RESUMEN
BACKGROUND: Curdione, a sesquiterpene compound isolated from the essential oil of Curcuma aromatica Salisb. inhibits platelet aggregation, suggesting its significant anticoagulant and antithrombotic effects. However, the mechanisms have not been fully elucidated. HYPOTHESIS: We hypothesized that curdione inhibits thrombin-induced platelet aggregation via regulating the AMP-activated protein kinase-vinculin/talin-integrin αIIbß3 signaling pathway. STUDY DESIGN: We performed in vitro assays to evaluate the effect of curdione on thrombin-induced expression levels of the AMPK signaling molecule and integrin αIIbß3 signaling pathway components. METHODS: Platelet proteins were extracted from washed human platelets, and the effects of curdione on thrombin-induced platelet aggregation were evaluated. The expression levels of the AMPK signaling molecule and integrin αIIbß3 signaling pathway-related proteins were examined using western blot and RT-PCR. The binding of vinculin and talin were studied using immunoprecipitation, double immunofluorescence staining and microscale thermophoresis. RESULTS: Platelet aggregation analysis showed that 0.02â¯U/ml thrombin significantly induces platelet aggregation. Western blot and RT-PCR analysis revealed that AMPK inhibits the vinculin/talin-mediated integrin αIIbß3 signaling pathway, and curdione downregulates the thrombin-induced expression of phosphorylated AMPK (P-AMPK) and P-integrin at both the protein and mRNA levels and downregulates vinculin and talin at the protein level. Furthermore, microscale thermophoresis experiments showed that curdione inhibits the binding of vinculin and talin. The results from the immunoprecipitation and double immunofluorescence staining were consistent with the results of the microscale thermophoresis experiments. CONCLUSION: Curdione inhibits thrombin-induced platelet aggregation via regulating the AMP-activated protein kinase-vinculin/talin-integrin αIIbß3 signaling pathway, which suggests its therapeutic potential in ethnomedicinal applications as an anti-platelet and anti-thrombotic compound to prevent thrombotic diseases.
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Inhibidores de Agregación Plaquetaria/farmacología , Agregación Plaquetaria/efectos de los fármacos , Sesquiterpenos de Germacrano/farmacología , Trombina/efectos adversos , Proteínas Quinasas Activadas por AMP/genética , Proteínas Quinasas Activadas por AMP/metabolismo , Curcuma/química , Evaluación Preclínica de Medicamentos , Humanos , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/genética , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Transducción de Señal/efectos de los fármacos , Talina/metabolismo , Vinculina/metabolismoRESUMEN
Rhizoma Curcumae, the dry rhizomes derived from Curcuma aromatica Salisb., are a classical Chinese medicinal herb used to activate blood circulation, remove blood stasis and alleviate pain. Our previous study proved that curdione, a sesquiterpene compound isolated from the essential oil of Curcuma aromatica Salisb. can inhibit platelet activation suggesting its significant anticoagulant and antithrombotic effects. However, the underlying mechanism of curdione mediated anti-platelet effect has not been fully elucidated. Platelet proteins extracted from washed human platelets, including normal group (treated with normal saline), thrombin group and curdione group were digested and analysed by nano ESI-LC-MS/MS. UniProt database and SIEVE software were employed to identify and reveal the differentially expressed proteins. Furthermore, possible mechanisms involved were explored by Ingenuity Pathway Analysis (IPA) Software and validated by western blot experiments. Twenty-two differentially expressed proteins between the normal and thrombin group were identified. Compared with the thrombin group, the curdione treatment was significantly down-regulated only 2 proteins (Talin1 and ß1-tubulin). Bioinformatics analysis showed that Talin1 and ß1-tubulin could be involved in the integrin signal pathway. The results of western blot analysis were consistent with that of the proteomics data. Vinculin, identified in IPA database was involved in the formation of cell cytoskeletal. The down-regulation of ß1-tubulin facilitated the decrease in vinculin/Talin1. Curdione regulated the expression of vinculin and Talin1 by ß1-tubulin affecting the integrin signalling pathway and eventually inhibiting platelet activation. The ß1-tubulin may be a potential target of curdione, which attenuates thrombin-induced human platelet activation.
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Curcuma/química , Activación Plaquetaria/efectos de los fármacos , Sesquiterpenos de Germacrano/farmacología , Trombina/farmacología , Tubulina (Proteína)/metabolismo , Plaquetas/efectos de los fármacos , Regulación hacia Abajo , Humanos , Aceites Volátiles , Proteoma , Rizoma/química , Transducción de Señal/efectos de los fármacos , Talina/metabolismo , Vinculina/metabolismoRESUMEN
OBJECTIVE: To investigate the role of aqueous extracts of Tribulus terrestris (TT) against oxidized low-density lipoprotein (ox-LDL)-induced human umbilical vein endothelial cells (HUVECs) dysfunction in vitro. METHODS: HUVECs were pre-incubated for 60 min with TT (30 and 3 µg/mL respectively) or 10(-5) mol/L valsartan (as positive controls) and then the injured endothelium model was established by applying 100 µg/mL ox-LDL for 24 h. Cell viability of HUVECs was observed by real-time cell electronic sensing assay and apoptosis rate by Annexin V/PI staining. The cell migration assay was performed with a transwell insert system. Cytoskeleton remodeling was observed by immunofluorescence assay. The content of endothelial nitric oxide synthase (eNOS) was measured by enzyme-linked immunosorbent assay. Intracellular reactive oxygen species (ROS) generation was assessed by immunofluorescence and flow cytometer. Key genes associated with the metabolism of ox-LDL were chosen for quantitative real-time polymerase chain reaction to explore the possible mechanism of TT against oxidized LDL-induced endothelial dysfunction. RESULTS: TT suppressed ox-LDL-induced HUVEC proliferation and apoptosis rates significantly (41.1% and 43.5% after treatment for 3 and 38 h, respectively; P<0.05). It also prolonged the HUVEC survival time and postponed the cell's decaying stage (from the 69th h to over 100 h). According to the immunofluorescence and transwell insert system assay, TT improved the endothelial cytoskeletal network, and vinculin expression and increased cell migration. Additionally, TT regulated of the synthesis of endothelial nitric oxide synthase and generation of intracellular reactive oxygen species (P<0.05). Both 30 and 3 µg/mL TT demonstrated similar efficacy to valsartan. TT normalized the increased mRNA expression of PI3Kα and Socs3. It also decreased mRNA expression of Akt1, AMPKα1, JAK2, LepR and STAT3 induced by ox-LDL. The most notable changes were JAK2, LepR, PI3Kα, Socs3 and STAT3. CONCLUSIONS: TT demonstrated potential lowering lipid benefits, anti-hypertension and endothelial protective effects. It also suggested that the JAK2/STAT3 and/or PI3K/AKT pathway might be a very important pathway which was involved in the pharmacological mechanism of TT as the vascular protective agent.
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Endotelio Vascular/fisiopatología , Lipoproteínas LDL/efectos adversos , Extractos Vegetales/farmacología , Sustancias Protectoras/farmacología , Tribulus/química , Agua/química , Apoptosis/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Citoesqueleto/efectos de los fármacos , Citoesqueleto/metabolismo , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/patología , Ensayo de Inmunoadsorción Enzimática , Técnica del Anticuerpo Fluorescente , Regulación de la Expresión Génica/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Humanos , Óxido Nítrico Sintasa de Tipo III/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Vinculina/metabolismoRESUMEN
Solid tumors arising from malignant transformation of glial cells are one of the leading causes of central nervous system tumor-related death in children. Recurrence in spite of rigorous surgical and chemoradiation therapies remains a major hurdle in management of these tumors. Here, we investigate the efficacy of the second-generation receptor tyrosine kinase inhibitor nilotinib as a therapeutic option for the management of pediatric gliomas. We have utilized two independent pediatric high-grade glioma cell lines with either high platelet-derived growth factor receptor alpha (PDGFRα) or high PDGFRß expression in in vitro assays to investigate the specific downstream effects of nilotinib treatment. Using in vitro cell-based assays we show that nilotinib inhibits PDGF-BB-dependent activation of PDGFRα. We further show that nilotinib is able to decrease cell proliferation and anchorage-independent growth via suppression of AKT and ERK1/2 signaling pathways. Our results suggest that nilotinib may be effective for management of a PDGFRα-dependent group of pediatric gliomas.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Glioma/patología , Pirimidinas/farmacología , Animales , Becaplermina , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Ensayo de Unidades Formadoras de Colonias , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Inhibidores Enzimáticos/uso terapéutico , Glioma/tratamiento farmacológico , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Ratones , Ratones SCID , Proteína Oncogénica v-akt/metabolismo , Proteínas Proto-Oncogénicas c-sis/metabolismo , Pirimidinas/uso terapéutico , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Factores de Tiempo , Vinculina/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
OBJECTIVE: To test whether tanshinone II A (Tan II A), a highly valued herb derivative to treat vascular diseases in Chinese medicine, could protect endothelial cells from bacterial endotoxin (lipopolysaccharides, LPS)-induced endothelial injury. METHODS: Endothelial cell injury was induced by treating human umbilical vein endothelial cells (HUVECs) with 0.2 µg/mL LPS for 24 h. Y27632 and valsartan were used as positive controls. The effects of tanshinone II A on the LPS-induced cell viability and apoptosis rate of HUVECs were tested by flow cytometry, cell migration by transwell, adhesion by a 96-well plate pre-coated with vitronectin and cytoskeleton reorganization by immunofluorescence assay. Rho/Rho kinase (ROCK) pathway-associated gene and protein expression were examined by microarray assay; quantitative real-time polymerase chain reaction and Western blotting were used to confirm the changes observed by microarray. RESULTS: Tan II A improved cell viability, suppressed apoptosis and protected cells from LPS-induced reductions in cell migration and adhesion at a comparable magnitude to that of Y27632 and valsartan. Tan II A, Y27632 and valsartan also normalized LPS-induced actomyosin contraction and vinculin protein aggregation. A microarray assay revealed increased levels of fibronectin, integrin A5 (ITG A5), Ras homolog gene family member A (RhoA), myosin light chain phosphatase, phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K, or PIP2 in Western blotting), focal adhesion kinase, vascular endothelial growth factor and vascular endothelial growth factor receptor 2 in the damaged HUVECs, which were attenuated to different degrees by Tan II A, Y27632 and valsartan. CONCLUSION: Tan II A exerted a strong protective effect on HUVECs, and the mechanism was caused, at least in part, by a blockade in the Rho/ROCK pathway, presumably through the down-regulation of ITG A5.
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Abietanos/farmacología , Citoprotección/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/patología , Sustancias Protectoras/farmacología , Transducción de Señal/efectos de los fármacos , Proteínas de Unión al GTP rho/metabolismo , Quinasas Asociadas a rho/metabolismo , Abietanos/química , Apoptosis/efectos de los fármacos , Adhesión Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Forma de la Célula/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Citoesqueleto/efectos de los fármacos , Citoesqueleto/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/genética , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/enzimología , Humanos , Integrina alfaV/metabolismo , Lipopolisacáridos , Cadenas Ligeras de Miosina/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Fosfatidilinositol 4,5-Difosfato/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/genética , Vinculina/metabolismoRESUMEN
Ginsenoside Rd is a protopanaxadiol-type ginsenoside found in ginseng and is the active ingredient in several Oriental herbal medicines. We investigated the effects of ginsenoside Rd on tumor invasion and metastasis in the human hepatocellular carcinoma HepG2 and its possible mechanism of action. HepG2 cells were treated with ginsenoside Rd at different concentrations. Scratch wound and Boyden chamber assays were used to determine the effects of ginsenoside Rd on the migration and invasiveness of HepG2 cells, respectively. The molecular mechanisms by which ginsenoside Rd inhibited the invasion and migration of HepG2 cells were investigated by RT-PCR, Western blotting, gelatin zymography, promoter assay, and treatment with inhibitors of MAPK signaling. Immunofluorescence analysis was conducted to evaluate the effect of ginsenoside Rd on focal adhesion formation in HepG2 cells. Treatment with ginsenoside Rd dose- and time-dependently inhibited the migration and invasion of HepG2 cells. It achieved this by reducing the expression of MMP-1, MMP-2, and MMP-7, by blocking MAPK signaling by inhibiting the phosphorylation of ERK and p38 MAPK, by inhibition of AP-1 activation, and by inducing focal adhesion formation and modulating vinculin localization and expression. Treatment of HepG2 cells with ginsenoside Rd significantly inhibited metastasis, most likely by blocking MMP activation and MAPK signaling pathways involved in cancer cell migration. These findings may be useful for the development of novel chemotherapeutic agents for the treatment of malignant cancers.
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Antineoplásicos Fitogénicos/farmacología , Adhesiones Focales/efectos de los fármacos , Ginsenósidos/farmacología , Sistema de Señalización de MAP Quinasas , Western Blotting , Movimiento Celular , Supervivencia Celular , Relación Dosis-Respuesta a Droga , Activación Enzimática/efectos de los fármacos , Técnica del Anticuerpo Fluorescente , Adhesiones Focales/metabolismo , Células Hep G2 , Humanos , Metaloproteinasa 1 de la Matriz/genética , Metaloproteinasa 1 de la Matriz/metabolismo , Metaloproteinasa 7 de la Matriz/genética , Metaloproteinasa 7 de la Matriz/metabolismo , Invasividad Neoplásica/prevención & control , Fosforilación , Inhibidores de Proteínas Quinasas/farmacología , Vinculina/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/genética , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
BACKGROUND AND OBJECTIVES: Dental lasers represent a promising therapeutic tool in the treatment of periodontal and peri-implant diseases. However, their clinical application remains still limited. Here, we investigated the potential biostimulatory effect of low pulse energy neodymium:yttrium-aluminum-garnet (Nd:YAG) laser irradiation on different cells representative of the oral microenvironment and elucidated the underlying molecular mechanisms. MATERIALS AND METHODS: Saos-2 osteoblasts, H-end endothelial cells, and NIH/3T3 fibroblasts pre-treated or not with photosensitizing dye methylene blue (MB), were irradiated with low pulse energy (20 mJ) and high repetition rate (50-70 Hz) Nd:YAG laser, and evaluated for cell viability and proliferation as well as for the expression of specific differentiation markers by confocal immunofluorescence and real-time RT-PCR. Changes in intracellular Ca(2+) levels after laser exposure were also evaluated in living osteoblasts. RESULTS: Nd:YAG laser irradiation did not affect cell viability in all the tested cell types, even when combined with pre-treatment with MB, and efficiently stimulated cell growth in the non-sensitized osteoblasts. Moreover, a significant induction in the expression of osteopontin, ALP, and Runx2 in osteoblasts, type I collagen in fibroblasts, and vinculin in endothelial cells could be observed in the irradiated cells. Pre-treatment with MB negatively affected cell differentiation in the unstimulated and laser-stimulated cells. Notably, laser irradiation also caused an increase in the intracellular Ca(2+) in osteoblasts through the activation of TRPC1 ion channels. Moreover, the pharmacologic or genetic inhibition of these channels strongly attenuated laser-induced osteopontin expression, suggesting a role for the laser-mediated Ca(2+) influx in regulating osteoblast differentiation. CONCLUSION: Low pulse energy and high repetition rate Nd:YAG laser irradiation may exert a biostimulative effect on different cells representative of the oral microenvironment, particularly osteoblasts. Pre-treatment with MB prior to irradiation hampers this effect and limits the potential clinical application of photosensitizing dyes in dental practice.
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Células Endoteliales/efectos de la radiación , Fibroblastos/efectos de la radiación , Terapia por Luz de Baja Intensidad , Mucosa Bucal/citología , Osteoblastos/efectos de la radiación , Fosfatasa Alcalina/metabolismo , Animales , Calcio/metabolismo , Proliferación Celular/efectos de la radiación , Supervivencia Celular/efectos de la radiación , Células Cultivadas , Colágeno Tipo I/metabolismo , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Células Endoteliales/metabolismo , Inhibidores Enzimáticos/farmacología , Fibroblastos/metabolismo , Técnica del Anticuerpo Fluorescente , Humanos , Láseres de Estado Sólido , Azul de Metileno/farmacología , Ratones , Osteoblastos/metabolismo , Osteopontina/metabolismo , Reacción en Cadena de la Polimerasa , Canales Catiónicos TRPC/fisiología , Vinculina/metabolismoRESUMEN
BACKGROUND: Recently, it was possible to show that human corneal endothelial cells (HCEC) can be cultured on thermo-responsive polymer substrates, and can be harvested as entire cell sheets without losing viability. We sought to study HCEC sheet cultivation on such cell culture carriers under serum-free conditions as the next consequential step in developing methods for generation of corneal endothelial cell transplants. METHODS: An immortalized heterogenous HCEC population and two immortalized, clonally grown HCEC lines (HCEC-B4G12 and HCEC-H9C1) were cultured on thermo-responsive substrates under serum-supplemented and serum-free culture conditions. Cell sheets were characterized by phase contrast microscopy and by immunofluorescent staining for ZO-1, Na(+),K(+)-ATPase, and vinculin. RESULTS: All tested HCEC populations were able to adhere, spread and proliferate on thermo-responsive substrates under serum-supplemented conditions. Under serum-free conditions, pre-coating of the polymer substrates with ECM proteins was necessary to facilitate attachment and spreading of the cells, except in the case of HCEC-B4G12 cells. The heterogenous HCEC population formed closed monolayers, properly localized ZO-1 to lateral cell borders, and had moderate vinculin levels under serum-free, and higher vinculin levels under serum-supplemented culture conditions. HCEC-B4G12 cells formed closed monolayers, showed proper localization of ZO-1 and Na(+),K(+)-ATPase to lateral cell borders, and had high vinculin levels irrespective of culture conditions. In contrast, HCEC-H9C1 cells had lowest vinculin levels under serum-supplemented, and higher vinculin levels under serum-free culture conditions. ZO-1 was detected throughout the cytoplasm under both culture conditions. These loosely adherent cells were only able to form a closed monolayer under serum-supplemented conditions. CONCLUSIONS: Serum-free production of HCEC sheets is possible. The extremely adherent clonal HCEC line B4G12 produced higher vinculin levels than the other two tested HCEC populations, and showed strong adherence to the thermo-responsive, polymeric culture substratum irrespective of culture conditions. This cell line closely resembles terminally differentiated HCEC in vivo, and was found to be particularly suitable for further studies on HCEC cell sheet engineering.
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Línea Celular Transformada , Técnicas Citológicas , Endotelio Corneal/citología , Sangre , Adhesión Celular , Proliferación Celular , Células Clonales , Medios de Cultivo , Medio de Cultivo Libre de Suero , Endotelio Corneal/metabolismo , Endotelio Corneal/fisiología , Técnica del Anticuerpo Fluorescente , Humanos , Proteínas de la Membrana/metabolismo , Microscopía de Contraste de Fase , Fosfoproteínas/metabolismo , Ácidos Polimetacrílicos , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Coloración y Etiquetado , Temperatura , Ingeniería de Tejidos/métodos , Vinculina/metabolismo , Proteína de la Zonula Occludens-1RESUMEN
Although studies in vivo revealed promising results in bone regeneration after implantation of scaffolds together with osteogenic progenitor cells, basic questions remain how material surfaces control the biology of mesenchymal stem cells (MSC). We used human MSC derived from bone marrow and studied the osteogenic differentiation on calcium phosphate surfaces. In osteogenic differentiation medium MSC differentiated to osteoblasts on hydroxyapatite and BONITmatrix, a degradable xerogel composite, within 14 days. Cells revealed a higher alkaline phosphatase (ALP) activity and increased RNA expression of collagen I and osteocalcin using real-time RTPCR compared with cells on tissue culture plastic. To test whether material surface characteristics alone are able to stimulate osteogenic differentiation, MSC were cultured on the materials in expansion medium without soluble additives for osteogenic differentiation. Indeed, cells on calcium phosphate without osteogenic differentiation additives developed to osteoblasts as shown by increased ALP activity and expression of osteogenic genes, which was not the case on tissue culture plastic. Because we reasoned that the stimulating effect on osteogenesis by calcium phosphate surfaces depends on an altered cell-extracellular matrix interaction we studied the dynamic behaviour of focal adhesions using cells transfected with GFP labelled vinculin. On BONITmatrix, an increased mobility of focal adhesions was observed compared with cells on tissue culture plastic. In conclusion, calcium phosphate surfaces are able to drive MSC to osteoblasts in the absence of osteogenic differentiation supplements in the medium. An altered dynamic behaviour of focal adhesions on calcium phosphate surfaces might be involved in the molecular mechanisms which promote osteogenic differentiation.
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Fosfatos de Calcio/química , Diferenciación Celular , Durapatita/química , Células Madre Mesenquimatosas/citología , Osteogénesis/fisiología , Dióxido de Silicio/química , Fosfatasa Alcalina/metabolismo , Células de la Médula Ósea/metabolismo , Adhesión Celular/fisiología , Movimiento Celular/fisiología , Células Cultivadas , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Osteoblastos/citología , Osteoblastos/metabolismo , Osteocalcina/metabolismo , Vinculina/metabolismoRESUMEN
Altered cellular adhesion and apoptotic signaling in cardiac remodeling requires coordinated regulation of multiple constituent proteins that comprise cytoskeletal focal adhesions. One such protein activated by cardiac remodeling is related adhesion focal tyrosine kinase (RAFTK, also known as pyk2). Adenoviral-mediated expression of RAFTK in neonatal rat cardiomyocytes involves concurrent increases in phosphorylation of Src, c-Jun N-terminal kinase, and p38 leading to characteristic apoptotic changes including cleavage of poly(ADP-ribose) polymerase, caspase-3 activation, and increased DNA laddering. DNA laddering was decreased by mutation of the Tyr(402) Src-binding site in RAFTK, suggesting a central role for Src activity in apoptotic cell death that was confirmed by adenoviral-mediated Src expression. Multiple apoptotic signaling cascades are recruited by RAFTK as demonstrated by prevention of apoptosis using caspase-3 inhibitor IV (caspase-3 specific inhibitor), PP2 (Src-specific kinase inhibitor), or Csk (cellular negative regulator for Src), as well as dominant negative constructs for p38beta or MKP-1. These RAFTK-mediated phenotypic characteristics are prevented by concurrent expression of wild-type or a phosphorylation-deficient paxillin mutated at Tyr(31) and Tyr(118). Wild-type or mutant paxillin protein accumulation in the cytoplasm has no overt effect upon cell structure, but paxillin accumulation prevents losses of myofibril organization as well as focal adhesion kinase, vinculin, and paxillin protein levels mediated by RAFTK. Apoptotic signaling cascade inhibition by paxillin indicates interruption of signaling proximal to but downstream of RAFTK activity. Chronic RAFTK activation in cardiac remodeling may represent a maladaptive reactive response that can be modulated by paxillin, opening up novel possibilities for inhibition of cardiomyocyte apoptosis and structural degeneration in heart failure.
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Apoptosis , Proteínas del Citoesqueleto/farmacología , Miocitos Cardíacos/patología , Fosfoproteínas/farmacología , Proteínas Tirosina Quinasas/metabolismo , Familia-src Quinasas/metabolismo , Adenoviridae/genética , Animales , Animales Recién Nacidos , Sitios de Unión , Caspasa 3 , Caspasas/metabolismo , Adhesión Celular , Proteínas de Ciclo Celular/metabolismo , Células Cultivadas , Citoplasma/metabolismo , Proteínas del Citoesqueleto/metabolismo , Citoesqueleto/metabolismo , ADN/metabolismo , Fragmentación del ADN , ADN Complementario/metabolismo , Fosfatasa 1 de Especificidad Dual , Inhibidores Enzimáticos/farmacología , Quinasa 2 de Adhesión Focal , Genes Dominantes , Proteínas Inmediatas-Precoces/metabolismo , Immunoblotting , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Proteínas de la Membrana/metabolismo , Microscopía Fluorescente , Modelos Biológicos , Mutación , Miocitos Cardíacos/metabolismo , Paxillin , Fenotipo , Fosfoproteínas Fosfatasas/metabolismo , Fosfoproteínas/metabolismo , Fosforilación , Proteína Fosfatasa 1 , Proteínas Tirosina Fosfatasas/metabolismo , Ratas , Transducción de Señal , Estaurosporina/farmacología , Factores de Tiempo , Tirosina/química , Vinculina/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Familia-src Quinasas/antagonistas & inhibidoresRESUMEN
Exposure of endothelial cells to vascular endothelial growth factor (VEGF) induced tyrosine phosphorylation of focal adhesion kinase (FAK) on site Tyr(407), an effect that required the association of VEGF receptor 2 (VEGFR2) with HSP90. The association of VEGFR2 with HSP90 involved the last 130 amino acids of VEGFR2 and was blocked by geldanamycin, a specific inhibitor of HSP90. Moreover, geldanamycin inhibited the VEGF-induced activation of the small GTPase RhoA, which resulted in an inhibition of phosphorylation of FAK on site Tyr(407). In this context, the inhibition of RhoA kinase (ROCK) with Y27632 or by expression of dominant negative forms of RhoA or ROCK impaired the VEGF-induced phosphorylation of Tyr(407) within FAK. In contrast to phosphorylation of Tyr(861), the phosphorylation of site Tyr(407) was insensitive to Src kinase inhibition by 4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo[3,4-d] pyrimidine (PP2). We also found that the recruitment of paxillin to FAK was inhibited by geldanamycin but not by PP2, whereas both geldanamycin and PP2 inhibited the recruitment of vinculin to FAK. In accordance, the recruitment of paxillin and vinculin to FAK was inhibited in cells that express the mutant FAK-Y407F, whereas the expression of the mutant Y861F inhibited the recruitment of paxillin but not of vinculin. Importantly, cell migration was abolished in cells in which the signal from the VEGFR2-HSP90 pathway was blocked by the expression of Delta130VEGFR2, a deletant of VEGFR2 that does not associate with HSP90. Our findings underscore for the first time the key role played by the VEGFR2-HSP90-RhoA-ROCK-FAK/Tyr(407) pathway in transducing the VEGF signal that leads to the assembly of focal adhesions and endothelial cell migration.
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Regulación de la Expresión Génica , Proteínas HSP90 de Choque Térmico/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Familia-src Quinasas/metabolismo , Amidas/farmacología , Benzoquinonas , Antígenos CD18/metabolismo , Movimiento Celular , Células Cultivadas , Proteínas del Citoesqueleto/metabolismo , ADN Complementario/metabolismo , Endotelio Vascular/citología , Quinasa 1 de Adhesión Focal , Proteína-Tirosina Quinasas de Adhesión Focal , Técnicas de Transferencia de Gen , Genes Dominantes , Proteínas Fluorescentes Verdes , Humanos , Lactamas Macrocíclicas , Proteínas Luminiscentes/metabolismo , Mutación , Paxillin , Fosfoproteínas/metabolismo , Fosforilación , Pruebas de Precipitina , Piridinas/farmacología , Quinonas/farmacología , Factores de Tiempo , Tirosina/química , Tirosina/metabolismo , Venas Umbilicales/citología , Vinculina/metabolismo , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
Associated with the metastatic progression of epithelial tumors is the dynamic regulation of cadherins. Whereas E-cadherin is expressed in most epithelium and carcinomas, recent studies suggest that the up-regulation of other cadherin subtypes in carcinomas, such as N-cadherin, may function in cancer progression. We demonstrate that a signal transduction cascade links the N-cadherin.catenin adhesion complex to up-regulation of the anti-apoptotic protein Bcl-2. In suspension, aggregates of DU-145 cells, an E-cadherin expressing human prostate carcinoma line, survive loss of integrin-dependent adhesion by a different anti-apoptotic signaling pathway than the N-cadherin expressing lines PC3 and PC3N. N-cadherin intercellular adhesion mediates a 3.5-fold increase in Bcl-2 protein expression, whereas the level of the proapoptotic protein Bax remains constant. Only N-cadherin ligation in PC3 cells, which express both N-cadherin and E-cadherin, is sufficient to induce activation of Akt/protein kinase B. N-cadherin homophilic ligation initiates phosphatidylinositol 3-kinase-dependent activation of Akt resulting in Akt phosphorylation of Bad on serine 136. Following N-cadherin homophilic adhesion phosphatidylinositol 3-kinase was identified in immunoprecipitates of the N-cadherin.catenin complex. The recruitment of phosphatidylinositol 3-kinase to the adhesion complex is dependent on ligation of N-cadherin and an organized actin cytoskeleton because cytochalasin D blocks the recruitment. We propose that N-cadherin homophilic adhesion can initiate anti-apoptotic signaling, which enhances the Akt cell survival pathway in metastatic cancer.
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Citoesqueleto de Actina/metabolismo , Cadherinas/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , Proteínas Proto-Oncogénicas/metabolismo , Transducción de Señal , Actinas/metabolismo , Apoptosis , Calcio/metabolismo , Adhesión Celular , Supervivencia Celular , ADN Complementario/metabolismo , Glutatión Transferasa/metabolismo , Humanos , Immunoblotting , Microscopía Fluorescente , Fosforilación , Plásmidos/metabolismo , Pruebas de Precipitina , Proteínas Proto-Oncogénicas c-akt , Factores de Tiempo , Transfección , Células Tumorales Cultivadas , Vinculina/metabolismo , Proteína X Asociada a bcl-2RESUMEN
PURPOSE: To understand the involvement of isoprenylated small guanosine triphosphatases (GTPases) in lovastatin-induced cataractogenesis, Rho- and Rac-mediated cell adhesion and actin cytoskeletal reorganization were investigated in lovastatin-treated lens epithelial cells. METHODS: The effects of lovastatin on F-actin reorganization (phalloidin staining), focal adhesion formation (paxillin or vinculin), cell-cell adhesions (cadherin and beta-catenin), and protein tyrosine phosphorylation were evaluated in human and porcine lens epithelial cells by immunocytochemical staining with specific antibodies. To explore the involvement of the Rho and Rac GTPases in lovastatin-mediated effects, changes in distribution of Rho and Rac GTPases were analyzed by Western blot analysis, and the effects of C3-exoenzyme on lovastatin-induced cytoskeletal changes were evaluated by immunocytochemical analysis. RESULTS: Lovastatin induced drastic changes in cell shape in both human and porcine lens epithelial cells, including a profound loss of actin stress fibers, focal adhesions, protein phosphotyrosine, and cell-cell adhesions. Lovastatin treatment also led to the accumulation of nonisoprenylated Rho and Rac GTPases in cytosolic fraction. Supplementation of culture media with geranylgeranyl pyrophosphate dramatically reversed the lovastatin-induced morphologic and cytoskeletal changes, whereas farnesyl pyrophosphate was ineffective. Treatment of cells with C3-exoenzyme (a Rho GTPase-specific inhibitor), however, abolished the geranylgeranyl-supplementation-induced recovery from the morphologic and cytoskeletal effects of lovastatin. CONCLUSIONS: This study demonstrates that inhibition of protein prenylation by lovastatin leads to disruption of actin cytoskeletal organization, and to loss of integrin-mediated focal adhesions and cadherin-mediated cell-cell adhesions in lens epithelial cells. Based on isoprenoid supplementation studies, it could be concluded that impairment of geranylgeranylated Rho and Rac GTPase function is most likely responsible for lovastatin-induced cytoskeletal changes in lens epithelial cells.
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Citoesqueleto/metabolismo , Células Epiteliales/efectos de los fármacos , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Cristalino/efectos de los fármacos , Lovastatina/farmacología , Transactivadores , Proteínas de Unión al GTP rho/fisiología , Actinas/metabolismo , Animales , Western Blotting , Cadherinas/metabolismo , Adhesión Celular , Tamaño de la Célula/efectos de los fármacos , Células Cultivadas , Proteínas del Citoesqueleto/metabolismo , Células Epiteliales/metabolismo , Humanos , Cristalino/metabolismo , Paxillin , Fosfoproteínas/metabolismo , Fosforilación , Prenilación de Proteína , Porcinos , Tirosina/metabolismo , Vinculina/metabolismo , beta Catenina , Proteínas de Unión al GTP rac/fisiologíaRESUMEN
The expression of drebrin A was induced in mouse fibroblasts (L cells) after transformation of cells with a vector that carried cDNA for rat drebrin A (developmentally regulated brain protein A) under the control of the promoter of the gene for metallothionein-I. When drebrin was expressed in the transformed cells (MTI-5 cells), the organization of actin filaments changed such that stress fibers were converted to a mesh-like structure. After subsequent treatment with 5 micrograms/ml cytochalasin D (a reagent that depolymerizes actin filaments), MTI-5 cells maintained their shape, while cells of a drebrin-negative cell line, MTI-11, formed retraction processes. Simultaneously, actin filaments changed into patchy dot-like aggregates in the cytoplasm of both MTI-5 and MTI-11 cells. These aggregates are known as cytoplasmic pools. In MTI-5 cells, adhesion plaques that were resistant to treatment with cytochalasin D appeared upon expression of drebrin. These adhesion plaques were immunostained with vinculin-specific antibodies, while those in MTI-11 cells were hardly immunostained. The amount of vinculin in MTI-5 cells increased in parallel with increase in the level of drebrin. These results suggest that expression of drebrin A induces changes in the assembly of actin filaments and adhesion plaques, with resultant modulation of cellular adhesion to the substratum.
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Fibroblastos/citología , Neuropéptidos/análisis , Western Blotting , Compuestos de Cadmio/farmacología , Adhesión Celular/fisiología , Citocalasina D/farmacología , ADN Complementario/farmacología , Fibroblastos/química , Proteínas de Choque Térmico/efectos de los fármacos , Humanos , Inmunohistoquímica , Uniones Intercelulares/química , Neuroblastoma , Neuropéptidos/efectos de los fármacos , Sulfatos/farmacología , Transfección , Células Tumorales Cultivadas/fisiología , Vinculina/efectos de los fármacos , Vinculina/metabolismoRESUMEN
Adherens-type junctions (AJ) are specialized intercellular contacts, mediated by cadherins and characterized by the association with actin filaments through a vinculin- and catenin-rich submembrane plaque. We describe here two mechanisms which potentiate AJ formation in mesenchymal cells. These include the augmentation of AJ by the co-expression of another adhesion molecule, namely NCAM, and the stimulation of tyrosine phosphorylation. These effects were obtained in NIH-3T3 cells, which, under normal conditions, have poor cadherin- and vinculin-containing intercellular junctions. The transfection of these cells with cDNA encoding the 140kD NCAM resulted in the extensive formation of cadherin- and vinculin-rich AJ, demonstrating a cooperativity between the two junctional systems. AJ could also be induced in 3T3, and in CEF and COS cells, upon a brief exposure to H2O2/vanadate, which elevates cellular levels of phosphotyrosine due to inhibition of tyrosine-specific phosphatases. This induction was, however, transient since prolonged exposure to H2O2/vanadate resulted in an overall destruction of AJ and detachment of cells from each other and from the extracellular matrix. AJ formation appears, therefore, to be modulated by a variety of factors including the level of expression of its intrinsic components, the cooperative effect of other adhesion molecules, and by tyrosine-phosphorylation.
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Moléculas de Adhesión Celular Neuronal/fisiología , Adhesión Celular/fisiología , Uniones Intercelulares/fisiología , Tirosina/metabolismo , Células 3T3 , Animales , Cadherinas/metabolismo , Adhesión Celular/efectos de los fármacos , Moléculas de Adhesión Celular Neuronal/genética , Línea Celular , Embrión de Pollo , Chlorocebus aethiops , ADN Complementario/genética , Matriz Extracelular/metabolismo , Técnica del Anticuerpo Fluorescente , Peróxido de Hidrógeno/farmacología , Uniones Intercelulares/efectos de los fármacos , Ratones , Fosforilación , Transfección , Vanadatos/farmacología , Vinculina/metabolismoRESUMEN
Paxillin is a cytoskeletal protein involved in actin-membrane attachment at sites of cell adhesion to the extracellular matrix. Extensive tyrosine phosphorylation of this protein occurs during integrin-mediated cell adhesion, embryonic development, fibroblast transformation and following stimulation of cells by mitogens that operate through the family of seven membrane-spanning G-protein-coupled receptors. Paxillin binds in vitro to the focal adhesion protein vinculin as well as to the SH3 domain of c-src and, when tyrosine phosphorylated, to the SH2 domain of v-crk. Here, we report the complementary DNA, and derived amino acid sequence, that codes for approximately 90% of the paxillin protein. We have identified a region in the amino-terminal half of the protein that supports the binding of both vinculin and the focal adhesion tyrosine kinase, pp125Fak. Although there is no significant overall homology with other identified proteins, the carboxyl third of paxillin contains one LIM domain and three LIM-like sequences. The LIM motif is common to a number of transcription factors and to two other focal adhesion proteins, zyxin and cysteine-rich protein. In addition to several potential tyrosine phosphorylation sites there are five tyrosine-containing sequences that conform to SH2-binding motifs. The protein also contains a short proline-rich region indicative of a SH3-binding domain. Taken together, these data suggest that paxillin is a unique cytoskeletal protein capable of interaction with a variety of intracellular signalling, and structural, molecules important in growth control and the regulation of cytoskeletal organization.(ABSTRACT TRUNCATED AT 250 WORDS)