together, these data suggest a novel model of the early stages of apoptosis where membrane flippage results in removal of the inhibitory effect of ANXA5 on K+ channel activity with the consequent amplification of Ca2+ influx and augmented activation of caspases.
Asunto(s)
Anexina A5/metabolismo , Apoptosis , Canales de Potasio de Gran Conductancia Activados por el Calcio/metabolismo , Anexina A5/antagonistas & inhibidores , Anexina A5/genética , Calcio/metabolismo , Caspasa 3/metabolismo , Caspasa 7/metabolismo , Membrana Celular/metabolismo , Células HEK293 , Humanos , Inmunohistoquímica , Inmunoprecipitación , Canales de Potasio de Gran Conductancia Activados por el Calcio/química , Canales de Potasio de Gran Conductancia Activados por el Calcio/genética , Microscopía Confocal , Técnicas de Placa-Clamp , Subunidades de Proteína/química , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismoRESUMEN
Extracellular vesicles (EVs) contain microRNAs (miRNAs). However, the exact molecular mechanisms of the recruitment of miRNAs in EVs are not well characterized. Based on proteomic analysis, we identified that silencing of Annexin A2 (ANXA2) significantly decreased the amount of miRNAs in EVs. In addition, microarray analysis revealed that ANXA2 regulated the loading of miRNAs into EVs in a sequence independent manner. Lastly, immunoprecipitation analysis confirmed that ANXA2 could bind miRNAs in EVs in the presence of Ca(2+). These observations demonstrate that ANXA2 plays an important role in the packaging process of miRNAs into EVs.
Asunto(s)
Anexina A2/metabolismo , Vesículas Extracelulares/metabolismo , MicroARNs/metabolismo , Proteínas de Neoplasias/metabolismo , ARN Neoplásico/metabolismo , Regulación hacia Arriba , Anexina A2/antagonistas & inhibidores , Anexina A2/genética , Anexina A5/antagonistas & inhibidores , Anexina A5/genética , Anexina A5/metabolismo , Transporte Biológico , Señalización del Calcio , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Humanos , Inmunoprecipitación , MicroARNs/química , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Tamaño de la Partícula , Proteómica/métodos , Interferencia de ARN , ARN Neoplásico/química , ARN Interferente Pequeño , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
The mechanical environment of the skeleton plays an important role in the establishment and maintenance of structurally competent bone. Biophysical signals induced by mechanical loading elicit a variety of cellular responses in bone cells, however, little is known about the underlying mechanotransduction mechanism. We hypothesized that bone cells detect and transduce biophysical signals into biological responses via a mechanism requiring annexin V (AnxV). AnxV, a calcium-dependent phospholipid binding protein, has several attributes, which suggest it is ideally suited for a role as a mechanosensor, possibly a mechanosensitive ion channel. These include the ability to function as a Ca2+ selective ion channel, and the ability to interact with both extracellular matrix proteins and cytoskeletal elements. To test the hypothesis that AnxV has a role in mechanosensing, we studied the response of osteoblastic cells to oscillating fluid flow, a physiologically relevant physical signal in bone, in the presence and absence of AnxV inhibitors. In addition, we investigated the effects of oscillating flow on the cellular location of AnxV. Oscillating fluid flow increased both [Ca2+]i levels and c-fos protein levels in osteoblasts. Disruption of AnxV with blocking antibodies or a pharmacological inhibitor, K201 (JTV-519), significantly inhibited both responses. Additionally, our data show that the cellular location of AnxV was modulated by oscillating fluid flow. Exposure to oscillating fluid flow resulted in a significant increase in AnxV at both the cell and nuclear membranes. In summary, our data suggest that AnxV mediates flow-induced Ca2+ signaling in osteoblastic cells. These data support the idea of AnxV as a Ca2+ channel, or a component of the signaling pathway, in the mechanism by which mechanical signals are transduced into cellular responses in the osteoblast. Furthermore, the presence of a highly mobile pool of AnxV may provide cells with a powerful mechanism by which cellular responses to mechanical loading might be amplified and regulated.
Asunto(s)
Anexina A5/antagonistas & inhibidores , Anexina A5/fisiología , Señalización del Calcio/fisiología , Osteoblastos/fisiología , Línea Celular , HumanosAsunto(s)
Anexina A5/metabolismo , Anticuerpos Antifosfolípidos/metabolismo , Inhibidor de Coagulación del Lupus/inmunología , Trombina/biosíntesis , Anexina A5/antagonistas & inhibidores , Anticuerpos Antifosfolípidos/inmunología , Síndrome Antifosfolípido/inmunología , Síndrome Antifosfolípido/metabolismo , Humanos , Trombina/inmunologíaRESUMEN
Biochemical studies have suggested that annexin 2 (A2) may participate in cytomegalovirus (CMV) infection. In the current work, effects of A2 monomer (p36) and heterotetramer (A2t; p36(2)p11(2)) were investigated. Demonstrating a role for endogenous A2, the four stages of infection that were followed were each inhibited by anti-p36 or anti-p11 at 37 degrees C. Immuno-inhibition was attenuated when the virus and cells were pre-incubated at 4 degrees C to coordinate virus entry initiated afterwards at 37 degrees C, reconciling controversy in the literature. As an explanation, CMV-induced phosphorylation of p36 was prevented by the 4 degrees C treatment. Supporting these immuno-inhibition data, purified A2t or p11 increased CMV infectious-progeny generation and CMV gene expression. A specific role for A2t was indicated by purified p36 having no effect. Unlike other steps, primary plaque formation was not enhanced by purified A2t or p11, possibly because of undetectable phosphorylation. As annexins 1 (A1) and 5 (A5) interact with A2, their effect on CMV was also tested. Both purified proteins inhibited CMV infection. In each experiment, the concentration of A1 required for half-maximal inhibition was five- to 10-fold lower than that of A5. Addition of A2 opposed A1- or A5-mediated inhibition of CMV, as did certain A2-specific antibodies that had no effect in the absence of added A1 or A5. Transfection of the p36-deficient cell line HepG2 increased CMV infection and was required for inhibition by the other annexins. These data suggest that CMV exploits A2t at physiological temperature to oppose the protection of cells conferred by A1 or A5.
Asunto(s)
Anexina A1/antagonistas & inhibidores , Anexina A2/farmacología , Anexina A5/antagonistas & inhibidores , Citomegalovirus/efectos de los fármacos , Anexina A2/aislamiento & purificación , Anexina A5/aislamiento & purificación , Citomegalovirus/fisiología , Infecciones por Citomegalovirus , Fibroblastos/virología , Humanos , Piel/citologíaRESUMEN
Many of the biological activities of IFN-gamma are mediated through the IFN-gammaR3-linked Jak-Stat1alpha pathway. However, regulation of IFN-gamma signaling is not fully understood, and not all responses to IFN-gamma are Stat1alpha dependent. To identify novel elements involved in IFN-gamma cell regulation, the cytoplasmic domain of the R2 subunit of the human IFN-gammaR was used as bait in a yeast two-hybrid screen of a human monocyte cDNA library. This identified annexin A5 (AxV) as a putative IFN-gammaR binding protein. The interaction was confirmed in pull-down experiments in which a GST-R2 cytoplasmic domain fusion protein was incubated with macrophage lysates. Furthermore, immunoprecipitation using anti-IFN-gammaR2 Abs showed that AxV interacted with IFN-gammaR2 to form a stable complex following incubation of cells with IFN-gamma. In 293T cells with reduced expression of AxV, brought about by small interfering RNA targeting, activation of Jak2 and Stat1alpha in response to IFN-gamma was enhanced. Inhibition of cell proliferation, a hallmark of the IFN-gamma response, also was potentiated in HeLa cells treated with small interfering RNA directed at AxV. Taken together, these results suggest that through an inducible association with the R2 subunit of the IFN-gammaR, AxV modulates cellular responses to IFN-gamma by modulating signaling through the Jak-Stat1 pathway.
Asunto(s)
Anexina A5/fisiología , Regulación hacia Abajo/inmunología , Interferón gamma/metabolismo , Receptores de Interferón/metabolismo , Transducción de Señal/inmunología , Anexina A5/antagonistas & inhibidores , Anexina A5/biosíntesis , Anexina A5/genética , Muerte Celular/inmunología , Línea Celular , Línea Celular Tumoral , Inhibidores de Crecimiento/metabolismo , Inhibidores de Crecimiento/fisiología , Humanos , Factor 3 de Genes Estimulados por el Interferón/metabolismo , Factor 3 de Genes Estimulados por el Interferón/fisiología , Interferón gamma/antagonistas & inhibidores , Interferón gamma/fisiología , Janus Quinasa 2 , Subunidades de Proteína/metabolismo , Subunidades de Proteína/fisiología , Proteínas Tirosina Quinasas/metabolismo , Proteínas Tirosina Quinasas/fisiología , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas/fisiología , ARN Interferente Pequeño/farmacología , Saccharomyces cerevisiae/genética , Técnicas del Sistema de Dos Híbridos , Receptor de Interferón gammaRESUMEN
Calphobindin I (CPB I) is a member of the family of Ca(2+)-dependent phospholipid binding proteins collectively termed as annexins. CPB I (Annexin V) has recently been shown to be an endogenous inhibitor of protein kinase C, a key enzyme in the cellular signal transduction and its inhibition by CPB I is presumed to be related ultimately to carcinogenesis. We therefore examined the level of production of CPB I in uterine cancer cells. Immunohistochemical analysis, northern blot, and in situ hybridization showed that the production of CPB I was markedly suppressed at the level of transcription in both cervical and endometrial carcinoma cells when compared to their normal counterparts. Decrease in production of CPB I may lead to dysregulated activation of protein kinase C and, accordingly, may be involved in a disorder of cell differentiation, proliferation, and carcinogenesis.
Asunto(s)
Anexina A5/antagonistas & inhibidores , Carcinoma/metabolismo , Endometrio/metabolismo , Neoplasias del Cuello Uterino/metabolismo , Anexina A5/biosíntesis , Secuencia de Bases , Northern Blotting , Femenino , Humanos , Inmunohistoquímica , Hibridación in Situ , Sondas Moleculares/genética , Datos de Secuencia Molecular , Valores de ReferenciaRESUMEN
The antiphospholipid syndrome is a thrombophilic condition marked by antibodies that recognize anionic phospholipid-protein cofactor complexes. We recently reported that exposure to IgG fractions from antiphospholipid patients reduces the level of annexin-V, a phospholipid-binding anticoagulant protein, on cultured trophoblasts and endothelial cells and accelerates coagulation of plasma exposed to these cells. Therefore, we asked whether antiphospholipid antibodies might directly reduce annexin-V binding to noncellular phospholipid substrates. Using ellipsometry, we found that antiphospholipid IgGs reduce the quantity of annexin-V bound to phospholipid bilayers; this reduction is dependent on the presence of beta2-glycoprotein I. Also, exposure to plasmas containing antiphospholipid antibodies reduces annexin-V binding to phosphatidyl serine-coated microtiter plates, frozen thawed washed platelets, activated partial thromboplastin time (aPTT) reagent and prothrombin time reagent and reduces the anticoagulant effect of the protein. These studies show that antiphospholipid antibodies interfere with the binding of annexin-V to anionic phospholipid and with its anticoagulant activity. This acceleration of coagulation, due to reduced binding of annexin V, stands in marked contrast to the "lupus anticoagulant effect" previously described in these patients. These results are the first direct demonstration of the displacement of annexin-V and the consequent acceleration of coagulation on noncellular phospholipid surfaces by antiphospholipid antibodies.
Asunto(s)
Anexina A5/metabolismo , Anticuerpos Antifosfolípidos/farmacología , Coagulación Sanguínea , Inhibidor de Coagulación del Lupus/farmacología , Fosfolípidos/metabolismo , Anexina A5/antagonistas & inhibidores , Plaquetas/metabolismo , Fluoresceína-5-Isotiocianato , Colorantes Fluorescentes , Humanos , Inmunoglobulina G/farmacología , Indicadores y Reactivos , Membrana Dobles de Lípidos/metabolismo , Tiempo de Tromboplastina Parcial , Fosfatidilserinas/metabolismo , Tiempo de Protrombina , Tromboplastina/metabolismoRESUMEN
Phosphatidate phosphohydrolase (PAPase) is a key enzyme involved in glycerolipid synthesis where it converts phosphatidic acid to diacylglycerol. Previous studies performed in lung have demonstrated the existence of 2 different forms of PAPases, namely PAP-1 and PAP-2. The former pulmonary Mg+2-dependent enzyme is N-ethylmaleimide (NEM)-sensitive, heat labile, and is involved in phospholipid biosynthesis. However, the function of the latter lung isozyme is unknown. PAP-2 activity was selectively assayed using NEM in the absence of Mg+2. Studies employing this assay and adult rat lung microsomal preparations demonstrated that PAP-2 activity was inhibited by amphiphilic amines, sphingoid bases, products of the PAP-2 reaction (monoacylglycerol [MAG] and diacylglycerol [DAG]), and substrate analogs such as lysophosphatidic acid (lyso-PA), ceramide-1-phosphate, and to a lesser extent, sphingosine-1-phosphate. Purified lung plasma membranes, prepared using discontinuous sucrose and Percoll gradients, showed that PAP-2 activity was enriched 6.9 +/- 1.6-fold over the whole homogenate and was between the enrichment for plasma membrane markers, 5'-nucleotidase (14.7 +/- 0.3) and Na+, K(+)-ATPase (4.0 +/- 0.2). Both phosphatidic acid and lysophosphatidic acid were good substrates for PAP-2 activity in this purified plasma membrane fraction. In contrast, sphingosine-1-phosphate was a relatively poor substrate. PAP-2 activity was slightly enriched in isolated type II cells and low in isolated rat lung fibroblasts. This study shows lung contains PAP-2 activity in plasma membranes and type II cells where it could play a role in signal transduction.