RESUMEN
Macropinocytosis is a type of endocytosis accompanied by actin rearrangement-driven membrane deformation, such as lamellipodia formation and membrane ruffling, followed by the formation of large vesicles, macropinosomes. Ras-transformed cancer cells efficiently acquire exogenous amino acids for their survival through macropinocytosis. Thus, inhibition of macropinocytosis is a promising strategy for cancer therapy. To date, few specific agents that inhibit macropinocytosis have been developed. Here, focusing on the mechanosensitive ion channel Piezo1, we found that Yoda1, a Piezo1 agonist, potently inhibits macropinocytosis induced by epidermal growth factor (EGF). The inhibition of ruffle formation by Yoda1 was dependent on the extracellular Ca2+ influx through Piezo1 and on the activation of the calcium-activated potassium channel KCa3.1. This suggests that Ca2+ ions can regulate EGF-stimulated macropinocytosis. We propose the potential for macropinocytosis inhibition through the regulation of a mechanosensitive channel activity using chemical tools.
Asunto(s)
Carcinoma de Células Escamosas , Factor de Crecimiento Epidérmico , Canales Iónicos , Pirazinas , Tiadiazoles , Transporte Biológico , Calcio/metabolismo , Línea Celular Tumoral , Factor de Crecimiento Epidérmico/farmacología , Humanos , Canales Iónicos/agonistas , Canales Iónicos/metabolismo , Pinocitosis/efectos de los fármacosRESUMEN
BACKGROUND: To promote understanding of the pathogenesis of cognitive impairment or dementia, we explored the potential interaction between transient cerebral ischemia and amyloid-ß (Aß) infusion in mediating cognitive decline and examined the possible ameliorative effect of angiotensin II type 2 (AT2) receptor activation in vascular smooth muscle cells (VSMC) on this cognitive deficit. METHODS: Adult male wild-type mice (WT) and mice with VSMC-specific AT2 receptor overexpression (smAT2) were subjected to intracerebroventricular (ICV) injection of Aß1-40. Transient cerebral ischemia was induced by 15 min of bilateral common carotid artery occlusion (BCCAO) 24 h after Aß injection. RESULTS: Aß injection in WT induced a cognitive decline, whereas BCCAO did not cause a significant cognitive deficit. In contrast, WT with BCCAO following Aß injection exhibited more marked cognitive decline compared to Aß injection alone, in concert with increases in superoxide anion production, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity, and expression of p22phox, p40phox, monocyte chemoattractant protein (MCP)-1 and interleukin (IL)-1ß in the hippocampus, and upregulation of RAGE (receptor for advanced glycation end product), an Aß transporter. BCCAO following Aß injection further enhanced neuronal pyknosis in the hippocampus, compared with BCCAO or Aß injection alone. In contrast, smAT2 did not show a cognitive decline, increase in oxidative stress, inflammation, and RAGE level or neuronal pyknosis, which were induced by BCCAO with/without Aß injection in WT. CONCLUSIONS: Transient cerebral ischemia might worsen Aß infusion-mediated cognitive decline and vice versa, with possible involvement of amplified oxidative stress and inflammation and impairment of the RAGE-mediated Aß clearance system, contributing to exaggerated neuronal degeneration. AT2 receptor activation in VSMC could play an inhibitory role in this cognitive deficit.
Asunto(s)
Péptidos beta-Amiloides/toxicidad , Cognición/fisiología , Disfunción Cognitiva/etiología , Ataque Isquémico Transitorio/complicaciones , Receptor de Angiotensina Tipo 2/metabolismo , Animales , Disfunción Cognitiva/metabolismo , Disfunción Cognitiva/patología , Ataque Isquémico Transitorio/metabolismo , Ataque Isquémico Transitorio/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Estrés Oxidativo/fisiologíaRESUMEN
OBJECTIVE: Estrogen deficiency caused by bilateral ovariectomy (OVX) has been reported to lead to morphological changes in otoconia. Thus, we examined the morphological changes in the otoconial layer after OVX. We also investigated whether micro-computed tomography (µCT) is useful for the detection of morphological changes in the otoconial layer. METHODS: The otic capsules of C57BL/6 J mice were removed and evaluated using histological techniques and µCT at 2, 4, and 8 weeks after OVX or sham surgery. The volume of the utricle otoconial layer was measured and compared between the OVX and sham groups. The µCT scan and histological study results were also compared. RESULTS: The volume of the utricle otoconial layer was significantly increased 4 weeks after OVX compared to the sham group in both histological and µCT studies (p < 0.05). The volume of the otoconial layer measured using µCT was significantly correlated with the histological study results (p < 0.05). CONCLUSION: The volume of the utricle otoconial layer increased after OVX. These morphological changes could be detected by µCT.
Asunto(s)
Membrana Otolítica/anatomía & histología , Ovariectomía , Microtomografía por Rayos X , Animales , Densidad Ósea , Femenino , Ratones , Ratones Endogámicos C57BL , Modelos Animales , Membrana Otolítica/diagnóstico por imagen , Útero/anatomía & histologíaRESUMEN
We conducted photo-activated delivery of drugs based on the fusion of liposomes with endocytic membranes, thus allowing the direct release of encapsulated drugs inside the cytoplasm. As described in our earlier works, liposomes can be photoresponsive and fusogenic following the incorporation of a malachite green derivative carrying a long alkyl chain (MGL) into the lipid membrane. We prepared MGL liposomes using 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine and encapsulated doxorubicin (DOX). Though the shape of MGL liposomes became elliptical after encapsulating DOX, UV irradiation did not enhance DOX leakage from MGL liposomes. We demonstrated the cellular uptake of MGL liposomes into murine cells derived from colon cancer (Colon 26 cells) using flow cytometry, and we found that the uptake was governed by a clathrin-dependent endocytosis pathway. Confocal fluorescence microscopic observations of Colon 26 cells treated with MGL liposomes encapsulating DOX revealed that DOX was localized in endosomes under dark conditions, while DOX was observed in the cytosol and nucleus after UV irradiation. The viability of Colon 26 cells treated with MGL liposomes encapsulating DOX was reduced by UV irradiation, indicating photo-induced enhancement of anti-cancer efficacy.
Asunto(s)
Antibióticos Antineoplásicos/farmacocinética , Neoplasias del Colon/tratamiento farmacológico , Doxorrubicina/farmacocinética , Sistemas de Liberación de Medicamentos , Endosomas/química , Liposomas/química , Colorantes de Rosanilina/química , Animales , Antibióticos Antineoplásicos/química , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Relación Dosis-Respuesta a Droga , Doxorrubicina/química , Portadores de Fármacos/química , Ensayos de Selección de Medicamentos Antitumorales , Ratones , Estructura Molecular , Relación Estructura-Actividad , Células Tumorales Cultivadas , Rayos UltravioletaRESUMEN
Chèdiak-Higashi syndrome is a rare autosomal recessive disease that causes hypopigmentation, recurrent infections, mild coagulation defects and neurological problems. Beige mice carry a mutation in the lysosome trafficking regulator (LYST) gene and display some of the key characteristics of human Chèdiak-Higashi syndrome, in particular, a high susceptibility to infection due to aberrant natural killer (NK) cell and polymorphonuclear leucocyte function. Morphological analysis of beige mice reveals the presence of enlarged lysosomes in a variety of cell types, including leucocytes, hepatocytes, fibroblasts and renal tubule cells. To examine the process of granule maturation and degranulation in beige mice mast cells, morphological studies have been conducted using a combination of electrophysiological techniques; however, few functional studies have been conducted with mast cells, such as mediator release. The aim of the present study was to determine the morphological and functional characteristics of skin and peritoneal mast cells and bone marrow-derived mast cells of homozygous (bg/bg) and heterozygous (bg/+) beige mice and wild-type (+/+) mice. The histamine concentration was lower in the peritoneal and bone marrow-derived mast cells of bg/bg mice compared with those of bg/+ and +/+ mice, but the histamine release response was potentiated. In vivo studies of passive cutaneous anaphylaxis showed no differences between bg/bg mice and either bg/+ or +/+ mice. Although bg/bg mast cells with enlarged granules display specific exocytotic processes in vitro, the consequences of mast cell activation in beige mice were similar to those of wild-type mice in vivo.
Asunto(s)
Síndrome de Chediak-Higashi/inmunología , Gránulos Citoplasmáticos/patología , Células Asesinas Naturales/inmunología , Lisosomas/patología , Mastocitos/fisiología , Neutrófilos/inmunología , Animales , Degranulación de la Célula , Células Cultivadas , Síndrome de Chediak-Higashi/genética , Modelos Animales de Enfermedad , Histamina/metabolismo , Homocigoto , Humanos , Péptidos y Proteínas de Señalización Intracelular , Mastocitos/patología , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Mutación/genética , Proteínas/genética , Proteínas de Transporte VesicularRESUMEN
Mast cells, in addition to endocrine cells and neurons, are typical secretory cells. Their function in allergic inflammation is to secrete inflammatory mediators from secretory vesicles. Intracellular synthesized inflammatory mediators are transported by vesicular monoamine transporters (VMATs) to vesicles where they are stored. After stimulation, the contents of the secretory vesicles are released via exocytosis. This study established a high throughput imaging screening system to monitor the functions of secretory vesicles in mast cells, including molecular uptake via VMAT2 and the exocytotic process, by using a novel fluorescent probe, FFN206, which was developed as a VMAT2 substrate. After loading with FFN206, the rapid uptake of FFN206 was observed and secretory vesicles in mouse bone marrow derived mast cells and a cultured mast cell line were clearly visualized. FFN206 uptake by secretory vesicles was time-dependent and was blocked by reserpine. Furthermore, exocytotic trafficking was monitored dynamically by real-time high-throughput fluorescence quantitation. In the present study, we verified the application of FFN206 for the monitoring of functional vesicles. This high-throughput screening system may benefit instinctive drug evaluation.
Asunto(s)
Células de la Médula Ósea/metabolismo , Exocitosis , Ensayos Analíticos de Alto Rendimiento/métodos , Mastocitos/metabolismo , Vesículas Secretoras/metabolismo , Animales , Línea Celular Tumoral , Evaluación Preclínica de Medicamentos/métodos , Femenino , Colorantes Fluorescentes/química , Colorantes Fluorescentes/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Cultivo Primario de Células , Ratas , Proteínas de Transporte Vesicular de Monoaminas/metabolismoRESUMEN
OBJECTIVE: Hydrogen-rich water, which is a potent antioxidant agent, was investigated for its protective effects against ischemic damage of the cochlea in gerbils. METHODS: The animals were subjected to transient cochlear ischemia by occluding the bilateral vertebral arteries for l5min. Five milliliters of hydrogen-rich saline was then intravenously administered immediately after the insult. Saline without hydrogen was used as a control. Effects of hydrogen were evaluated using the auditory brainstem response (ABR) and histological studies of the inner ear. RESULTS: In non-ischemia animals, ABR thresholds and histological findings of the cochlea did not change by administration of saline or hydrogen-rich saline. In contrast, transient cochlear ischemia caused a 24.2±3.8dB increase in the ABR threshold at 8kHz, and a decrease of 14.1%±1.8% in the number of inner hair cells (IHCs) at the basal turn on day 7. Ischemic damage was more severe at 16 and 32kHz. When the animals were treated with hydrogen-rich saline, cochlear damage was significantly reduced: the increase in ABR threshold was 11.7±2.6dB at 8kHz and the IHC loss was 7.5%±2.1% at the basal turn on day 7. The effects of hydrogen-rich saline were more prominent at higher frequencies. CONCLUSIONS: Intravenous administration of hydrogen-rich saline was effective in preventing acute hearing loss due to transient cochlear ischemia.
Asunto(s)
Sordera/prevención & control , Pérdida Auditiva/prevención & control , Hidrógeno/farmacología , Isquemia/complicaciones , Cloruro de Sodio/farmacología , Administración Intravenosa/métodos , Animales , Sordera/patología , Potenciales Evocados Auditivos del Tronco Encefálico/efectos de los fármacos , Gerbillinae , Células Ciliadas Auditivas/efectos de los fármacos , Células Ciliadas Auditivas Internas/patología , Pérdida Auditiva/patología , MasculinoRESUMEN
PURPOSE: The increasing amounts of evidence with abnormal aging process have been involved in the pathogenesis of chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF). Mice with deficient protein L-isoaspartate (D-aspartate) O-methyl transferase 1 (PCMT1) expression reveal acceleration of aging and result in the increased proportion of D-aspartate (D-Asp) residues and dysfunction in proteins. Furthermore, mitochondrial morphology and functions are associated with COPD and IPF pathogenesis. The purpose of the current study was to investigate the role of PCMT1 on mitochondrial morphology using A549 cells. MATERIALS AND METHODS: We investigated PCMT1, prohibitin1 (PHB1), mitochondrial membrane proteins expression, mitochondrial morphology, and the proportion of D-Asp residues in PHB1 in A549 cells with (PCMT1-KD) and without the context of decreased PCMT1 expression (PCMT1-Cont) using electron microscopy, fluorescence staining, Western blot analysis, and the ATP content per cells. To investigate the effects of the PCMT1-KD cells, we developed double-transfected cell lines containing either the cytosolic or the endoplasmic isoform of PCMT1. RESULTS: We found a significantly higher proportion of D-Asp residues in PHB1 in PCMT1-KD cells than that in PCMT1-Cont cells. The PCMT1-KD cells without cigarette smoke extract exposure were characterized by a significantly increased proportion of the D-Asp residues in PHB1, damaged mitochondrial ultrastructure, and a tendency toward the fission direction of the mitochondrial dynamics followed by a significant decrease in the cellular ATP content. CONCLUSIONS: The increased proportion of the D-Asp residues may contribute to COPD pathogenesis, via irreversible protein conformational changes, followed by mitochondrial dysfunction.
Asunto(s)
Mitocondrias/enzimología , Proteína D-Aspartato-L-Isoaspartato Metiltransferasa/metabolismo , Proteínas Represoras/metabolismo , Células A549 , Adenosina Trifosfato/metabolismo , Estrés del Retículo Endoplásmico , Humanos , Mitocondrias/ultraestructura , Dinámicas Mitocondriales , Estrés Oxidativo , ProhibitinasRESUMEN
Lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1), a type I transmembrane glycoprotein, is known as one of the most specific lymphatic vessel markers in the skin. In this study, we found that the ectodomain of LYVE-1 undergoes proteolytic cleavage, and this process produces soluble LYVE-1. We further identified the cleavage site for ectodomain shedding and generated an uncleavable mutant of LYVE-1. In lymphatic endothelial cells, ectodomain shedding of LYVE-1 was induced by vascular endothelial growth factor (VEGF)-A, an important factor for angiogenesis and lymphangiogenesis under pathological conditions. VEGF-A-induced LYVE-1 ectodomain shedding was mediated via the extracellular signal-regulated kinase (ERK) and a disintegrin and metalloproteinase (ADAM) 17. Wild-type LYVE-1, but not uncleavable LYVE-1, promoted migration of lymphatic endothelial cells in response to VEGF-A. Immunostaining analyses in human psoriasis skin lesions and VEGF-A transgenic mouse skin suggested that the ectodomain shedding of LYVE-1 occurred in lymphatic vessels undergoing chronic inflammation. These results indicate that the ectodomain shedding of LYVE-1 might be involved in promoting pathological lymphangiogenesis.
Asunto(s)
Glicoproteínas/metabolismo , Vasos Linfáticos/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Proteína ADAM17/genética , Proteína ADAM17/metabolismo , Animales , Línea Celular , Micropartículas Derivadas de Células/metabolismo , Modelos Animales de Enfermedad , Células Endoteliales/metabolismo , Femenino , Glicoproteínas/genética , Humanos , Linfangiogénesis/fisiología , Sistema de Señalización de MAP Quinasas , Proteínas de Transporte de Membrana , Ratones , Ratones Transgénicos , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Psoriasis/etiología , Psoriasis/metabolismo , Psoriasis/patología , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Factor A de Crecimiento Endotelial Vascular/genética , Proteínas de Transporte Vesicular/genéticaRESUMEN
Systemic lupus erythematosus (SLE) is characterized by production of a variety of autoantibodies. Although anti-double-stranded DNA (anti-dsDNA) antibodies contribute to the pathogenesis of lupus nephritis (LN), they are not sufficient for diagnosis and evaluation of disease activity. To obtain other autoantibodies associated with LN, we screened autoantigens reacting with the sera of LN patients by using an N-terminal biotinylated protein library created from a wheat cell-free protein production system. We screened 17 proteins that showed higher positive signals in the active phase than in the inactive phase of SLE, and higher positive signals in the serum of SLE patient with nephritis than in that of patient without nephritis. Of these, two LN-associated autoantigens, ribosomal RNA-processing protein 8 (RRP8) and spermatid nuclear transition protein 1 (TNP1) were identified by immunoprecipitation and immunofluorescence of renal tissues. Circulating anti-RRP8 and anti-TNP1 autoantibodies were recognized and deposited as an immune complex (IC) in glomeruli. IC was deposited preferentially in glomeruli rather than in other organs in C57BL/6 mice injected with RRP8 or TNP1. ELISA analysis of sera from patients with various rheumatic diseases demonstrated reactivity for RRP8 and TNP1 in 20% and 14.7% of SLE patients, respectively, whereas there was little or no reactivity in patients with other rheumatic diseases. Among SLE patients, 63.6% and 45.5% of those with LN were positive for anti-RRP8 and anti-TNP1 antibodies, compared with 12.5% and 9.4% of SLE patients without nephritis, respectively. Both proteins are cationic, and their respective antibodies did not cross-react with dsDNA. These proteins released from apoptotic cells form ICs with each autoantibody, and their ICs may become trapped at anionic sites in the glomerular basement membrane, leading to deposition in glomeruli. These autoantibodies may be useful for prediction of LN in subsets of SLE patients who are negative for anti-dsDNA antibodies.
Asunto(s)
Autoanticuerpos/inmunología , Proteínas Cromosómicas no Histona/inmunología , Complejo Multienzimático de Ribonucleasas del Exosoma/inmunología , Nefritis Lúpica/inmunología , Metiltransferasas/inmunología , Proteínas Nucleares/inmunología , Animales , Anticuerpos Antinucleares/inmunología , Complejo Antígeno-Anticuerpo/metabolismo , Apoptosis , Autoantígenos/inmunología , ADN/inmunología , Femenino , Humanos , Corteza Renal/patología , Glomérulos Renales/inmunología , Glomérulos Renales/patología , Nefritis Lúpica/diagnóstico , Ratones , Ratones Endogámicos C57BL , Proteínas de Unión al ARNRESUMEN
The regulated control of Ca(2+) influx is essential for the activation and function of the adaptive immune response, as Ca(2+) is a key regulator of important transcription factors. To determine whether Ca(2+) release-activated Ca(2+) (CRAC) channels contribute to the abnormal behaviour of T cells in patients with rheumatoid arthritis (RA), we performed a cross-sectional study to characterize the expression and functional status of CRACM1 channels in RA patients. Peripheral blood was obtained from 50 RA patients, 50 osteoarthritis (OA) patients and healthy donors. We measured Ca(2+) influx and CRAC currents in naïve and memory CD4(+) T cells. CRACM1 expression was evaluated in T cells from each of the three groups. These cells were further characterized by flow cytometric analysis of interleukin-4 (IL-4), IL-17, interferon-γ and tumour necrosis factor-α. These cytokines were also measured in naïve CD4(+) T cells following the lentivirus-mediated silencing of CRACM1.There was a significant positive correlation between Ca(2+) influx in naïve T cells and RA activity. Functionally aberrant naïve CD4(+) T cells from patients with active RA showed the different cytokine release pattern and exhibited increased Ca(2+) influx as well as increased CRACM1 protein expression and function. Specific lentiviral-induced gene silencing of CRACM1 reversed the alterations in T-cell cytokine production. The data presented here indicate that an upregulation of CRACM1 expression and function may be responsible for the abnormal cytokine release of naïve CD4(+) T cells in RA patients. CRACM1 might therefore represent a new molecular target for RA therapies.
Asunto(s)
Artritis Reumatoide/metabolismo , Linfocitos T CD4-Positivos/metabolismo , Calcio/metabolismo , Citocinas/metabolismo , Regulación hacia Arriba/fisiología , Adulto , Anciano , Canales de Calcio/metabolismo , Estudios de Casos y Controles , Estudios Transversales , Femenino , Humanos , Interferón gamma/metabolismo , Interleucina-17/metabolismo , Interleucina-4/metabolismo , Masculino , Persona de Mediana Edad , Proteína ORAI1 , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
We reported previously that an angiotensin II type 1 receptor blocker, telmisartan, improved cognitive decline with peroxisome proliferator-activated receptor-γ activation; however, the detailed mechanisms are unclear. Enhanced blood-brain barrier (BBB) permeability with alteration of tight junctions is suggested to be related to diabetes mellitus. Therefore, we examined the possibility that telmisartan could attenuate BBB impairment with peroxisome proliferator-activated receptor-γ activation to improve diabetes mellitus-induced cognitive decline. Type 2 diabetic mice KKA(y) exhibited impairment of cognitive function, and telmisartan treatment attenuated this. Cotreatment with GW9662, a peroxisome proliferator-activated receptor-γ antagonist, interfered with these protective effects of telmisartan against cognitive function. BBB permeability was increased in both the cortex and hippocampus in KKA(y) mice. Administration of telmisartan attenuated this increased BBB permeability. Coadministration of GW9662 reduced this effect of telmisartan. Significant decreases in expression of tight junction proteins and increases in matrix metalloproteinase expression, oxidative stress, and proinflammatory cytokine production were observed in the brain, and treatment with telmisartan restored these changes. Swollen astroglial end-feet in BBB were observed in KKA(y) mice, and this change in BBB ultrastructure was decreased in telmisartan. These effects of telmisartan were weakened by cotreatment with GW9662. In contrast, administration of another angiotensin II type 1 receptor blocker, losartan, was less effective compared with telmisartan in terms of preventing BBB permeability and astroglial end-foot swelling, and coadministration of GW9662 did not affect the effects of losartan. These findings are consistent with the possibility that, in type 2 diabetic mice, angiotensin II type 1 receptor blockade with peroxisome proliferator-activated receptor-γ activation by telmisartan may help with protection against cognitive decline by preserving the integrity of the BBB.
Asunto(s)
Bloqueadores del Receptor Tipo 1 de Angiotensina II/farmacología , Barrera Hematoencefálica/efectos de los fármacos , Trastornos del Conocimiento/prevención & control , Diabetes Mellitus Experimental/tratamiento farmacológico , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , PPAR gamma/biosíntesis , Análisis de Varianza , Anilidas/farmacología , Animales , Bencimidazoles/farmacología , Benzoatos/farmacología , Glucemia/análisis , Western Blotting , Trastornos del Conocimiento/etiología , Trastornos del Conocimiento/metabolismo , Diabetes Mellitus Experimental/complicaciones , Diabetes Mellitus Tipo 2/complicaciones , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Modelos Animales de Enfermedad , Losartán/farmacología , Masculino , Aprendizaje por Laberinto , Ratones , Ratones Endogámicos C57BL , PPAR gamma/antagonistas & inhibidores , Distribución Aleatoria , Sensibilidad y Especificidad , TelmisartánRESUMEN
Muscle-specific kinase (MuSK), a receptor tyrosine kinase, is required for the formation and maintenance of neuromuscular junctions (NMJs). Although autoantibodies against MuSK have been demonstrated to cause myasthenia gravis (MG), the underlying pathogenic mechanism remains unclear because a major subclass of these antibodies is functionally monovalent. We investigated the pathogenic role of MuSK antibodies in the onset of MG in vivo and in vitro. Ultrastructural visualization of NMJs in paretic rabbits with MuSK antibodies indicated that postsynaptic membranes were preserved, despite a significant loss of complexity in the convoluted synaptic folds. In addition, an in vitro assay indicated that both divalent and monovalent antibodies from paretic rabbits could interfere with agrin-induced acetylcholine receptor (AChR) clustering in cultured myotubes. Furthermore, in the absence of agrin, divalent antibodies induced MuSK phosphorylation and accelerated downregulation of Dok-7, an essential intracellular MuSK binding protein, while monovalent antibodies inhibited agrin-induced phosphorylation of MuSK, thus demonstrating distinct molecular mechanisms underlying the MuSK dysfunction induced by these two types of antibodies. Taken together, these findings suggest that complement activation is not necessary for the MG onset and that both divalent and monovalent antibodies may cause MG in vivo by inducing MuSK dysfunction.
Asunto(s)
Autoanticuerpos/inmunología , Músculo Esquelético/enzimología , Miastenia Gravis/inmunología , Proteínas Tirosina Quinasas Receptoras/inmunología , Agrina/farmacología , Animales , Modelos Animales de Enfermedad , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/ultraestructura , Unión Neuromuscular/efectos de los fármacos , Unión Neuromuscular/inmunología , Conejos , Receptores Colinérgicos/metabolismoRESUMEN
Mucosal mast cells (MMCs) have an important role in allergic inflammation, and effective antagonists are required for their regulation. To discover a possible mechanism of controlling the activation of MMCs, we investigated the expression and function of syntaxin4, one of the soluble membrane N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins, in RBL-2H3 cells, which is a rat mucosal mast cell line. Syntaxin4 silencing was induced by transfection of short interfering RNAs (siRNAs). Syntaxin4 was knocked down in mast cells at both the mRNA and protein levels. The release of granule contents that are involved in inflammation, such as histamine and hexosaminidase, was significantly suppressed by the gene silencing of syntaxin4. Silencing of this gene was also induced in the trachea and bronchi of rats by intratracheal application of the siRNAs using an atelocollagen delivery system. The activation of MMCs, which was monitored by the level of rat mast cell protease-II (RMCPII) in the bronchoalveolar lavage fluid (BALF), was inhibited, and asthmatic airway constriction was prevented by administration of the syntaxin/atelocollagen complex. These results indicate that siRNAs targeting syntaxin4 can stabilize mucosal mast cells and may have beneficial therapeutic effects on the asthmatic response.
Asunto(s)
Asma/inmunología , Degranulación de la Célula , Mastocitos/inmunología , Proteínas Qa-SNARE/metabolismo , ARN Interferente Pequeño/metabolismo , Mucosa Respiratoria/inmunología , Obstrucción de las Vías Aéreas/etiología , Obstrucción de las Vías Aéreas/prevención & control , Animales , Animales Modificados Genéticamente , Asma/complicaciones , Bronquios/patología , Degranulación de la Célula/genética , Línea Celular , Quimasas/genética , Quimasas/metabolismo , Hexosaminidasas/genética , Hexosaminidasas/metabolismo , Histamina/genética , Histamina/metabolismo , Mastocitos/patología , Proteínas Qa-SNARE/genética , Proteínas Qa-SNARE/inmunología , ARN Interferente Pequeño/genética , Ratas , Ratas Wistar , Tráquea/patologíaRESUMEN
Angiogenesis is required for physiological tissue repair processes, such as cutaneous wound healing. However, recent studies indicate that endogenous angiogenic factors may enhance photo-induced skin alterations in response to experimental ultraviolet (UV)-B exposure. Angiopoietin-related growth factor (AGF), also known as angiopoietin-like protein 6 (Angptl6), is known to promote new blood vessel formation and vascular hyperpermeability. Importantly, epidermal overexpression of Angptl6/AGF in mice promotes wound healing in the skin. However, it remains unclear whether overexpression of Angptl6/AGF facilitates tissue repair processes in response to UV-B irradiation. To test this hypothesis, we subjected Angptl6/AGF transgenic mice to acute or chronic UV-B exposure. Surprisingly, transgenic mice showed enhanced photosensitivity to subthreshold doses of UV-B that did not induce skin alterations in wild-type littermates. Marked enlargement of blood vessels was observed after a single exposure to UV-B in Angptl6/AGF transgenic mice, although no epidermal changes were observed. Chronic UV-B exposure over 14 weeks promoted cutaneous skin damage in Angptl6/AGF transgenic mice, whereas wild-type mice showed little or no macroscopic skin alteration. In addition to pronounced angiogenesis and epidermal hyperplasia, marked enlargement of dermal lymphatic vessels was observed in UV-B-exposed Angptl6/AGF transgenic mice. Electron microscopy analysis further revealed that the number and size of collagen bundles in the dermis was markedly reduced after chronic UV-B exposure in Angptl6/AGF transgenic mice. Taken together, these results indicate that ectopic expression of Angptl6/AGF in mice likely promotes UV-B-induced skin alterations, and that angiogenesis could be a therapeutic target in prevention of skin photo-aging.
Asunto(s)
Angiopoyetinas/genética , Angiopoyetinas/fisiología , Piel/irrigación sanguínea , Piel/efectos de la radiación , Proteína 6 similar a la Angiopoyetina , Proteínas Similares a la Angiopoyetina , Animales , Linfangiogénesis/genética , Linfangiogénesis/fisiología , Linfangiogénesis/efectos de la radiación , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Transgénicos , Neovascularización Fisiológica/genética , Neovascularización Fisiológica/fisiología , Neovascularización Fisiológica/efectos de la radiación , Trastornos por Fotosensibilidad/etiología , Trastornos por Fotosensibilidad/genética , Piel/anatomía & histología , Rayos Ultravioleta , Regulación hacia Arriba , Cicatrización de Heridas/genética , Cicatrización de Heridas/fisiología , Cicatrización de Heridas/efectos de la radiaciónRESUMEN
During an allergic inflammatory response in the airway, if a failure of the epithelial cell barrier occurs before the systemic immune response is triggered by allergens, more allergens can invade. Using a rat model of asthma, we previously found that mucosal mast cells, which localise to the epithelial layer of the airways, are activated to promote a pro-asthmatic immune response. In this study, we developed a neonatal rat model of allergic airway hypersensitivity that mimics some features of childhood asthma. Airway hypersensitivity was measured using unrestrained whole-body plethysmography after analysis of the serum IgE titre. Inflammatory cells and inflammatory mediators in bronchoalveolar lavage fluid samples were examined. Two mast cell-specific proteases were detected using PCR. In addition, we analysed the phenotype and the number of mast cells in the airways by immunohistochemistry, and we found that the number of mucosal mast cells and the expression level of the proteases increased 2 weeks after sensitisation. Changes in the IgE titre, airway hypersensitivity and the activation of other inflammatory cells were delayed, appearing during the 4 weeks after sensitisation. Our results indicate that the activation of mucosal mast cells contributes to the pro-asthmatic immune response. This activation may be a biomarker allowing early intervention that could help prevent allergic airway inflammation.
Asunto(s)
Asma/inmunología , Inmunidad/inmunología , Mastocitos/inmunología , Mucosa Respiratoria/inmunología , Mucosa Respiratoria/patología , Animales , Animales Recién Nacidos , Asma/sangre , Asma/complicaciones , Asma/patología , Bronquios/enzimología , Bronquios/patología , Líquido del Lavado Bronquioalveolar/inmunología , Recuento de Células , Niño , Modelos Animales de Enfermedad , Humanos , Inmunoglobulina E/sangre , Inmunohistoquímica , Mediadores de Inflamación/metabolismo , Mastocitos/enzimología , Péptido Hidrolasas/metabolismo , Neumonía/sangre , Neumonía/complicaciones , Neumonía/inmunología , Neumonía/patología , Ratas , Ratas Wistar , Triptasas/metabolismoRESUMEN
Amphiregulin (AR), a member of the EGF family, is synthesized as a type I transmembrane protein precursor (proAR) and expressed on the cell surface. Shedding of proAR yields a transmembrane-cytoplasmic fragment (AR-CTF), as well as a soluble AR. Here we demonstrate that the proAR-shedding stimuli trigger endocytosis of both AR-CTF and un-shed proAR. ProAR translocates from the plasma membrane to the inner nuclear membrane, whereas AR-CTF is translocated to the lysosome via retrograde membrane trafficking. Nuclear envelope localization of proAR involves truncation of the C-terminus, which subsequently activates the ER-retrieval signal. The truncated form of proAR interacts with A-type lamin and is retained at the inner nuclear membrane. Heterochromatin formation is then induced and global transcription is transiently suppressed. This study gives new insight into epigenetic chromatin organization in mammalian cells: a plasma-membrane-anchored growth factor is targeted to the inner nuclear membrane where it participates in dynamic chromatin organization and control of transcription.
Asunto(s)
Membrana Celular/metabolismo , Glicoproteínas/fisiología , Péptidos y Proteínas de Señalización Intercelular/fisiología , Lamina Tipo A/fisiología , Transcripción Genética , Anfirregulina , Transporte Biológico , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Familia de Proteínas EGF , Endocitosis , Glicoproteínas/metabolismo , Células HeLa , Heterocromatina/química , Humanos , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Lamina Tipo A/química , Lisosomas/metabolismo , Modelos Biológicos , Transporte de Proteínas , ARN Polimerasa II/metabolismoRESUMEN
Different types of nonionic vesicles were prepared from commercial Span 80 (also called sorbitan monooleate), as an inexpensive, biocompatible alternative to conventional phospholipid-based vesicles (liposomes). The vesicles were characterized by different techniques and comparison was made with vesicles formed from POPC (1-palmitoyl-2-oleoyl- sn-glycero-3-phosphocholine) or DOPC (1,2-dioleoyl- sn-glycero-3-phosphocholine). Dynamic light scattering measurements, electron microscopy analyses, and two types of fusion assays indicate that Span 80 vesicles are stable for at least 7 days at 4 or 25 degrees C, while storage at 42 degrees C causes irreversible vesicle fusion. This indicates that Span 80 vesicles are thermoresponsive with vesicle fusion occurring at elevated temperature. This property may be related to headgroup dehydration and is certainly not directly linked to the phase transition temperature (Tm) of the vesicles, since the Tm is below -30 degrees C, as determined by differential scanning calorimetry (DSC). The measured Tm value for Span 80 vesicles is lower than in the case of DOPC or POPC, correlating with a higher fluidity of Span 80 vesicles as compared to POPC or DOPC vesicles, as determined with DPH (1,6-diphenyl-1,3,5-hexatriene) as fluorescent membrane probe. High fluidity correlates with increased leakage of entrapped water-soluble dye molecules. Addition of cholesterol and soybean phosphatidylcholine lowers the extent of leakage, allowing a tuning of the bilayer permeability.
Asunto(s)
Hexosas/química , Temperatura , Rastreo Diferencial de Calorimetría , Cromatografía Líquida de Alta Presión , Microscopía por Crioelectrón , Iones/química , Microscopía Electrónica de Transmisión , Fosfatidilcolinas/química , Sensibilidad y EspecificidadRESUMEN
Heparin-binding EGF-like growth factor (HB-EGF) is synthesized as a type I transmembrane protein (proHB-EGF) and expressed on the cell surface. The ectodomain shedding of proHB-EGF at the extracellular region on the plasma membrane yields a soluble EGF receptor ligand and a transmembrane-cytoplasmic fragment (HB-EGF-CTF). The cytoplasmic domain of proHB-EGF (HB-EGF-cyto) interacts with transcriptional repressors to reverse their repressive activities. However, how HB-EGF-cyto accesses transcriptional repressors is yet unknown. The present study demonstrates that, after exposure to shedding stimuli, both HB-EGF-CTF and unshed proHB-EGF translocate to the nuclear envelope. Immunoelectron microscopy and digitonin-permeabilized cells showed that HB-EGF-cyto signals are at the inner nuclear membrane. A short sequence element within the HB-EGF-cyto allows a transmembrane protein to localize to the nuclear envelope. The dominant-active form of Rab5 and Rab11 suppressed nuclear envelope targeting. Collectively, these data demonstrate that membrane-anchored HB-EGF is targeted to the inner nuclear membrane via a retrograde membrane trafficking pathway.