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Endothelial YAP/TAZ (YAP is also known as YAP1, and TAZ as WWTR1) signaling is crucial for sprouting angiogenesis and vascular homeostasis. However, the underlying molecular mechanisms that explain how YAP/TAZ control the vasculature remain unclear. This study reveals that the focal adhesion protein deleted-in-liver-cancer 1 (DLC1) is a direct transcriptional target of the activated YAP/TAZ-TEAD complex. We find that substrate stiffening and VEGF stimuli promote expression of DLC1 in endothelial cells. In turn, DLC1 expression levels are YAP and TAZ dependent, and constitutive activation of YAP is sufficient to drive DLC1 expression. DLC1 is needed to limit F-actin fiber formation, integrin-based focal adhesion lifetime and integrin-mediated traction forces. Depletion of endothelial DLC1 strongly perturbs cell polarization in directed collective migration and inhibits the formation of angiogenic sprouts. Importantly, ectopic expression of DLC1 is sufficient to restore migration and angiogenic sprouting in YAP-depleted cells. Together, these findings point towards a crucial and prominent role for DLC1 in YAP/TAZ-driven endothelial adhesion remodeling and collective migration during angiogenesis.This article has an associated First Person interview with the first author of the paper.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Células Endoteliais , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Células Endoteliais/metabolismo , Proteínas Ativadoras de GTPase/genética , Humanos , Morfogênese , Neovascularização Patológica , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Transdução de Sinais , Proteínas Supressoras de Tumor/genéticaRESUMO
During inflammation, endothelial cells are bombarded with cytokines and other stimuli from surrounding cells. Leukocyte extravasation and vascular leakage are both prominent but believed to be uncoupled as they occur in separate spatiotemporal patterns. In this study, we investigated a "double-hit" approach on primary human endothelial cells primed with LPS followed by histamine. Using neutrophil transendothelial migration (TEM) under physiological flow assays, we found that an LPS-primed endothelium synergistically enhanced neutrophil TEM when additionally treated with histamine, whereas the effects on neutrophil TEM of the individual stimuli were moderate to undetectable. Interestingly, the double-hit-induced TEM increase was not due to decreased endothelial barrier, increased adhesion molecule expression, or Weibel-Palade body release. Instead, we found that it was directly correlated with junctional remodeling. Compounds that increased junctional "linearity" (i.e., stability) counteracted the double-hit effect on neutrophil TEM. We conclude that a compound, in this case histamine (which has a short primary effect on vascular permeability), can have severe secondary effects on neutrophil TEM in combination with an inflammatory stimulus. This effect is due to synergic modifications of the endothelial cytoskeleton and junctional remodeling. Therefore, we hypothesize that junctional linearity is a better and more predictive readout than endothelial resistance for compounds aiming to attenuate inflammation.
Assuntos
Junções Aderentes/metabolismo , Endotélio Vascular/fisiologia , Histamina/metabolismo , Inflamação/patologia , Leucócitos/fisiologia , Lipopolissacarídeos/metabolismo , Neutrófilos/fisiologia , Permeabilidade Capilar , Adesão Celular , Moléculas de Adesão Celular/metabolismo , Movimento Celular , Células Cultivadas , Citocinas/metabolismo , Citoesqueleto/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Migração Transendotelial e TransepitelialRESUMO
Sprouting angiogenesis is key to many pathophysiological conditions, and is strongly regulated by vascular endothelial growth factor (VEGF) signaling through VEGF receptor 2 (VEGFR2). Here we report that the early endosomal GTPase Rab5C and its activator RIN2 prevent lysosomal routing and degradation of VEGF-bound, internalized VEGFR2 in human endothelial cells. Stabilization of endosomal VEGFR2 levels by RIN2/Rab5C is crucial for VEGF signaling through the ERK and PI3-K pathways, the expression of immediate VEGF target genes, as well as specification of angiogenic 'tip' and 'stalk' cell phenotypes and cell sprouting. Using overexpression of Rab mutants, knockdown and CRISPR/Cas9-mediated gene editing, and live-cell imaging in zebrafish, we further show that endosomal stabilization of VEGFR2 levels is required for developmental angiogenesis in vivo. In contrast, the premature degradation of internalized VEGFR2 disrupts VEGF signaling, gene expression, and tip cell formation and migration. Thus, an endosomal feedforward mechanism maintains receptor signaling by preventing lysosomal degradation, which is directly linked to the induction of target genes and cell fate in collectively migrating cells during morphogenesis.
Assuntos
Proteínas de Transporte/metabolismo , Regulação da Expressão Gênica , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Neovascularização Fisiológica , Proteólise , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Peixe-Zebra/metabolismo , Proteínas rab5 de Ligação ao GTP/metabolismo , Animais , Proteínas de Transporte/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Humanos , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Peixe-Zebra/genética , Proteínas rab5 de Ligação ao GTP/genéticaRESUMO
Endothelial barrier disruption and vascular leak importantly contribute to organ dysfunction and mortality during inflammatory conditions like sepsis and acute respiratory distress syndrome. We identified the kinase Arg/Abl2 as a mediator of endothelial barrier disruption, but the role of Arg in endothelial monolayer regulation and its relevance in vivo remain poorly understood. Here we show that depletion of Arg in endothelial cells results in the activation of both RhoA and Rac1, increased cell spreading and elongation, redistribution of integrin-dependent cell-matrix adhesions to the cell periphery, and improved adhesion to the extracellular matrix. We further show that Arg is activated in the endothelium during inflammation, both in murine lungs exposed to barrier-disruptive agents, and in pulmonary microvessels of septic patients. Importantly, Arg-depleted endothelial cells were less sensitive to barrier-disruptive agents. Despite the formation of F-actin stress fibers and myosin light chain phosphorylation, Arg depletion diminished adherens junction disruption and intercellular gap formation, by reducing the disassembly of cell-matrix adhesions and cell retraction. In vivo, genetic deletion of Arg diminished vascular leak in the skin and lungs, in the presence of a normal immune response. Together, our data indicate that Arg is a central and non-redundant regulator of endothelial barrier integrity, which contributes to cell retraction and gap formation by increasing the dynamics of adherens junctions and cell-matrix adhesions in a Rho GTPase-dependent fashion. Therapeutic inhibition of Arg may provide a suitable strategy for the treatment of a variety of clinical conditions characterized by vascular leak.
Assuntos
Matriz Extracelular/metabolismo , Junções Comunicantes/enzimologia , Células Endoteliais da Veia Umbilical Humana/enzimologia , Proteínas Tirosina Quinases/metabolismo , Alvéolos Pulmonares/enzimologia , Animais , Adesão Celular/genética , Ativação Enzimática , Matriz Extracelular/genética , Junções Comunicantes/genética , Humanos , Inflamação/enzimologia , Inflamação/genética , Camundongos , Camundongos Knockout , Proteínas Tirosina Quinases/genéticaRESUMO
OBJECTIVE: Vascular endothelial-cadherin- and integrin-based cell adhesions are crucial for endothelial barrier function. Formation and disassembly of these adhesions controls endothelial remodeling during vascular repair, angiogenesis, and inflammation. In vitro studies indicate that vascular cytokines control adhesion through regulation of the actin cytoskeleton, but it remains unknown whether such regulation occurs in human vessels. We aimed to investigate regulation of the actin cytoskeleton and cell adhesions within the endothelium of human arteries and veins. APPROACH AND RESULTS: We used an ex vivo protocol for immunofluorescence in human vessels, allowing detailed en face microscopy of endothelial monolayers. We compared arteries and veins of the umbilical cord and mesenteric, epigastric, and breast tissues and find that the presence of central F-actin fibers distinguishes the endothelial phenotype of adult arteries from veins. F-actin in endothelium of adult veins as well as in umbilical vasculature predominantly localizes cortically at the cell boundaries. By contrast, prominent endothelial F-actin fibers in adult arteries anchor mostly to focal adhesions containing integrin-binding proteins paxillin and focal adhesion kinase and follow the orientation of the extracellular matrix protein fibronectin. Other arterial F-actin fibers end in vascular endothelial-cadherin-based endothelial focal adherens junctions. In vitro adhesion experiments on compliant substrates demonstrate that formation of focal adhesions is strongly induced by extracellular matrix rigidity, irrespective of arterial or venous origin of endothelial cells. CONCLUSIONS: Our data show that F-actin-anchored focal adhesions distinguish endothelial phenotypes of human arteries from veins. We conclude that the biomechanical properties of the vascular extracellular matrix determine this endothelial characteristic.
Assuntos
Actinas/análise , Artérias/citologia , Endotélio Vascular/citologia , Adesões Focais , Veias/citologia , Citoesqueleto de Actina/ultraestrutura , Adulto , Antígenos CD/análise , Mama/irrigação sanguínea , Caderinas/análise , Células Cultivadas , Células Endoteliais , Artérias Epigástricas/citologia , Matriz Extracelular/fisiologia , Proteínas da Matriz Extracelular/fisiologia , Feminino , Humanos , Recém-Nascido , Artérias Mesentéricas/citologia , Veias Mesentéricas/citologia , Microscopia Confocal , Microscopia de Fluorescência , Fenótipo , Artérias Umbilicais/citologia , Veias Umbilicais/citologiaRESUMO
Nuclear receptor Nur77 (NR4A1, TR3, or NGFI-B) has been shown to play an anti-inflammatory role in macrophages, which have a crucial function in defense against peritonitis. The function of Nur77 in Escherichia coli-induced peritoneal sepsis has not yet been investigated. Wild-type and Nur77-knockout mice were inoculated with E. coli, and bacterial outgrowth, cell recruitment, cytokine profiles, and tissue damage were investigated. We found only a minor transient decrease in bacterial loads in lung and liver of Nur77-knockout compared to wild-type mice at 14 h postinfection, yet no changes were found in the peritoneal lavage fluid or blood. No differences in inflammatory cytokine levels or neutrophil/macrophage numbers were observed, and bacterial loads were equal in wild-type and Nur77-knockout mice at 20 h postinfection in all body compartments tested. Also, isolated peritoneal macrophages did not show any differences in cytokine expression patterns in response to E. coli. In endothelial cells, Nur77 strongly downregulated both protein and mRNA expression of claudin-5, VE-cadherin, occludin, ZO-1, and ß-catenin, and accordingly, these genes were upregulated in lungs of Nur77-deficient mice. Functional permeability tests pointed toward a strong role for Nur77 in endothelial barrier function. Indeed, tissue damage in E. coli-induced peritonitis was notably modulated by Nur77; liver necrosis and plasma aspartate aminotransferase (ASAT)/alanine aminotransferase (ALAT) levels were lower in Nur77-knockout mice. These data suggest that Nur77 does not play a role in the host response to E. coli in the peritoneal and blood compartments. However, Nur77 does modulate bacterial influx into the organs via increased vascular permeability, thereby aggravating distant organ damage.
Assuntos
Infecções por Escherichia coli/metabolismo , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/fisiologia , Periodontite/microbiologia , Animais , Carga Bacteriana , Citocinas/metabolismo , Modelos Animais de Doenças , Infecções por Escherichia coli/patologia , Fígado/citologia , Fígado/microbiologia , Pulmão/citologia , Pulmão/microbiologia , Macrófagos/citologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neutrófilos/citologia , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/deficiência , Periodontite/metabolismo , Periodontite/patologia , Cavidade Peritoneal/microbiologiaRESUMO
Integrin-dependent cell-extracellular matrix adhesion is essential for wound healing, embryonic development, immunity, and tissue organization. Here, we present a protocol for the imaging and quantitative analysis of integrin-dependent cell-matrix adhesions. We describe steps for cell culture; virus preparation; lentiviral transduction; imaging with widefield, confocal, and total internal reflection fluorescence microscopy; and using a script for their quantitative analysis. We then detail procedures for analyzing adhesion dynamics by live-cell imaging and fluorescence recovery after photobleaching (FRAP). For complete details on the use and execution of this protocol, please refer to Margadant et al. (2012),1 van der Bijl et al. (2020),2 Amado-Azevedo et al. (2021).3.
Assuntos
Técnicas de Cultura de Células , Microscopia , Feminino , Gravidez , Humanos , Junções Célula-Matriz , Desenvolvimento Embrionário , IntegrinasRESUMO
Patients whose hematopoietic system is compromised by chemo- and/or radiotherapy require transplantation of hematopoietic stem and progenitor cells (HSPCs) to restore hematopoiesis. Successful homing of transplanted HSPCs to the bone marrow (BM) largely depends on their migratory potential, which is critically regulated by the chemokine CXCL12. In this study, we have investigated the expression and function of Slit proteins and their corresponding Roundabout (Robo) receptors in human HSPC migration. Slit proteins are extracellular matrix proteins that can modulate the (chemoattractant-induced) migration of mature leukocytes. We show that mRNAs for all Slits (Slit1-3) are expressed in primary BM stroma and BM-derived endothelial and stromal cell lines, but not in CD34⺠HSPCs. Human CD34⺠HSPCs expressed mRNAs for all Robos (Robo1-4), but only the Robo1 protein was detected on their cell surface. Functionally, Slit3 treatment increased the in vivo homing efficiency of CD34⺠HSPCs to the BM in NOD/SCID mice, whereas Slit3-exposed HSPC migration in vitro was inhibited. These effects do not appear to result from modulated CXCL12 responsiveness as CXCR4 expression, CXCL12-induced actin polymerization or the basal and CXCL12-induced adhesion to fibronectin or BM-derived endothelial cells of CD34⺠HSPC were not altered by Slit3 exposure. However, we show that Slit3 rapidly reduced the levels of active RhoA in HL60 cells and primary CD34⺠HSPC, directly affecting a pathway involved in actin cytoskeleton remodeling and HSPC migration. Together, our results support a role for Slit3 in human HSPC migration in vitro and homing in vivo and might contribute to the design of future approaches aimed at improving transplantation efficiency of human CD34⺠HSPCs.
Assuntos
Movimento Celular/fisiologia , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Proteínas de Membrana/metabolismo , Animais , Antígenos CD/metabolismo , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Adesão Celular/fisiologia , Linhagem Celular Tumoral , Células Cultivadas , Quimiocina CXCL12/genética , Quimiocina CXCL12/metabolismo , Citometria de Fluxo , Humanos , Proteínas de Membrana/genética , Camundongos , Camundongos SCID , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Receptores CXCR4/genética , Receptores CXCR4/metabolismo , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismo , Baço/citologia , Transplante de Células-Tronco , Proteína rhoA de Ligação ao GTP/metabolismo , Proteínas RoundaboutRESUMO
Acute respiratory distress syndrome (ARDS) is a major clinical problem without available therapies. Known risks for ARDS include severe sepsis, SARS-CoV-2, gram-negative bacteria, trauma, pancreatitis, and blood transfusion. During ARDS, blood fluids and inflammatory cells enter the alveoli, preventing oxygen exchange from air into blood vessels. Reduced pulmonary endothelial barrier function, resulting in leakage of plasma from blood vessels, is one of the major determinants in ARDS. It is, however, unknown why systemic inflammation particularly targets the pulmonary endothelium, as endothelial cells (ECs) line all vessels in the vascular system of the body. In this study, we examined ECs of pulmonary, umbilical, renal, pancreatic, and cardiac origin for upregulation of adhesion molecules, ability to facilitate neutrophil (PMN) trans-endothelial migration (TEM) and for endothelial barrier function, in response to the gram-negative bacterial endotoxin LPS. Interestingly, we found that upon LPS stimulation, pulmonary ECs showed increased levels of adhesion molecules, facilitated more PMN-TEM and significantly perturbed the endothelial barrier, compared to other types of ECs. These observations could partly be explained by a higher expression of the adhesion molecule ICAM-1 on the pulmonary endothelial surface compared to other ECs. Moreover, we identified an increased expression of Cadherin-13 in pulmonary ECs, for which we demonstrated that it aids PMN-TEM in pulmonary ECs stimulated with LPS. We conclude that pulmonary ECs are uniquely sensitive to LPS, and intrinsically different, compared to ECs from other vascular beds. This may add to our understanding of the development of ARDS upon systemic inflammation.
Assuntos
COVID-19 , Síndrome do Desconforto Respiratório , Moléculas de Adesão Celular/metabolismo , Células Endoteliais/metabolismo , Endotélio Vascular/metabolismo , Humanos , Inflamação/metabolismo , Lipopolissacarídeos/metabolismo , Lipopolissacarídeos/farmacologia , SARS-CoV-2RESUMO
We describe an optimized, cost-effective, reproducible, and robust protocol to study sprouting angiogenesis in glass-bottom 96-well plates by confocal microscopy, ideal for screening of drug or shRNA libraries. Effective and stable knockdown of gene expression in primary endothelial cells is achieved by lentiviral transduction. Dynamic behavior of individual cells and fluorescent proteins is analyzed by time-lapse imaging, while competitive advantages in tip cell formation are assessed using mixtures of differentially labeled cell populations. Finally, we present a macro for high-throughput analysis. For complete information on the use and execution of this protocol, please refer to van der Bijl et al. (2020) and Kempers et al. (2021).
Assuntos
Ensaios de Triagem em Larga Escala/métodos , Microscopia Confocal/métodos , Neovascularização Fisiológica/fisiologia , Células Endoteliais/metabolismo , Humanos , MorfogêneseRESUMO
Collective cell behaviour during embryogenesis and tissue repair requires the coordination of intercellular junctions, cytoskeleton-dependent shape changes controlled by Rho GTPases, and integrin-dependent cell-matrix adhesion. Many different integrins are simultaneously expressed during wound healing, embryonic development, and sprouting angiogenesis, suggesting that there is extensive integrin/integrin cross-talk to regulate cell behaviour. Here, we show that fibronectin-binding ß1 and ß3 integrins do not act synergistically, but rather antagonize each other during collective cell processes in neuro-epithelial cells, placental trophoblasts, and endothelial cells. Reciprocal ß1/ß3 antagonism controls RhoA activity in a kindlin-2-dependent manner, balancing cell spreading, contractility, and intercellular adhesion. In this way, reciprocal ß1/ß3 antagonism controls cell cohesion and cellular plasticity to switch between extreme and opposing states, including epithelial versus mesenchymal-like phenotypes and collective versus individual cell migration. We propose that integrin/integrin antagonism is a universal mechanism to effectuate social cellular interactions, important for tissue morphogenesis, endothelial barrier function, trophoblast invasion, and sprouting angiogenesis.
Assuntos
Integrina beta1/metabolismo , Integrina beta3/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Células Neuroepiteliais/citologia , Proteína rhoA de Ligação ao GTP/metabolismo , Movimento Celular , Plasticidade Celular , Citoplasma/metabolismo , Desenvolvimento Embrionário , Células HEK293 , Células Endoteliais da Veia Umbilical Humana , Humanos , Células Neuroepiteliais/metabolismo , FenótipoRESUMO
Major efforts are made to characterize the presence of microRNA (miRNA) and messenger RNA in blood plasma to discover novel disease-associated biomarkers. MiRNAs in plasma are associated to several types of macromolecular structures, including extracellular vesicles (EV), lipoprotein particles (LPP) and ribonucleoprotein particles (RNP). RNAs in these complexes are recovered at variable efficiency by commonly used EV- and RNA isolation methods, which causes biases and inconsistencies in miRNA quantitation. Besides miRNAs, various other non-coding RNA species are contained in EV and present within the pool of plasma extracellular RNA. Members of the Y-RNA family have been detected in EV from various cell types and are among the most abundant non-coding RNA types in plasma. We previously showed that shuttling of full-length Y-RNA into EV released by immune cells is modulated by microbial stimulation. This indicated that Y-RNAs could contribute to the functional properties of EV in immune cell communication and that EV-associated Y-RNAs could have biomarker potential in immune-related diseases. Here, we investigated which macromolecular structures in plasma contain full length Y-RNA and whether the levels of three Y-RNA subtypes in plasma (Y1, Y3 and Y4) change during systemic inflammation. Our data indicate that the majority of full length Y-RNA in plasma is stably associated to EV. Moreover, we discovered that EV from different blood-related cell types contain cell-type-specific Y-RNA subtype ratios. Using a human model for systemic inflammation, we show that the neutrophil-specific Y4/Y3 ratios and PBMC-specific Y3/Y1 ratios were significantly altered after induction of inflammation. The plasma Y-RNA ratios strongly correlated with the number and type of immune cells during systemic inflammation. Cell-type-specific "Y-RNA signatures" in plasma EV can be determined without prior enrichment for EV, and may be further explored as simple and fast test for diagnosis of inflammatory responses or other immune-related diseases.
RESUMO
Leukocytes follow the well-defined steps of rolling, spreading, and crawling prior to diapedesis through endothelial cells (ECs). We found increased expression of DLC-1 in stiffness-associated diseases like atherosclerosis and pulmonary arterial hypertension. Depletion of DLC-1 in ECs cultured on stiff substrates drastically reduced cell stiffness and mimicked leukocyte transmigration kinetics observed for ECs cultured on soft substrates. Mechanistic studies revealed that DLC-1-depleted ECs or ECs cultured on soft substrates failed to recruit the actin-adaptor proteins filamin B, α-actinin-4, and cortactin to clustered ICAM-1, thereby preventing the ICAM-1 adhesome formation and impairing leukocyte spreading. This was rescued by overexpressing DLC-1, resulting in ICAM-1 adhesome stabilization and leukocyte spreading. Our results reveal an essential role for substrate stiffness-regulated endothelial DLC-1, independent of its GAP domain, in locally stabilizing the ICAM-1 adhesome to promote leukocyte spreading, essential for efficient leukocyte transendothelial migration.
Assuntos
Proteínas Ativadoras de GTPase/genética , Leucócitos/fisiologia , Migração Transendotelial e Transepitelial , Proteínas Supressoras de Tumor/genética , Rigidez Vascular , Células Cultivadas , Proteínas Ativadoras de GTPase/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/fisiologia , Humanos , Molécula 1 de Adesão Intercelular/metabolismo , Leucócitos/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas Supressoras de Tumor/metabolismoRESUMO
Vascular homoeostasis, development and disease critically depend on the regulation of endothelial cell-cell junctions. Here we uncover a new role for the F-BAR protein pacsin2 in the control of VE-cadherin-based endothelial adhesion. Pacsin2 concentrates at focal adherens junctions (FAJs) that are experiencing unbalanced actomyosin-based pulling. FAJs move in response to differences in local cytoskeletal geometry and pacsin2 is recruited consistently to the trailing end of fast-moving FAJs via a mechanism that requires an intact F-BAR domain. Photoconversion, photobleaching, immunofluorescence and super-resolution microscopy reveal polarized dynamics, and organization of junctional proteins between the front of FAJs and their trailing ends. Interestingly, pacsin2 recruitment inhibits internalization of the VE-cadherin complex from FAJ trailing ends and is important for endothelial monolayer integrity. Together, these findings reveal a novel junction protective mechanism during polarized trafficking of VE-cadherin, which supports barrier maintenance within dynamic endothelial tissue.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Junções Aderentes/metabolismo , Antígenos CD/metabolismo , Caderinas/metabolismo , Actomiosina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Antígenos CD/genética , Caderinas/genética , Adesões Focais/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Microscopia de Fluorescência/métodosRESUMO
Nuclear receptor Nur77, also referred to as NR4A1 or TR3, plays an important role in innate and adaptive immunity. Nur77 is crucial in regulating the T helper 1/regulatory T-cell balance, is expressed in macrophages and drives M2 macrophage polarization. In this study we aimed to define the function of Nur77 in inflammatory bowel disease. In wild-type and Nur77-/- mice, colitis development was studied in dextran sodium sulphate (DSS)- and 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced models. To understand the underlying mechanism, Nur77 was overexpressed in macrophages and gut epithelial cells. Nur77 protein is expressed in colon tissues from Crohn's disease and Ulcerative colitis patients and colons from colitic mice in inflammatory cells and epithelium. In both mouse colitis models inflammation was increased in Nur77-/- mice. A higher neutrophil influx and enhanced IL-6, MCP-1 and KC production was observed in Nur77-deficient colons after DSS-treatment. TNBS-induced influx of T-cells and inflammatory monocytes into the colon was higher in Nur77-/- mice, along with increased expression of MCP-1, TNFα and IL-6, and decreased Foxp3 RNA expression, compared to wild-type mice. Overexpression of Nur77 in lipopolysaccharide activated RAW macrophages resulted in up-regulated IL-10 and downregulated TNFα, MIF-1 and MCP-1 mRNA expression through NFκB repression. Nur77 also strongly decreased expression of MCP-1, CXCL1, IL-8, MIP-1α and TNFα in gut epithelial Caco-2 cells. Nur77 overexpression suppresses the inflammatory status of both macrophages and gut epithelial cells and together with the in vivo mouse data this supports that Nur77 has a protective function in experimental colitis. These findings may have implications for development of novel targeted treatment strategies regarding inflammatory bowel disease and other inflammatory diseases.
Assuntos
Colite Ulcerativa/metabolismo , Colite/metabolismo , Doença de Crohn/metabolismo , Macrófagos/metabolismo , NF-kappa B/metabolismo , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/imunologia , Animais , Linhagem Celular , Colite/induzido quimicamente , Colite/imunologia , Colite Ulcerativa/patologia , Colo/metabolismo , Colo/patologia , Doença de Crohn/patologia , Citocinas/biossíntese , Citocinas/genética , Sulfato de Dextrana/toxicidade , Modelos Animais de Doenças , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Feminino , Fatores de Transcrição Forkhead/biossíntese , Fatores de Transcrição Forkhead/genética , Regulação da Expressão Gênica , Humanos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/deficiência , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/genética , Células RAW 264.7 , Ácido Trinitrobenzenossulfônico/toxicidadeRESUMO
INTRODUCTION: The Rho family GTPase Rac1 regulates cytoskeletal rearrangements crucial for the recruitment, extravasation and activation of leukocytes at sites of inflammation. Rac1 signaling also promotes the activation and survival of lymphocytes and osteoclasts. Therefore, we assessed the ability of a cell-permeable Rac1 carboxy-terminal inhibitory peptide to modulate disease in mice with collagen-induced arthritis (CIA). METHODS: CIA was induced in DBA/1 mice, and in either early or chronic disease, mice were treated three times per week by intraperitoneal injection with control peptide or Rac1 inhibitory peptide. Effects on disease progression were assessed by measurement of paw swelling. Inflammation and joint destruction were examined by histology and radiology. Serum levels of anti-collagen type II antibodies were measured by enzyme-linked immunosorbent assay. T-cell phenotypes and activation were assessed by fluorescence-activated cell sorting analysis. Results were analyzed using Mann-Whitney U and unpaired Student t tests. RESULTS: Treatment of mice with Rac1 inhibitory peptide resulted in a decrease in paw swelling in early disease and to a lesser extent in more chronic arthritis. Of interest, while joint destruction was unaffected by Rac1 inhibitory peptide, anti-collagen type II antibody production was significantly diminished in treated mice, in both early and chronic arthritis. Ex vivo, Rac1 inhibitory peptide suppressed T-cell receptor/CD28-dependent production of tumor necrosis factor alpha, interferon gamma and interleukin-17 by T cells from collagen-primed mice, and reduced induction of ICOS and CD154, T-cell costimulatory proteins important for B-cell help. CONCLUSIONS: The data suggest that targeting of Rac1 with the Rac1 carboxy-terminal inhibitory peptide may suppress T-cell activation and autoantibody production in autoimmune disease. Whether this could translate into clinically meaningful improvement remains to be shown.
Assuntos
Antirreumáticos/uso terapêutico , Artrite Experimental/tratamento farmacológico , Artrite Reumatoide/tratamento farmacológico , Autoanticorpos/efeitos dos fármacos , Neuropeptídeos/antagonistas & inibidores , Proteínas rac de Ligação ao GTP/antagonistas & inibidores , Animais , Formação de Anticorpos/efeitos dos fármacos , Artrite Experimental/imunologia , Artrite Experimental/patologia , Artrite Reumatoide/imunologia , Artrite Reumatoide/patologia , Autoanticorpos/biossíntese , Autoanticorpos/imunologia , Linfócitos B/efeitos dos fármacos , Linfócitos B/imunologia , Linfócitos B/metabolismo , Colágeno Tipo II/imunologia , Citocinas/biossíntese , Citocinas/efeitos dos fármacos , Edema/tratamento farmacológico , Ensaio de Imunoadsorção Enzimática , Membro Posterior/efeitos dos fármacos , Membro Posterior/patologia , Ativação Linfocitária/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos DBA , Peptídeos , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Linfócitos T/metabolismo , Proteínas rac1 de Ligação ao GTPRESUMO
Cadherin-mediated cell-cell adhesion controls the morphology and function of epithelial cells and is a critical component of the pathology of chronic inflammatory disorders. Dynamic interactions between cadherins and the actin cytoskeleton are required for stable cell-cell contact. Besides actin, microtubules also target intercellular, cadherin-based junctions and contribute to their formation and stability. Here, we studied the role of microtubules in conjunction with Rho-like GTPases in the regulation of lung epithelial barrier function using real-time monitoring of transepithelial electrical resistance. Unexpectedly, we found that disruption of microtubules promotes epithelial cell-cell adhesion. This increase in epithelial barrier function is accompanied by the accumulation of beta-catenin at cell-cell junctions, as detected by immunofluorescence. Moreover, we found that the increase in cell-cell contact, induced by microtubule depolymerization, requires signaling through a RhoA/Rho kinase pathway. The Rac-1 GTPase counteracts this pathway, because inhibition of Rac-1 signaling rapidly promotes epithelial barrier function, in a microtubule- and RhoA-independent fashion. Together, our data suggest that microtubule-RhoA-mediated signaling and Rac-1 control lung epithelial integrity through counteracting independent pathways.