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1.
J Neuroinflammation ; 20(1): 25, 2023 Feb 04.
Artículo en Inglés | MEDLINE | ID: mdl-36739425

RESUMEN

BACKGROUND: Clustering of microglia around the vasculature has been reported in the retina and the brain after systemic administration of lipopolysaccharides (LPS) in mice. LPS acts via activation of Toll-like receptor 4 (TRL4), which is expressed in several cell types including microglia, monocytes and vascular endothelial cells. The purpose of this study was to investigate the effect of systemic LPS in the pigmented mouse retina and the involvement of endothelial TLR4 in LPS-induced retinal microglia activation. METHODS: C57BL/6J, conditional knockout mice that lack Tlr4 expression selectively on endothelial cells (TekCre-posTlr4loxP/loxP) and TekCre-negTlr4loxP/loxP mice were used. The mice were injected with 1 mg/kg LPS via the tail vein once per day for a total of 4 days. Prior to initiation of LPS injections and approximately 5 h after the last injection, in vivo imaging using fluorescein angiography and spectral-domain optical coherence tomography was performed. Immunohistochemistry, flow cytometry, electroretinography and transmission electron microscopy were utilized to investigate the role of endothelial TLR4 in LPS-induced microglia activation and retinal function. RESULTS: Activation of microglia, infiltration of monocyte-derived macrophages, impaired ribbon synapse organization and retinal dysfunction were observed after the LPS exposure in C57BL/6J and TekCre-negTlr4loxP/loxP mice. None of these effects were observed in the retinas of conditional Tlr4 knockout mice after the LPS challenge. CONCLUSIONS: The findings of the present study suggest that systemic LPS exposure can have detrimental effects in the healthy retina and that TLR4 expressed on endothelial cells is essential for retinal microglia activation and retinal dysfunction upon systemic LPS challenge. This important finding provides new insights into the role of microglia-endothelial cell interaction in inflammatory retinal disease.


Asunto(s)
Lipopolisacáridos , Microglía , Animales , Ratones , Células Endoteliales/metabolismo , Lipopolisacáridos/toxicidad , Ratones Endogámicos C57BL , Ratones Noqueados , Microglía/metabolismo , Retina/metabolismo , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/metabolismo
2.
EMBO Rep ; 22(2): e48961, 2021 02 03.
Artículo en Inglés | MEDLINE | ID: mdl-33512764

RESUMEN

Endothelial tip cells are essential for VEGF-induced angiogenesis, but underlying mechanisms are elusive. The Ena/VASP protein family, consisting of EVL, VASP, and Mena, plays a pivotal role in axon guidance. Given that axonal growth cones and endothelial tip cells share many common features, from the morphological to the molecular level, we investigated the role of Ena/VASP proteins in angiogenesis. EVL and VASP, but not Mena, are expressed in endothelial cells of the postnatal mouse retina. Global deletion of EVL (but not VASP) compromises the radial sprouting of the vascular plexus in mice. Similarly, endothelial-specific EVL deletion compromises the radial sprouting of the vascular plexus and reduces the endothelial tip cell density and filopodia formation. Gene sets involved in blood vessel development and angiogenesis are down-regulated in EVL-deficient P5-retinal endothelial cells. Consistently, EVL deletion impairs VEGF-induced endothelial cell proliferation and sprouting, and reduces the internalization and phosphorylation of VEGF receptor 2 and its downstream signaling via the MAPK/ERK pathway. Together, we show that endothelial EVL regulates sprouting angiogenesis via VEGF receptor-2 internalization and signaling.


Asunto(s)
Moléculas de Adhesión Celular/fisiología , Células Endoteliales , Neovascularización Fisiológica , Receptor 2 de Factores de Crecimiento Endotelial Vascular , Animales , Células Endoteliales/metabolismo , Ratones , Morfogénesis , Transducción de Señal , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genética , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo
3.
J Mol Cell Cardiol ; 129: 13-26, 2019 04.
Artículo en Inglés | MEDLINE | ID: mdl-30771306

RESUMEN

Cardiac functionality is dependent on a balanced protein turnover. Accordingly, regulated protein decay is critical to maintain cardiac function. Here we demonstrate that deficiency of SPRED2, an intracellular repressor of ERK-MAPK signaling markedly expressed in human heart, resulted in impaired autophagy, heart failure, and shortened lifespan. SPRED2-/- mice showed cardiomyocyte hypertrophy, cardiac fibrosis, impaired electrical excitability, and severe arrhythmias. Mechanistically, cardiomyocyte dysfunction resulted from ERK hyperactivation and dysregulated autophagy, observed as accumulation of vesicles, vacuolar structures, and degenerated mitochondria. The diminished autophagic flux in SPRED2-/- hearts was reflected by a reduced LC3-II/LC3-I ratio and by decreased Atg7, Atg4B and Atg16L expression. Furthermore, the autophagosomal adaptors p62/SQSTM1 and NBR1 and lysosomal Cathepsin D accumulated in SPRED2-/- hearts. In wild-type hearts, SPRED2 interacted physically with p62/SQSTM1, NBR1, and Cathepsin D, indicating that SPRED2 is required for autophagolysosome formation in regular autophagy. Restored inhibition of MAPK signaling by selumetinib led to an increase in autophagic flux in vivo. Therefore, our study identifies SPRED2 as a novel, indispensable regulator of cardiac autophagy. Vice versa, SPRED2 deficiency impairs autophagy, leading to cardiac dysfunction and life-threatening arrhythmias.


Asunto(s)
Arritmias Cardíacas/metabolismo , Autofagia , Mortalidad Prematura , Proteínas Represoras/deficiencia , Adulto , Aldosterona/farmacología , Animales , Arritmias Cardíacas/complicaciones , Arritmias Cardíacas/patología , Arritmias Cardíacas/fisiopatología , Autofagosomas/metabolismo , Autofagosomas/ultraestructura , Biomarcadores/metabolismo , Presión Sanguínea , Cardiomegalia/complicaciones , Cardiomegalia/metabolismo , Cardiomegalia/patología , Cardiomegalia/fisiopatología , Catepsina D/metabolismo , Colágeno/metabolismo , Fenómenos Electrofisiológicos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Sistema de Conducción Cardíaco/fisiopatología , Hemodinámica , Humanos , Lisosomas/metabolismo , Lisosomas/ultraestructura , Ratones Endogámicos C57BL , Ratones Noqueados , Miocardio/metabolismo , Miocardio/patología , Miocardio/ultraestructura , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/ultraestructura , Fosforilación/efectos de los fármacos , Fosfotreonina/metabolismo , Proteínas Represoras/metabolismo , Vacuolas/metabolismo , Vacuolas/ultraestructura
4.
Basic Res Cardiol ; 113(2): 11, 2018 01 17.
Artículo en Inglés | MEDLINE | ID: mdl-29344719

RESUMEN

Ischemic preconditioning (IP) is a well-known strategy to protect organs against cell death following ischemia. The previous work has shown that vasodilator-stimulated phosphoprotein (VASP) is involved in cytoskeletal reorganization and that it holds significant importance for the extent of myocardial ischemia reperfusion injury. Yet, the role of VASP during myocardial IP is, to date, not known. We report here that VASP phosphorylation at serine157 and serine239 is induced during hypoxia in vitro and during IP in vivo. The preconditioning-induced VASP phosphorylation inactivates the GP IIb/IIIa integrin receptor on platelets, which results in the reduced formation of organ compromising platelet neutrophil complexes. Experiments in chimeric mice confirmed the importance of VASP phosphorylation during myocardial IP. When studying this in VASP-/- animals and in an isolated heart model, we were able to confirm the important role of VASP on myocardial IP. In conclusion, we were able to show that IP-induced VASP phosphorylation in platelets is a protective mechanism against the deleterious effects of ischemia.


Asunto(s)
Plaquetas/metabolismo , Moléculas de Adhesión Celular/sangre , Precondicionamiento Isquémico Miocárdico/métodos , Proteínas de Microfilamentos/sangre , Infarto del Miocardio/prevención & control , Miocardio/metabolismo , Neutrófilos/metabolismo , Fosfoproteínas/sangre , Adhesividad Plaquetaria , Animales , Moléculas de Adhesión Celular/deficiencia , Moléculas de Adhesión Celular/genética , Hipoxia de la Célula , Modelos Animales de Enfermedad , Preparación de Corazón Aislado , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas de Microfilamentos/deficiencia , Proteínas de Microfilamentos/genética , Infarto del Miocardio/sangre , Infarto del Miocardio/genética , Infarto del Miocardio/patología , Miocardio/patología , Fosfoproteínas/deficiencia , Fosfoproteínas/genética , Fosforilación , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Transducción de Señal
5.
J Cell Sci ; 127(Pt 16): 3425-33, 2014 Aug 15.
Artículo en Inglés | MEDLINE | ID: mdl-24963131

RESUMEN

At the early stages of carcinogenesis, transformation occurs in single cells within tissues. In an epithelial monolayer, such mutated cells are recognized by their normal neighbors and are often apically extruded. The apical extrusion requires cytoskeletal reorganization and changes in cell shape, but the molecular switches involved in the regulation of these processes are poorly understood. Here, using stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative mass spectrometry, we have identified proteins that are modulated in transformed cells upon their interaction with normal cells. Phosphorylation of VASP at serine 239 is specifically upregulated in Ras(V12)-transformed cells when they are surrounded by normal cells. VASP phosphorylation is required for the cell shape changes and apical extrusion of Ras-transformed cells. Furthermore, PKA is activated in Ras-transformed cells that are surrounded by normal cells, leading to VASP phosphorylation. These results indicate that the PKA-VASP pathway is a crucial regulator of tumor cell extrusion from the epithelium, and they shed light on the events occurring at the early stage of carcinogenesis.


Asunto(s)
Moléculas de Adhesión Celular/metabolismo , Transformación Celular Neoplásica , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Epitelio/metabolismo , Proteínas de Microfilamentos/metabolismo , Fosfoproteínas/metabolismo , Moléculas de Adhesión Celular/genética , Línea Celular Transformada , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Células Epiteliales/enzimología , Células Epiteliales/metabolismo , Epitelio/enzimología , Humanos , Proteínas de Microfilamentos/genética , Fosfoproteínas/genética , Fosforilación , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo
6.
Cell Commun Signal ; 14(1): 22, 2016 09 17.
Artículo en Inglés | MEDLINE | ID: mdl-27639852

RESUMEN

Under physiological conditions, endothelial cells and the endothelial nitric oxide (NO) synthase (eNOS) are the main source of NO in the cardiovascular system. However, several other cell types have also been implicated in the NO-dependent regulation of cell function, including erythrocytes. NO derived from red blood cells has been proposed to regulate erythrocyte membrane fluidity, inhibit platelet activation and induce vasodilation in hypoxic areas, but these proposals are highly controversial. In the current issue of Cell Communication and Signaling, an elegant study by Gambaryan et al., assayed NO production by erythrocytes by monitoring the activation of the platelet intracellular NO receptor, soluble guanylyl cyclase, and its downstream kinase protein kinase G. After systematically testing different combinations of erythrocyte/platelet suspensions, the authors found no evidence for platelet soluble guanylyl cyclase/protein kinase G activation by erythrocytes and conclude that erythrocytes do not release biologically active NO to inhibit platelet activation.


Asunto(s)
Proteínas Quinasas Dependientes de GMP Cíclico , Óxido Nítrico , GMP Cíclico , Eritrocitos , Guanilato Ciclasa , Transducción de Señal , Vasodilatación
7.
Cell Commun Signal ; 14(1): 21, 2016 09 13.
Artículo en Inglés | MEDLINE | ID: mdl-27620165

RESUMEN

BACKGROUND: Vasodilator-Stimulated Phosphoprotein (VASP) is involved in the inhibition of agonist-induced platelet aggregation by cyclic nucleotides and the adhesion of platelets to the vascular wall. αIIbß3 is the main integrin responsible for platelet activation and Rap1b plays a key role in integrin signalling. We investigated whether VASP is involved in the regulation of Rap1b in platelets since VASP-null platelets exhibit augmented adhesion to endothelial cells in vivo. METHODS: Washed platelets from wild type and VASP-deficient mice were stimulated with thrombin, the purinergic receptors agonist ADP, or the thromboxane A2 receptor agonist U46619 and Rap1b activation was measured using the GST-RalGDS-RBD binding assay. Interaction of VASP and Crkl was investigated by co-immunoprecipitation, confocal microscopy, and pull-down assays using Crkl domains expressed as GST-fusion proteins. RESULTS: Surprisingly, we found that activation of Rap1b in response to thrombin, ADP, or U46619 was significantly reduced in platelets from VASP-null mice compared to platelets from wild type mice. However, inhibition of thrombin-induced activation of Rap1b by nitric oxide (NO) was similar in platelets from wild type and VASP-null mice indicating that the NO/cGMP/PKG pathway controls inhibition of Rap1b independently from VASP. To understand how VASP regulated Rap1b, we investigated association between VASP and the Crk-like protein (Crkl), an adapter protein which activates the Rap1b guanine nucleotide exchange factor C3G. We demonstrated the formation of a Crkl/VASP complex by showing that: 1) Crkl co-immunoprecipitated VASP from platelet lysates; 2) Crkl and VASP dynamically co-localized at actin-rich protrusions reminiscent of focal adhesions, filopodia, and lamellipodia upon platelet spreading on fibronectin; 3) recombinant VASP bound directly to the N-terminal SH3 domain of Crkl; 4) Protein Kinase A (PKA) -mediated VASP phosphorylation on Ser157 abrogated the binding of Crkl. CONCLUSIONS: We identified Crkl as a novel protein interacting with VASP in platelets. We propose that the C3G/Crkl/VASP complex plays a role in the regulation of Rap1b and this explains, at least in part, the reduced agonist-induced activation of Rap1b in VASP-null platelets. In addition, the fact that PKA-dependent VASP phosphorylation abrogated its interaction with Crkl may provide, at least in part, a rationale for the PKA-dependent inhibition of Rap1b and platelet aggregation.


Asunto(s)
Plaquetas/metabolismo , Moléculas de Adhesión Celular/metabolismo , Proteínas de Microfilamentos/metabolismo , Fosfoproteínas/metabolismo , Proteínas de Unión al GTP rap/metabolismo , Animales , Plaquetas/efectos de los fármacos , Moléculas de Adhesión Celular/genética , Células Cultivadas , Factor 2 Liberador de Guanina Nucleótido/metabolismo , Humanos , Ratones , Proteínas de Microfilamentos/genética , Fosfoproteínas/genética , Unión Proteica , Proteínas Proto-Oncogénicas c-crk/metabolismo , Agonistas Purinérgicos/farmacología , Trombina/farmacología
8.
Cell Commun Signal ; 11: 56, 2013 Aug 12.
Artículo en Inglés | MEDLINE | ID: mdl-23937664

RESUMEN

BACKGROUND: In the heart, cytoplasmic actin networks are thought to have important roles in mechanical support, myofibrillogenesis, and ion channel function. However, subcellular localization of cytoplasmic actin isoforms and proteins involved in the modulation of the cytoplasmic actin networks are elusive. Mena and VASP are important regulators of actin dynamics. Due to the lethal phenotype of mice with combined deficiency in Mena and VASP, however, distinct cardiac roles of the proteins remain speculative. In the present study, we analyzed the physiological functions of Mena and VASP in the heart and also investigated the role of the proteins in the organization of cytoplasmic actin networks. RESULTS: We generated a mouse model, which simultaneously lacks Mena and VASP in the heart. Mena/VASP double-deficiency induced dilated cardiomyopathy and conduction abnormalities. In wild-type mice, Mena and VASP specifically interacted with a distinct αII-Spectrin splice variant (SH3i), which is in cardiomyocytes exclusively localized at Z- and intercalated discs. At Z- and intercalated discs, Mena and ß-actin localized to the edges of the sarcomeres, where the thin filaments are anchored. In Mena/VASP double-deficient mice, ß-actin networks were disrupted and the integrity of Z- and intercalated discs was markedly impaired. CONCLUSIONS: Together, our data suggest that Mena, VASP, and αII-Spectrin assemble cardiac multi-protein complexes, which regulate cytoplasmic actin networks. Conversely, Mena/VASP deficiency results in disrupted ß-actin assembly, Z- and intercalated disc malformation, and induces dilated cardiomyopathy and conduction abnormalities.


Asunto(s)
Actinas/metabolismo , Moléculas de Adhesión Celular/metabolismo , Proteínas del Citoesqueleto/metabolismo , Proteínas de Microfilamentos/metabolismo , Miocitos Cardíacos/metabolismo , Fosfoproteínas/metabolismo , Espectrina/metabolismo , Animales , Cardiomiopatía Dilatada/metabolismo , Moléculas de Adhesión Celular/genética , Citoplasma/metabolismo , Proteínas del Citoesqueleto/genética , Sistema de Conducción Cardíaco/metabolismo , Masculino , Ratones , Ratones Transgénicos , Proteínas de Microfilamentos/genética , Fosfoproteínas/genética , Espectrina/química , Dominios Homologos src
9.
Cells ; 12(13)2023 06 28.
Artículo en Inglés | MEDLINE | ID: mdl-37443774

RESUMEN

Actin binding proteins are of crucial importance for the spatiotemporal regulation of actin cytoskeletal dynamics, thereby mediating a tremendous range of cellular processes. Since their initial discovery more than 30 years ago, the enabled/vasodilator-stimulated phosphoprotein (Ena/VASP) family has evolved as one of the most fascinating and versatile family of actin regulating proteins. The proteins directly enhance actin filament assembly, but they also organize higher order actin networks and link kinase signaling pathways to actin filament assembly. Thereby, Ena/VASP proteins regulate dynamic cellular processes ranging from membrane protrusions and trafficking, and cell-cell and cell-matrix adhesions, to the generation of mechanical tension and contractile force. Important insights have been gained into the physiological functions of Ena/VASP proteins in platelets, leukocytes, endothelial cells, smooth muscle cells and cardiomyocytes. In this review, we summarize the unique and redundant functions of Ena/VASP proteins in cardiovascular cells and discuss the underlying molecular mechanisms.


Asunto(s)
Actinas , Células Endoteliales , Actinas/metabolismo , Células Endoteliales/metabolismo , Proteínas de Microfilamentos/metabolismo , Fosfoproteínas/metabolismo
10.
J Biol Chem ; 286(11): 9477-88, 2011 Mar 18.
Artículo en Inglés | MEDLINE | ID: mdl-21199868

RESUMEN

Sprouty-related proteins with EVH1 (enabled/vasodilator-stimulated phosphoprotein homology 1) domain (SPREDs) are inhibitors of MAPK signaling. To elucidate SPRED2 in vivo function, we characterized body homeostasis in SPRED2(-/-) mice. They showed a doubled daily water uptake, induced by elevated serum osmolality, originating from increased blood salt load. Accordingly, serum aldosterone was doubled, accompanied by augmented adrenal aldosterone synthase (AS) expression. Surprisingly, serum vasopressin (AVP) was unaltered, and, as evidenced by halved angiotensin II (Ang II) levels, the renin angiotensin system (RAS) was down-regulated. Adrenocorticotropic hormone (ACTH) was significantly elevated in SPRED2(-/-) mice, together with its secretagogue corticotropin-releasing hormone (CRH) and its downstream target corticosterone. ERK phosphorylation in brains was augmented, and hypothalamic CRH mRNA levels were elevated, both contributing to the increased CRH release. Our data were supported by CRH promoter reporter assays in hypothalamic mHypoE-44 cells, revealing a SPRED-dependent inhibition of Ets (ERK/E-twenty-six)-dependent transcription. Furthermore, SPRED suppressed CRH production in these cells. In conclusion, our study suggests that SPRED2 deficiency leads to an increased MAPK signaling, which results in an augmented CRH promoter activity. The subsequent CRH overproduction causes an up-regulation of downstream hypothalamic-pituitary-adrenal (HPA) hormone secretion. This constitutes a possible trigger for the observed compulsive grooming in SPRED2(-/-) mice and may, together with hyperplasia of aldosterone-producing cells, contribute to the hyperaldosteronism and homeostatic imbalances.


Asunto(s)
Sistema Hipotálamo-Hipofisario/metabolismo , Hipotálamo/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Sistema Hipófiso-Suprarrenal/metabolismo , Proteínas Represoras/metabolismo , Animales , Hormona Liberadora de Corticotropina/genética , Hormona Liberadora de Corticotropina/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/genética , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Ratones , Ratones Noqueados , Proteínas del Tejido Nervioso/genética , Fosforilación/fisiología , Proteínas Represoras/genética
11.
Ocul Immunol Inflamm ; : 1-12, 2022 Nov 28.
Artículo en Inglés | MEDLINE | ID: mdl-36441988

RESUMEN

This study aims to investigate the effect of a systemic lipopolysaccharide (LPS) stimulus in the course of laser-induced choroidal neovascularization (CNV) in C57BL/6 J mice. A group of CNV-subjected mice received 1 mg/kg LPS via the tail vein immediately after CNV induction. Mouse eyes were monitored in vivo with fluorescein angiography for 2 weeks. In situ hybridization and flow cytometry were performed in the retina at different time points. LPS led to increased fluorescein leakage 3 days after CNV, correlated with a large influx of monocyte-derived macrophages and increase of pro-inflammatory microglia/macrophages in the retina. Additionally, LPS enhanced Vegfα mRNA expression by Glul-expressing cells but not Aif1 positive microglia/macrophages in the laser lesion. These findings suggest that systemic LPS exposure has transient detrimental effects in the course of CNV through activation of microglia/macrophages to a pro-inflammatory phenotype and supports the important role of these cells in the CNV course.

12.
J Cell Sci ; 122(Pt 21): 3954-65, 2009 Nov 01.
Artículo en Inglés | MEDLINE | ID: mdl-19825941

RESUMEN

Proteins of the Enabled/vasodilator-stimulated phosphoprotein (Ena/VASP) family link signal transduction pathways to actin cytoskeleton dynamics. VASP is substrate of cAMP-dependent, cGMP-dependent and AMP-activated protein kinases that primarily phosphorylate the sites S157, S239 and T278, respectively. Here, we systematically analyzed functions of VASP phosphorylation patterns for actin assembly and subcellular targeting in vivo and compared the phosphorylation effects of Ena/VASP family members. Methods used were the reconstitution of VASP-null cells with ;locked' phosphomimetic VASP mutants, actin polymerization of VASP mutants in vitro and in living cells, site-specific kinase-mediated VASP phosphorylation, and analysis of the endogenous protein with phosphorylation-status-specific antibodies. Phosphorylation at S157 influenced VASP localization, but had a minor impact on F-actin assembly. Phosphorylation of the S157-equivalent site in the Ena/VASP family members Mena and EVL had no effect on the ratio of cellular F-actin to G-actin. By contrast, VASP phosphorylation at S239 (and the equivalent site in Mena) or T278 impaired VASP-driven actin filament formation. The data show that VASP functions are precisely regulated by differential phosphorylation and provide new insights into cytoskeletal control by serine/threonine kinase-dependent signaling pathways.


Asunto(s)
Actinas/metabolismo , Moléculas de Adhesión Celular/metabolismo , Citoesqueleto/metabolismo , Proteínas de Microfilamentos/metabolismo , Fosfoproteínas/metabolismo , Actinas/genética , Animales , Moléculas de Adhesión Celular/química , Moléculas de Adhesión Celular/genética , Línea Celular , Citoesqueleto/genética , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas de Microfilamentos/química , Proteínas de Microfilamentos/genética , Fosfoproteínas/química , Fosfoproteínas/genética , Fosforilación , Transporte de Proteínas
13.
J Neurosci ; 29(21): 6809-18, 2009 May 27.
Artículo en Inglés | MEDLINE | ID: mdl-19474308

RESUMEN

Photoreceptor ribbon synapses release glutamate in response to graded changes in membrane potential evoked by vast, logarithmically scalable light intensities. Neurotransmitter release is modulated by intracellular calcium levels. Large Ca(2+)-dependent chloride currents are important regulators of synaptic transmission from photoreceptors to second-order neurons; the molecular basis underlying these currents is unclear. We cloned human and mouse TMEM16B, a member of the TMEM16 family of transmembrane proteins, and show that it is abundantly present in the photoreceptor synaptic terminals in mouse retina. TMEM16B colocalizes with adaptor proteins PSD95, VELI3, and MPP4 at the ribbon synapses and contains a consensus PDZ class I binding motif capable of interacting with PDZ domains of PSD95. Furthermore, TMEM16B is lost from photoreceptor membranes of MPP4-deficient mice. This suggests that TMEM16B is a novel component of a presynaptic protein complex recruited to specialized plasma membrane domains of photoreceptors. TMEM16B confers Ca(2+)-dependent chloride currents when overexpressed in mammalian cells as measured by halide sensitive fluorescent protein assays and whole-cell patch-clamp recordings. The compartmentalized localization and the electrophysiological properties suggest TMEM16B to be a strong candidate for the long sought-after Ca(2+)-dependent chloride channel in the photoreceptor synapse.


Asunto(s)
Canales de Cloruro/fisiología , Proteínas de la Membrana/metabolismo , Neuronas/citología , Células Fotorreceptoras/citología , Terminales Presinápticos/metabolismo , Sinapsis/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Anoctaminas , Calcio/metabolismo , Línea Celular Transformada , Clonación Molecular , Homólogo 4 de la Proteína Discs Large , Estimulación Eléctrica , Ojo/citología , Expresión Génica , Guanilato-Quinasas , Humanos , Técnicas In Vitro , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Luminiscentes/genética , Potenciales de la Membrana/genética , Potenciales de la Membrana/fisiología , Proteínas de la Membrana/química , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Moleculares , Dominios PDZ/fisiología , Técnicas de Placa-Clamp , Retina/citología , Transfección
14.
Acta Physiol (Oxf) ; 228(1): e13325, 2020 01.
Artículo en Inglés | MEDLINE | ID: mdl-31162891

RESUMEN

AIM: Protein kinase (PK) A anchoring protein (AKAP) 12 is a scaffolding protein that anchors PKA to compartmentalize cyclic AMP signalling. This study assessed the consequences of the downregulation or deletion of AKAP12 on endothelial cell migration and angiogenesis. METHODS: The consequences of siRNA-mediated downregulation AKAP12 were studied in primary cultures of human endothelial cells as well as in endothelial cells and retinas from wild-type versus AKAP12-/- mice. Molecular interactions were investigated using a combination of immunoprecipitation and mass spectrometry. RESULTS: AKAP12 was expressed at low levels in confluent endothelial cells but its expression was increased in actively migrating cells, where it localized to lamellipodia. In the postnatal retina, AKAP12 was expressed by actively migrating tip cells at the angiogenic front, and its deletion resulted in defective extension of the vascular plexus. In migrating endothelial cells, AKAP12 was co-localized with the PKA type II-α regulatory subunit as well as multiple key regulators of actin dynamics and actin filament-based movement; including components of the Arp2/3 complex and the vasodilator-stimulated phosphoprotein (VASP). Fitting with the evidence of a physical VASP/AKAP12/PKA complex, it was possible to demonstrate that the VEGF-stimulated and PKA-dependent phosphorylation of VASP was dependent on AKAP12. Indeed, AKAP12 colocalized with phospho-Ser157 VASP at the leading edge of migrating endothelial cells. CONCLUSION: The results suggest that compartmentalized AKAP12/PKA signalling mediates VASP phosphorylation at the leading edge of migrating endothelial cells to translate angiogenic stimuli into altered actin dynamics and cell movement.


Asunto(s)
Proteínas de Anclaje a la Quinasa A/metabolismo , Proteínas de Ciclo Celular/metabolismo , Células Endoteliales/efectos de los fármacos , Factor A de Crecimiento Endotelial Vascular/farmacología , Proteínas de Anclaje a la Quinasa A/genética , Animales , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/metabolismo , Proteínas de Ciclo Celular/genética , Movimiento Celular/efectos de los fármacos , Movimiento Celular/fisiología , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Células Endoteliales/fisiología , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/fisiología , Ratones , Ratones Noqueados , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilación , Factor A de Crecimiento Endotelial Vascular/administración & dosificación
15.
Int Immunopharmacol ; 8(2): 319-24, 2008 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-18182247

RESUMEN

In pathological conditions, the inflammatory mediator prostaglandin E2 (PGE2) has been shown to induce apoptosis through a cAMP-dependent pathway. However, underlying mechanisms have remained illusive. Irrespective whether apoptosis is induced by the intrinsic or extrinsic pathway, the cysteine protease caspase-3 becomes activated and cleaves many key proteins including spectrins. Cleavage of the plasma membrane-associated spectrins leads to cell shrinkage, membrane blebbing, the formation of apoptotic bodies, and irreversible cell death. Recently, we identified a novel interaction between alpha II-spectrin and vasodilator-stimulated phosphoprotein (VASP), which is abrogated by the cAMP-dependent protein kinase (PKA)-mediated phosphorylation of VASP. In the present study we investigated whether VASP binding to alpha II-spectrin affects spectrin breakdown in PGE2-induced apoptosis. PGE2 dose- and time-dependently triggered VASP phosphorylation. Following induction of apoptosis, caspase-3-mediated alpha II-spectrin breakdown and membrane blebbing were markedly delayed in wild-type as compared to VASP-deficient endothelial cells. This suggests that VASP binding to alpha II-spectrin attenuates alpha II-spectrin cleavage in apoptotic cells and that PGE2-induced VASP phosphorylation regulates this process. Our findings may therefore provide the molecular basis for PGE2-induced apoptosis in pathological events.


Asunto(s)
Apoptosis , Moléculas de Adhesión Celular/metabolismo , Dinoprostona/farmacología , Proteínas de Microfilamentos/metabolismo , Fosfoproteínas/metabolismo , Espectrina/metabolismo , Animales , Caspasa 3/fisiología , Relación Dosis-Respuesta a Droga , Ratones , Fosforilación
16.
J Cell Biol ; 217(4): 1503-1519, 2018 04 02.
Artículo en Inglés | MEDLINE | ID: mdl-29507126

RESUMEN

In ischemic vascular diseases, leukocyte recruitment and polarization are crucial for revascularization and tissue repair. We investigated the role of vasodilator-stimulated phosphoprotein (VASP) in vascular repair. After hindlimb ischemia induction, blood flow recovery, angiogenesis, arteriogenesis, and leukocyte infiltration into ischemic muscles in VASP-/- mice were accelerated. VASP deficiency also elevated the polarization of the macrophages through increased signal transducer and activator of transcription (STAT) signaling, which augmented the release of chemokines, cytokines, and growth factors to promote leukocyte recruitment and vascular repair. Importantly, VASP deletion in bone marrow-derived cells was sufficient to mimic the increased blood flow recovery of global VASP-/- mice. In chemotaxis experiments, VASP-/- neutrophils/monocytes were significantly more responsive to M1-related chemokines than wild-type controls. Mechanistically, VASP formed complexes with the chemokine receptor CCR2 and ß-arrestin-2, and CCR2 receptor internalization was significantly reduced in VASP-/- leukocytes. Our data indicate that VASP is a major regulator of leukocyte recruitment and polarization in postischemic revascularization and support a novel role of VASP in chemokine receptor trafficking.


Asunto(s)
Moléculas de Adhesión Celular/metabolismo , Quimiotaxis de Leucocito , Isquemia/metabolismo , Leucocitos/metabolismo , Proteínas de Microfilamentos/metabolismo , Músculo Esquelético/irrigación sanguínea , Neovascularización Fisiológica , Peritonitis/metabolismo , Fosfoproteínas/metabolismo , Citoesqueleto de Actina/metabolismo , Animales , Moléculas de Adhesión Celular/deficiencia , Moléculas de Adhesión Celular/genética , Quimiocinas/metabolismo , Modelos Animales de Enfermedad , Miembro Posterior , Isquemia/genética , Isquemia/patología , Isquemia/fisiopatología , Macrófagos/metabolismo , Ratones Noqueados , Proteínas de Microfilamentos/deficiencia , Proteínas de Microfilamentos/genética , Neuropéptidos/metabolismo , Peritonitis/genética , Peritonitis/patología , Peritonitis/fisiopatología , Fenotipo , Fosfoproteínas/deficiencia , Fosfoproteínas/genética , Transporte de Proteínas , Receptores CCR2/metabolismo , Factores de Transcripción STAT/metabolismo , Transducción de Señal , Factores de Tiempo , Arrestina beta 2/metabolismo , Proteína de Unión al GTP rac1/metabolismo
17.
Cell Death Dis ; 8(6): e2898, 2017 06 29.
Artículo en Inglés | MEDLINE | ID: mdl-28661475

RESUMEN

Chemotherapy-induced thrombocytopenia is a common bleeding risk in cancer patients and limits chemotherapy dose and frequency. Recent data from mouse and human platelets revealed that activation of protein kinase A/G (PKA/PKG) not only inhibited thrombin/convulxin-induced platelet activation but also prevented the platelet pro-coagulant state. Here we investigated whether or not PKA/PKG activation could attenuate caspase-dependent apoptosis induced by the anti-cancer drugs ABT-737 (the precursor of navitoclax) and thymoquinone (TQ), thereby potentially limiting chemotherapy-induced thrombocytopenia. This is particularly relevant as activation of cyclic nucleotide signalling in combination chemotherapy is an emerging strategy in cancer treatment. However, PKA/PKG-activation, as monitored by phosphorylation of Vasodilator-stimulated phosphoprotein (VASP), did not block caspase-3-dependent platelet apoptosis induced by the compounds. In contrast, both substances induced PKA activation themselves and PKA activation correlated with platelet inhibition and apoptosis. Surprisingly, ABT-737- and TQ-induced VASP-phosphorylation was independent of cAMP levels and neither cyclases nor phosphatases were affected by the drugs. In contrast, however, ABT-737- and TQ-induced PKA activation was blocked by caspase-3 inhibitors. In conclusion, we show that ABT-737 and TQ activate PKA in a caspase-3-dependent manner, which correlates with platelet inhibition and apoptosis and therefore potentially contributes to the bleeding risk in chemotherapy patients.


Asunto(s)
Plaquetas/metabolismo , Caspasa 3/genética , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Neoplasias/tratamiento farmacológico , Trombocitopenia/genética , Animales , Apoptosis/efectos de los fármacos , Benzoquinonas/administración & dosificación , Benzoquinonas/efectos adversos , Compuestos de Bifenilo/administración & dosificación , Compuestos de Bifenilo/efectos adversos , Plaquetas/efectos de los fármacos , AMP Cíclico/antagonistas & inhibidores , AMP Cíclico/genética , Proteínas Quinasas Dependientes de GMP Cíclico/genética , Humanos , Ratones , Neoplasias/genética , Neoplasias/patología , Nitrofenoles/administración & dosificación , Nitrofenoles/efectos adversos , Piperazinas/administración & dosificación , Piperazinas/efectos adversos , Activación Plaquetaria/efectos de los fármacos , Sulfonamidas/administración & dosificación , Sulfonamidas/efectos adversos , Trombocitopenia/inducido químicamente , Trombocitopenia/patología
18.
J Cell Biol ; 180(1): 205-19, 2008 Jan 14.
Artículo en Inglés | MEDLINE | ID: mdl-18195108

RESUMEN

Directed cortical actin assembly is the driving force for intercellular adhesion. Regulated by phosphorylation, vasodilator-stimulated phosphoprotein (VASP) participates in actin fiber formation. We screened for endothelial proteins, which bind to VASP, dependent on its phosphorylation status. Differential proteomics identified alphaII-spectrin as such a VASP-interacting protein. alphaII-Spectrin binds to the VASP triple GP(5)-motif via its SH3 domain. cAMP-dependent protein kinase-mediated VASP phosphorylation at Ser157 inhibits alphaII-spectrin-VASP binding. VASP is dephosphorylated upon formation of cell-cell contacts and in confluent, but not in sparse cells, alphaII-spectrin colocalizes with nonphosphorylated VASP at cell-cell junctions. Ectopic expression of the alphaII-spectrin SH3 domain at cell-cell contacts translocates VASP, initiates cortical actin cytoskeleton formation, stabilizes cell-cell contacts, and decreases endothelial permeability. Conversely, the permeability of VASP-deficient endothelial cells (ECs) and microvessels of VASP-null mice increases. Reconstitution of VASP-deficient ECs rescues barrier function, whereas alphaII-spectrin binding-deficient VASP mutants fail to restore elevated permeability. We propose that alphaII-spectrin-VASP complexes regulate cortical actin cytoskeleton assembly with implications for vascular permeability.


Asunto(s)
Citoesqueleto de Actina/metabolismo , Proteínas Portadoras/fisiología , Moléculas de Adhesión Celular/fisiología , Células Endoteliales/ultraestructura , Uniones Intercelulares/metabolismo , Proteínas de Microfilamentos/fisiología , Fosfoproteínas/fisiología , Secuencia de Aminoácidos , Animales , Sitios de Unión , Proteínas Portadoras/análisis , Proteínas Portadoras/química , Adhesión Celular , Moléculas de Adhesión Celular/análisis , Moléculas de Adhesión Celular/química , Células Endoteliales/citología , Células Endoteliales/metabolismo , Ratones , Proteínas de Microfilamentos/análisis , Proteínas de Microfilamentos/química , Datos de Secuencia Molecular , Fosfoproteínas/análisis , Fosfoproteínas/química , Fosforilación , Dominios y Motivos de Interacción de Proteínas , Mapeo de Interacción de Proteínas
19.
J Biol Chem ; 282(7): 4601-4612, 2007 Feb 16.
Artículo en Inglés | MEDLINE | ID: mdl-17082196

RESUMEN

Vasodilator-stimulated phosphoprotein (VASP) is an actin regulatory protein that links signaling pathways to remodeling of the cytoskeleton. VASP functions are modulated by protein kinases, which phosphorylate the sites Ser-157, Ser-239, and Thr-278. The kinase responsible for Thr-278 phosphorylation, biological functions of the phosphorylation, and association with disease states have remained enigmatic. Using VASP phosphorylation status-specific antibodies, we identified AMP-activated protein kinase (AMPK), a serine-threonine kinase and fundamental sensor of energy homeostasis, in a screen for kinases that phosphorylate the Thr-278 site of VASP in endothelial cells. Pharmacological AMPK inhibitors and activators and AMPK mutants revealed that the kinase specifically targets residue Thr-278 but not Ser-157 or Ser-239. Quantitative fluorescence-activated cell sorter analysis and serum response factor transcriptional reporter assays, which quantify the cellular F-/G-actin equilibrium, indicated that AMPK-mediated VASP phosphorylation impaired actin stress fiber formation and altered cell morphology. In the Zucker Diabetic Fatty (ZDF) rat model for type II diabetes, AMPK activity and Thr-278 phosphorylation were substantially reduced in arterial vessel walls. These findings suggest that VASP is a new AMPK substrate, that VASP Thr-278 phosphorylation translates metabolic signals into actin cytoskeleton rearrangements, and that this signaling system becomes down-regulated in diabetic vessels.


Asunto(s)
Moléculas de Adhesión Celular/metabolismo , Citoesqueleto/metabolismo , Células Endoteliales/enzimología , Proteínas de Microfilamentos/metabolismo , Complejos Multienzimáticos/metabolismo , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Fibras de Estrés/metabolismo , Proteínas Quinasas Activadas por AMP , Animales , Moléculas de Adhesión Celular/genética , Citoesqueleto/genética , Diabetes Mellitus Experimental/enzimología , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Tipo 2/enzimología , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/patología , Células Endoteliales/patología , Humanos , Masculino , Proteínas de Microfilamentos/genética , Complejos Multienzimáticos/antagonistas & inhibidores , Fosfoproteínas/genética , Fosforilación/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Procesamiento Proteico-Postraduccional/genética , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Ratas , Ratas Zucker , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Fibras de Estrés/genética , Especificidad por Sustrato
20.
Biol Chem ; 387(2): 173-8, 2006 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-16497149

RESUMEN

Blood coagulation factor XII (FXII, Hageman factor) is a plasma serine protease which is autoactivated following contact with negatively charged surfaces in a reaction involving plasma kallikrein and high-molecular-weight kininogen (contact phase activation). Active FXII has the ability to initiate blood clotting via the intrinsic pathway of coagulation and inflammatory reactions via the kallikrein-kinin system. Here we have determined FXII-mediated bradykinin formation and clotting in plasma. Western blotting analysis with specific antibodies against various parts of the contact factors revealed that limited activation of FXII is sufficient to promote plasma kallikrein activation, resulting in the conversion of high-molecular-weight kininogen and bradykinin generation. The presence of platelets significantly promoted FXII-initiated bradykinin formation. Similarly, in vitro clotting assays revealed that platelets critically promoted FXII-driven thrombin and fibrin formation. In summary, our data suggest that FXII-initiated protease cascades may proceed on platelet surfaces, with implications for inflammation and clotting.


Asunto(s)
Coagulación Sanguínea/fisiología , Plaquetas/fisiología , Factor XII/fisiología , Sistema Calicreína-Quinina/fisiología , Bradiquinina/biosíntesis , Humanos , Quininógenos/biosíntesis , Peso Molecular , Valores de Referencia , Trombina/biosíntesis , Factores de Tiempo
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