RESUMEN
The molecular mechanisms by which lymphatic vessels induce cell contact inhibition are not understood. Here, we identify the cGMP-dependent phosphodiesterase 2A (PDE2A) as a selective regulator of lymphatic but not of blood endothelial contact inhibition. Conditional deletion of Pde2a in mouse embryos reveals severe lymphatic dysplasia, whereas blood vessel architecture remains unaltered. In the absence of PDE2A, human lymphatic endothelial cells fail to induce mature junctions and cell cycle arrest, whereas cGMP levels, but not cAMP levels, are increased. Loss of PDE2A-mediated cGMP hydrolysis leads to the activation of p38 signaling and downregulation of NOTCH signaling. However, DLL4-induced NOTCH activation restores junctional maturation and contact inhibition in PDE2A-deficient human lymphatic endothelial cells. In postnatal mouse mesenteries, PDE2A is specifically enriched in collecting lymphatic valves, and loss of Pde2a results in the formation of abnormal valves. Our data demonstrate that PDE2A selectively finetunes a crosstalk of cGMP, p38, and NOTCH signaling during lymphatic vessel maturation.
Asunto(s)
Fosfodiesterasas de Nucleótidos Cíclicos Tipo 2 , Vasos Linfáticos , Animales , Humanos , Ratones , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 2/genética , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 2/metabolismo , Regulación hacia Abajo , Células Endoteliales/metabolismo , Vasos Linfáticos/metabolismo , Transducción de SeñalRESUMEN
BACKGROUND: During eye development the lens placode invaginates to form the lens pit. Further bending of lens epithelium and separation from ectoderm leads eventually to a spherical lens vesicle with enclosed extracellular fluid. Changes in epithelial morphology involve the actin cytoskeleton and its regulators. The myosin Myo9b is simultaneously an actin-based motor and Rho GTPase-activating protein that regulates actin cytoskeleton organization. Myo9b-deficient adult mice and embryos were analyzed for eye malformations and alterations in lens development. RESULTS: Myo9b-deficient mice showed a high incidence of microphthalmia and cataracts with occasional blepharitis. Formation of the lens vesicle during embryonic lens development was disordered in virtually all embryos. Lens placode invagination was less deep and gave rise to a conical structure instead of a spherical pit. At later stages either no lens vesicle was formed or a significantly smaller one that was not enclosed by the optic cup. Expression of the cell fate marker Pax6 was not altered. Staining of adherens junctions and F-actin was most intense at the tip of conical invaginations, suggesting that mechanical forces are not properly coordinated between epithelial cells that form the pit. CONCLUSIONS: Myo9b is a critical regulator of ocular lens vesicle morphogenesis during eye development.
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Cristalino , Morfogénesis , Miosinas , Animales , Ratones , Actinas/fisiología , Ojo , Cristalino/embriología , Miosinas/fisiologíaRESUMEN
Inflammation and fibrosis are important components of diseases that contribute to the malfunction of epithelia and endothelia. The Rho guanine nucleotide exchange factor (GEF) GEF-H1/ARHGEF-2 is induced in disease and stimulates inflammatory and fibrotic processes, cell migration, and metastasis. Here, we have generated peptide inhibitors to block the function of GEF-H1. Inhibitors were designed using a structural in silico approach or by isolating an inhibitory sequence from the autoregulatory C-terminal domain. Candidate inhibitors were tested for their ability to block RhoA/GEF-H1 binding in vitro, and their potency and specificity in cell-based assays. Successful inhibitors were then evaluated in models of TGFß-induced fibrosis, LPS-stimulated endothelial cell-cell junction disruption, and cell migration. Finally, the most potent inhibitor was successfully tested in an experimental retinal disease mouse model, in which it inhibited blood vessel leakage and ameliorated retinal inflammation when treatment was initiated after disease diagnosis. Thus, an antagonist that blocks GEF-H1 signaling effectively inhibits disease features in in vitro and in vivo disease models, demonstrating that GEF-H1 is an effective therapeutic target and establishing a new therapeutic approach.
Asunto(s)
Enfermedades de la Retina , Transducción de Señal , Animales , Fibrosis , Inflamación , Ratones , Factores de Intercambio de Guanina Nucleótido Rho/genética , Factores de Intercambio de Guanina Nucleótido Rho/metabolismoRESUMEN
BACKGROUND: TP (thromboxane A2 receptor) plays an eminent role in the pathophysiology of endothelial dysfunction and cardiovascular disease. Moreover, its expression is reported to increase in the intimal layer of blood vessels of cardiovascular high-risk individuals. Yet it is unknown, whether TP upregulation per se has the potential to affect the homeostasis of the vascular endothelium. METHODS: We combined global transcriptome analysis, lipid mediator profiling, functional cell analyses, and in vivo angiogenesis assays to study the effects of endothelial TP overexpression or knockdown/knockout on the angiogenic capacity of endothelial cells in vitro and in vivo. RESULTS: Here we report that endothelial TP expression induces COX-2 (cyclooxygenase-2) in a Gi/o- and Gq/11-dependent manner, thereby promoting its own activation via the auto/paracrine release of TP agonists, such as PGH2 (prostaglandin H2) or prostaglandin F2 but not TxA2 (thromboxane A2). TP overexpression induces endothelial cell tension and aberrant cell morphology, affects focal adhesion dynamics, and inhibits the angiogenic capacity of human endothelial cells in vitro and in vivo, whereas TP knockdown or endothelial-specific TP knockout exerts opposing effects. Consequently, this TP-dependent feedback loop is disrupted by pharmacological TP or COX-2 inhibition and by genetic reconstitution of PGH2-metabolizing prostacyclin synthase even in the absence of functional prostacyclin receptor expression. CONCLUSIONS: Our work uncovers a TP-driven COX-2-dependent feedback loop and important effector mechanisms that directly link TP upregulation to angiostatic TP signaling in endothelial cells. By these previously unrecognized mechanisms, pathological endothelial upregulation of the TP could directly foster endothelial dysfunction, microvascular rarefaction, and systemic hypertension even in the absence of exogenous sources of TP agonists.
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Células Endoteliales , Receptores de Tromboxanos , Ciclooxigenasa 2/genética , Ciclooxigenasa 2/metabolismo , Ciclooxigenasa 2/farmacología , Células Endoteliales/metabolismo , Retroalimentación , Homeostasis , Humanos , Receptores de Tromboxanos/metabolismo , Receptores de Tromboxano A2 y Prostaglandina H2/genética , Tromboxano A2/metabolismo , Tromboxanos/metabolismo , Tromboxanos/farmacologíaRESUMEN
Myosin XIX (Myo19) is an actin-based motor that competes with adaptors of microtubule-based motors for binding to the outer mitochondrial transmembrane proteins Miro1 and Miro2 (collectively Miro, also known as RhoT1 and RhoT2, respectively). Here, we investigate which mitochondrial and cellular processes depend on the coordination of Myo19 and microtubule-based motor activities. To this end, we created Myo19-deficient HEK293T cells. Mitochondria in these cells were not properly fragmented at mitosis and were partitioned asymmetrically to daughter cells. Respiratory functions of mitochondria were impaired and ROS generation was enhanced. On a cellular level, cell proliferation, cytokinesis and cell-matrix adhesion were negatively affected. On a molecular level, Myo19 regulates focal adhesions in interphase, and mitochondrial fusion and mitochondrially associated levels of fission protein Drp1 and adaptor proteins dynactin and TRAK1 at prometaphase. These alterations were due to a disturbed coordination of Myo19 and microtubule-based motor activities by Miro.
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Actinas , Miosinas , Actinas/genética , Actinas/metabolismo , Células HEK293 , Humanos , Mitocondrias/metabolismo , Dinámicas Mitocondriales , Membranas Mitocondriales/metabolismo , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Miosinas/metabolismo , Proteínas de Unión al GTP rho/metabolismoRESUMEN
To migrate, cells assume a polarized morphology, extending forward with a leading edge with their trailing edge retracting back toward the cell body. Both cell extension and retraction critically depend on the organization and dynamics of the actin cytoskeleton, and the small, monomeric GTPases Rac and Rho are important regulators of actin. Activation of Rac induces actin polymerization and cell extension, whereas activation of Rho enhances acto-myosin II contractility and cell retraction. To coordinate migration, these processes must be carefully regulated. The myosin Myo9b, a Rho GTPase-activating protein (GAP), negatively regulates Rho activity and deletion of Myo9b in leukocytes impairs cell migration through increased Rho activity. However, it is not known whether cell motility is regulated by global or local inhibition of Rho activity by Myo9b. Here, we addressed this question by using Myo9b-deficient macrophage-like cells that expressed different recombinant Myo9b constructs. We found that Myo9b accumulates in lamellipodial extensions generated by Rac-induced actin polymerization as a function of its motor activity. Deletion of Myo9b in HL-60-derived macrophages altered cell morphology and impaired cell migration. Reintroduction of Myo9b or Myo9b motor and GAP mutants revealed that local GAP activity rescues cell morphology and migration. In summary, Rac activation leads to actin polymerization and recruitment of Myo9b, which locally inhibits Rho activity to enhance directional cell migration.
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Citoesqueleto de Actina/metabolismo , Proteínas Activadoras de GTPasa/metabolismo , Macrófagos/citología , Macrófagos/metabolismo , Miosinas/metabolismo , Seudópodos/metabolismo , Movimiento Celular/fisiología , Células Cultivadas , Proteínas Activadoras de GTPasa/genética , Humanos , Miosinas/genéticaRESUMEN
Macrophage filopodia, finger-like membrane protrusions, were first implicated in phagocytosis more than 100 years ago, but little is still known about the involvement of these actin-dependent structures in particle clearance. Using spinning disk confocal microscopy to image filopodial dynamics in mouse resident Lifeact-EGFP macrophages, we show that filopodia, or filopodia-like structures, support pathogen clearance by multiple means. Filopodia supported the phagocytic uptake of bacterial (Escherichia coli) particles by (i) capturing along the filopodial shaft and surfing toward the cell body, the most common mode of capture; (ii) capturing via the tip followed by retraction; (iii) combinations of surfing and retraction; or (iv) sweeping actions. In addition, filopodia supported the uptake of zymosan (Saccharomyces cerevisiae) particles by (i) providing fixation, (ii) capturing at the tip and filopodia-guided actin anterograde flow with phagocytic cup formation, and (iii) the rapid growth of new protrusions. To explore the role of filopodia-inducing Cdc42, we generated myeloid-restricted Cdc42 knock-out mice. Cdc42-deficient macrophages exhibited rapid phagocytic cup kinetics, but reduced particle clearance, which could be explained by the marked rounded-up morphology of these cells. Macrophages lacking Myo10, thought to act downstream of Cdc42, had normal morphology, motility, and phagocytic cup formation, but displayed markedly reduced filopodia formation. In conclusion, live-cell imaging revealed multiple mechanisms involving macrophage filopodia in particle capture and engulfment. Cdc42 is not critical for filopodia or phagocytic cup formation, but plays a key role in driving macrophage lamellipodial spreading.
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Proteína Quinasa CDC2/fisiología , Miosinas/fisiología , Fagocitosis , Seudópodos/metabolismo , Animales , Proteína Quinasa CDC2/genética , Quimiotaxis , Eliminación de Gen , Genotipo , Proteínas Fluorescentes Verdes/metabolismo , Concentración de Iones de Hidrógeno , Macrófagos Peritoneales/metabolismo , Ratones , Ratones Noqueados , Microscopía Confocal , Mutación , Miosinas/genética , Miosinas/metabolismo , Fenotipo , Saccharomyces cerevisiae/metabolismo , Receptor Toll-Like 4/metabolismo , Proteína de Unión al GTP cdc42/metabolismoRESUMEN
Directed migration of stimulated dendritic cells (DCs) to secondary lymphoid organs and their interaction with Ag-specific T cells is a prerequisite for the induction of primary immune responses. In this article, we show that murine DCs that lack myosin IXB (Myo9b), a motorized negative regulator of RhoA signaling, exhibit increased Rho signaling activity and downstream acto-myosin contractility, and inactivation of the Rho target protein cofilin, an actin-depolymerizing factor. On a functional level, Myo9b(-/-) DCs showed impaired directed migratory activity both in vitro and in vivo. Moreover, despite unaltered Ag presentation and costimulatory capabilities, Myo9b(-/-) DCs were poor T cell stimulators in vitro in a three-dimensional collagen matrix and in vivo, associated with altered DC-T cell contact dynamics and T cell polarization. Accordingly, Myo9b(-/-) mice showed an attenuated ear-swelling response in a model of contact hypersensitivity. The impaired migratory and T cell stimulatory capacity of Myo9b(-/-) DCs was restored in large part by pharmacological activation of cofilin. Taken together, these results identify Myo9b as a negative key regulator of the Rho/RhoA effector Rho-kinase [Rho-associated coiled-coil-forming kinase (ROCK)]/LIM domain kinase signaling pathway in DCs, which controls cofilin inactivation and myosin II activation and, therefore may control, in part, the induction of adaptive immune responses.
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Factores Despolimerizantes de la Actina/metabolismo , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Miosinas/metabolismo , Transducción de Señal , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Quinasas Asociadas a rho/metabolismo , Animales , Células de la Médula Ósea/inmunología , Células de la Médula Ósea/metabolismo , Comunicación Celular/inmunología , Diferenciación Celular , Movimiento Celular/inmunología , Células Dendríticas/citología , Dermatitis por Contacto/genética , Dermatitis por Contacto/inmunología , Dermatitis por Contacto/metabolismo , Proteínas Activadoras de GTPasa/metabolismo , Activación de Linfocitos/inmunología , Ratones , Ratones Noqueados , Miosinas/genéticaRESUMEN
MarvelD3 is a transmembrane component of tight junctions, but there is little evidence for a direct involvement in the junctional permeability barrier. Tight junctions also regulate signaling mechanisms that guide cell proliferation; however, the transmembrane components that link the junction to such signaling pathways are not well understood. In this paper, we show that MarvelD3 is a dynamic junctional regulator of the MEKK1-c-Jun NH2-terminal kinase (JNK) pathway. Loss of MarvelD3 expression in differentiating Caco-2 cells resulted in increased cell migration and proliferation, whereas reexpression in a metastatic tumor cell line inhibited migration, proliferation, and in vivo tumor formation. Expression levels of MarvelD3 inversely correlated with JNK activity, as MarvelD3 recruited MEKK1 to junctions, leading to down-regulation of JNK phosphorylation and inhibition of JNK-regulated transcriptional mechanisms. Interplay between MarvelD3 internalization and JNK activation tuned activation of MEKK1 during osmotic stress, leading to junction dissociation and cell death in MarvelD3-depleted cells. MarvelD3 thus couples tight junctions to the MEKK1-JNK pathway to regulate cell behavior and survival.
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Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Quinasa 1 de Quinasa de Quinasa MAP/metabolismo , Sistema de Señalización de MAP Quinasas , Proteínas de la Membrana/fisiología , Uniones Estrechas/metabolismo , Células CACO-2 , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Supervivencia Celular , Humanos , Proteínas de la Membrana/metabolismo , Presión OsmóticaRESUMEN
STUDY QUESTION: Is post-implantation embryonic development after blastocyst transfer affected by exposure to different assisted reproduction technology (ART) culture media? SUMMARY ANSWER: Fetal development and placental histology of ART embryos cultured in vitro in different ART media was not impaired compared with embryos grown in vivo. WHAT IS KNOWN ALREADY: The application of different in vitro culture (IVC) media for human ART has an effect on birthweight of newborns. In the mouse model, differences in blastocyst formation were reported after culture in different ART media. Moreover, abnormalities in the liver and heart have been detected as a result of suboptimal IVC conditions. STUDY DESIGN, SIZE, DURATION: Fertilized oocytes from inbred and outbred breeding schemes were retrieved and either immediately transferred to foster mothers or incubated in control or human ART culture media up to the blastocyst stage prior to transfer. Placental and fetal anatomy and particularly bone development were evaluated. PARTICIPANTS/MATERIALS, SETTING, METHODS: B6C3F1 female mice were used as oocyte donors after ovulation induction. C57Bl/6 and CD1 males were used for mating and CD1 females as foster mothers for embryo transfer. Fertilized oocytes were recovered from mated females and incubated in sequential human ART media (ISM1/ISM2 and HTF/Multiblast), in control media [KSOM(aa) and Whitten's medium] or grown in utero without IVC (zygote control). As in vivo, control B6C3F1 females were superovulated and left untreated. Fetuses and placentae were isolated by Caesarean section and analysed at 18.5 days post-coitum (dpc) for placenta composition and at 15.5 dpc for body weight, crown-rump length (CRL), fetal organ development, morphological development, total bone length and extent of bone ossification. MAIN RESULTS AND THE ROLE OF CHANCE: No major differences in the number of implantation sites or in histological appearance of the placentae were detected. CRL of KSOM(aa) fetuses was higher compared with zygote control and Whitten's medium. Histological analysis of tissue sections revealed no gross morphological differences compared with the in vitro groups or in vivo controls. Furthermore, no changes in skeletal development and degree of ossification were observed. However, fibula and tibia of ISM1/ISM2 fetuses were longer than the respective ones from in vivo fetuses. LIMITATIONS, REASONS FOR CAUTION: Findings in the mouse embryo and fetus may not be fully transferable to humans. In addition to skeletal development and placentation, there may be other parameters, e.g. on the molecular level which respond to IVC in ART media. Some comparisons have limited statistical power. WIDER IMPLICATIONS OF THE FINDINGS: Our data suggest that once implantation is achieved, subsequent post-implantation development unfolds normally, resulting in healthy fetuses. With mouse models, we gather information for the safety of human ART culture media. Our mouse study is reassuring for the safety of ART conditions on human embryonic development, given the lack of bold detrimental effects observed in the mouse model. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the Deutsche Forschungsgemeinschaft (BO 2540/4-1 and SCHL 394/9-1) and by the Nederlandse Organisatie voor Wetenschappelijk Onderzoek (S.L.G.); Bilateral grant NWO-DFG 63-258. None of the authors has any conflict of interest to declare. TRIAL REGISTRATION NUMBER: Not applicable.