RESUMEN
Tight junctions (TJs) firmly seal epithelial cells and are key players in the epithelial barrier. TJs consist of several proteins, including those of the transmembrane claudin family and the scaffold zonula occludens (ZO) family. Epithelial tissues are exposed to different conditions: to air in the stratified epithelium of the skin and to liquids in the monolayer of the intestine. The TJs in stratified oral mucosal epithelium have remained insufficiently elucidated in terms of distributions, appearances and barrier functions of TJ proteins in normal buccal mucosa. We investigated these and ZO-1 and claudin-1 were found to be expressed in the top third and in the bottom three quarters of the mucosal epithelium. ZO-1 in the buccal mucosa was found to have an irregular linear appearance. ZO-1 in the buccal mucosa continuously existed in several layers. Electron microscopy revealed the buccal mucosa to have kissing points. In a biotin permeation assay that sought to investigate inside-outside barrier function, the biotin tracer penetrated several ZO-1 layers but did not pass through all the ZO-1 layers. We found that the oral mucosal cell knockdown of TJP1 or CLDN1 resulted in decreases of TER but no significant change in FITC-dextran leakage. Our results suggest that the distribution and appearance of ZO-1 in the buccal mucosa differ from those in the skin. We were unable to prove barrier function in this study but we did show barrier function against small molecules in vivo and against ions in vitro.
Asunto(s)
Claudina-1/metabolismo , Células Epiteliales , Mucosa Bucal , Proteína de la Zonula Occludens-1/fisiología , Anciano , Células Epiteliales/citología , Células Epiteliales/metabolismo , Femenino , Humanos , Masculino , Persona de Mediana Edad , Mucosa Bucal/citología , Mucosa Bucal/metabolismoRESUMEN
AIMS: Defective tight junctions (TJs) can induce intestinal epithelial dysfunction, which participates in various diseases such as irritable bowel syndrome. However, the mechanisms of TJ defects remain unclear. Our study revealed the role of Piezo1 in regulating intestinal epithelial function and TJs. MATERIALS AND METHODS: The human colonic adenocarcinoma cell line Caco-2 were cultured on Transwell plate to form an epithelial barrier in vitro, and Piezo1 expression was manipulated using a lentivirus vector. Epithelial function was evaluated by measuring transepithelial electronic resistance (TEER) and 4-kDa FITC-dextran (FD4) transmission. TJ proteins (claudin-1, occludin, ZO-1) were evaluated by RT-PCR, western blot, and immunostaining analysis. Potential signal pathways, including the ROCK and Erk pathways, were detected. Moreover, to explore the regulatory effect of Piezo1 activity on epithelial function, inhibitors (ruthenium red, GsMTx4) and an agonist (Yoda1) were introduced both ex vivo and in vitro. KEY FINDINGS: Alteration of Piezo1 expression altered epithelial function and the expression of the tight junction protein claudin-1. Piezo1 expression regulated phosphorylated ROCK1/2 expression, whereas interference on ROCK1/2 prevented the regulation of claudin-1 by Piezo1. In both Caco-2 monolayer and mouse colon epithelium, Piezo1 activity directly modulated epithelial function and permeability. SIGNIFICANCE: Piezo1 negatively regulates epithelial barrier function by affecting the expression of claudin-1. Such regulation may be achieved partially via the ROCK1/2 pathway. Moreover, activating Piezo1 can induce epithelial dysfunction.
Asunto(s)
Claudina-1/fisiología , Mucosa Intestinal/fisiología , Canales Iónicos/fisiología , Transducción de Señal , Quinasas Asociadas a rho/metabolismo , Animales , Western Blotting , Células CACO-2 , Claudina-1/metabolismo , Humanos , Mucosa Intestinal/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ocludina/metabolismo , Ocludina/fisiología , Reacción en Cadena en Tiempo Real de la Polimerasa , Transducción de Señal/fisiología , Proteína de la Zonula Occludens-1/metabolismo , Proteína de la Zonula Occludens-1/fisiologíaRESUMEN
The barrier function of epithelia and endothelia depends on tight junctions, which are formed by the polymerization of claudins on a scaffold of ZO proteins. Two differentially spliced isoforms of ZO-1 have been described, depending on the presence of the α domain, but the function of this domain is unclear. ZO-1 also contains a C-terminal ZU5 domain, which is involved in a mechano-sensitive intramolecular interaction with the central (ZPSG) region of ZO-1. Here we use immunoblotting and immunofluorescence to map the binding sites for commercially available monoclonal and polyclonal antibodies against ZO-1, and for a new polyclonal antibody (R3) that we developed against the ZO-1 C-terminus. We demonstrate that antibody R40.76 binds to the α domain, and the R3 antibody binds to the ZU5 domain. The (α+) isoform of ZO-1 shows higher expression in epithelial versus endothelial cells, and in differentiated versus undifferentiated primary keratinocytes, suggesting a link to epithelial differentiation and a potential molecular adaptation to junctions subjected to stronger mechanical forces. These results provide new tools and hypotheses to investigate the role of the α and ZU5 domains in ZO-1 mechano-sensing and dynamic interactions with the cytoskeleton and junctional ligands.
Asunto(s)
Epitelio/metabolismo , Queratinocitos/metabolismo , Uniones Estrechas/metabolismo , Proteína de la Zonula Occludens-1/fisiología , Animales , Diferenciación Celular , HumanosRESUMEN
The selective permeability of the blood-brain barrier (BBB) is controlled by tight junction-expressing brain endothelial cells. The integrity of these junctional proteins, which anchor to actin via zonula occludens (e.g., ZO-1), plays a vital role in barrier function. While disrupted junctions are linked with several neurodegenerative diseases, the mechanisms underlying disruption are not fully understood. This is largely due to the lack of appropriate models and efficient techniques to quantify edge-localized protein. Here, we developed a novel junction analyzer program (JAnaP) to semi-automate the quantification of junctional protein presentation. Because significant evidence suggests a link between myosin-II mediated contractility and endothelial barrier properties, we used the JAnaP to investigate how biochemical and physical cues associated with altered contractility influence ZO-1 presentation in brain endothelial cells. Treatment with contractility-decreasing agents increased continuous ZO-1 presentation; however, this increase was greatest on soft gels of brain-relevant stiffness, suggesting improved barrier maturation. This effect was reversed by biochemically inhibiting protein phosphatases to increase cell contractility on soft substrates. These results promote the use of brain-mimetic substrate stiffness in BBB model design and motivates the use of this novel JAnaP to provide insight into the role of junctional protein presentation in BBB physiology and pathologies.
Asunto(s)
Encéfalo/citología , Células Endoteliales/fisiología , Uniones Intercelulares/fisiología , Proteína de la Zonula Occludens-1/fisiología , Células Cultivadas , Humanos , FenotipoRESUMEN
Glomerular podocytes in the kidney originate from columnar epithelial cells possessing tight junctions. During podocyte differentiation, tight junctions are replaced by slit diaphragms, which are formed between foot processes and function as a blood filtration barrier. Although the expression of most tight junction components is suppressed during podocyte differentiation, several components, including ZO-1 and ZO-2, are consistently expressed. We recently showed that podocyte-specific deletion of ZO-1 gene impaired slit diaphragm formation, leading to proteinuria and glomerular sclerosis. Here, we address the relevance of ZO-2, whose sequence is highly similar to ZO-1, in the maintenance of the structure and function of podocytes. In glomerular development, the spatiotemporal expression of ZO-2 was similar to that of ZO-1 until the capillary loop stage. Subsequently, the distribution patterns of ZO-1 and ZO-2 diverged at the maturation stage, when slit diaphragms are formed. This divergence could partly rely on the ability of ZO-2 to interact with the slit diaphragm membrane proteins. Podocyte-specific deletion of the ZO-2 gene did not cause overt defects; however, double knockout of ZO-1 and ZO-2 genes accelerated the defects observed in ZO-1 knockout mice. These results suggest that ZO-2 plays supportive roles in the ZO-1-dependent regulation of podocyte filtration barrier.
Asunto(s)
Podocitos/metabolismo , Proteína de la Zonula Occludens-1/fisiología , Proteína de la Zonula Occludens-2/fisiología , Animales , Células COS , Diferenciación Celular , Línea Celular , Chlorocebus aethiops , Células Epiteliales/metabolismo , Regulación de la Expresión Génica/genética , Uniones Intercelulares , Riñón/metabolismo , Enfermedades Renales/metabolismo , Glomérulos Renales/metabolismo , Proteínas de la Membrana/metabolismo , Ratones , Ratones Noqueados , Podocitos/fisiología , Uniones Estrechas/genética , Uniones Estrechas/metabolismo , Uniones Estrechas/fisiología , Proteína de la Zonula Occludens-1/genética , Proteína de la Zonula Occludens-1/metabolismo , Proteína de la Zonula Occludens-2/genética , Proteína de la Zonula Occludens-2/metabolismoRESUMEN
Mucosal surfaces are lined by epithelial cells. In the intestine, the epithelium establishes a selectively permeable barrier that supports nutrient absorption and waste secretion while preventing intrusion by luminal materials. Intestinal epithelia therefore play a central role in regulating interactions between the mucosal immune system and luminal contents, which include dietary antigens, a diverse intestinal microbiome, and pathogens. The paracellular space is sealed by the tight junction, which is maintained by a complex network of protein interactions. Tight junction dysfunction has been linked to a variety of local and systemic diseases. Two molecularly and biophysically distinct pathways across the intestinal tight junction are selectively and differentially regulated by inflammatory stimuli. This review discusses the mechanisms underlying these events, their impact on disease, and the potential of using these as paradigms for development of tight junction-targeted therapeutic interventions.
Asunto(s)
Uniones Estrechas/fisiología , Animales , Humanos , Inmunidad Mucosa , Interleucina-13/fisiología , Mucosa Intestinal/anatomía & histología , Membrana Mucosa/anatomía & histología , Quinasa de Cadena Ligera de Miosina/fisiología , Permeabilidad , Uniones Estrechas/química , Proteína de la Zonula Occludens-1/fisiologíaRESUMEN
OBJECTIVES/HYPOTHESIS: The vocal fold epithelium that includes tight junction (TJ)-based barrier function protects underlying connective tissues from external insults. TJs play an important role to control paracellular permeability of not only solutes but also ions, and preserve the vocal fold homeostasis. However, the distribution of TJs and paracellular diffusion barrier across the entire vocal fold epithelium are still unknown. The aim of this study was to identify the distribution of TJs in the vocal fold epithelium and to characterize the recovery process of TJ-based paracellular diffusion barrier in a rat model of vocal fold injury. STUDY DESIGN: Animal experiments with controls. METHODS: Normal and vocal fold-injured rats were used. Larynges were harvested for immunohistochemical examination of TJ proteins. For functional analysis, a tracer permeability assay was performed using EZ-Link Sulfo-NHS-LC-Biotin. RESULTS: TJ proteins occludin and zonula occludens 1 signals were localized to the junctional regions of the most luminal cell layers of the vocal fold epithelium. The injured region had been recovered with epithelium at 5 days postinjury, but the paracellular diffusion barrier assays revealed that biotinylation reagents diffused into the lamina propria at 5 days postinjury, and were blocked at the epithelium at 14 and 28 days postinjury. CONCLUSIONS: It was strongly suggested that TJs in the vocal fold epithelium exist at the junctional regions of the first layer of stratified squamous epithelium. TJ-based paracellular diffusion barrier following vocal fold injury is recovered by 14 days postinjury, and this period corresponds with the time course of structural changes in the regenerating epithelium layer. LEVEL OF EVIDENCE: NA. Laryngoscope, 128:E150-E156, 2018.
Asunto(s)
Uniones Estrechas/fisiología , Pliegues Vocales/lesiones , Pliegues Vocales/metabolismo , Animales , Epitelio/lesiones , Epitelio/metabolismo , Masculino , Ocludina/fisiología , Permeabilidad , Ratas , Ratas Sprague-Dawley , Proteína de la Zonula Occludens-1/fisiologíaRESUMEN
Neuronal synaptic connections are either chemical or electrical, and these two types of synapses work together to dynamically define neural circuit function [1]. Although we know a great deal about the molecules that support chemical synapse formation and function, we know little about the macromolecular complexes that regulate electrical synapses. Electrical synapses are created by gap junction (GJ) channels that provide direct ionic communication between neurons [2]. Although they are often molecularly and functionally symmetric, recent work has found that pre- and postsynaptic neurons can contribute different GJ-forming proteins, creating molecularly asymmetric channels that are correlated with functional asymmetry at the synapse [3, 4]. Associated with the GJs are structures observed by electron microscopy termed the electrical synapse density (ESD) [5]. The ESD has been suggested to be critical for the formation and function of the electrical synapse, yet the biochemical makeup of these structures is poorly understood. Here we find that electrical synapse formation in vivo requires an intracellular scaffold called Tight Junction Protein 1b (Tjp1b). Tjp1b is localized to the electrical synapse, where it is required for the stabilization of the GJs and for electrical synapse function. Strikingly, we find that Tjp1b protein localizes and functions asymmetrically, exclusively on the postsynaptic side of the synapse. Our findings support a novel model of electrical synapse molecular asymmetry at the level of an intracellular scaffold that is required for building the electrical synapse. We propose that such ESD asymmetries could be used by all nervous systems to support molecular and functional asymmetries at electrical synapses.
Asunto(s)
Sinapsis/fisiología , Proteína de la Zonula Occludens-1/metabolismo , Proteína de la Zonula Occludens-1/fisiología , Animales , Conexinas/metabolismo , Sinapsis Eléctricas/fisiología , Uniones Comunicantes/metabolismo , Canales Iónicos/metabolismo , Sistema Nervioso , Neuronas/fisiología , Uniones Estrechas/metabolismo , Uniones Estrechas/fisiología , Vertebrados/metabolismo , Pez Cebra/crecimiento & desarrollo , Pez Cebra/fisiología , Proteínas de Pez Cebra/metabolismoRESUMEN
To investigate whether connexin phosphorylation regulates the known role of zonula occludens-1 protein (ZO-1) in gap junction (GJ) function, we generated and analyzed a series of phosphomimetic and phosphorylation-dead mutants by mutating known conserved regulatory serine (S) residues 255, 279/282, 365, 368, and 373 located in the C-terminal domain of connexin43 (Cx43) into glutamic acid (E) or alanine (A) residues. All connexin mutants were translated into stable, full-length proteins and assembled into GJs when expressed in HeLa or Madin-Darby canine kidney epithelial cells. However, mutants with S residues exchanged at positions 365, 368, and 373 exhibited a significantly altered ZO-1 interaction profile, while mutants with S residues exchanged at 255 and 279/282 did not. Unlike wild-type Cx43, in which ZO-1 binding is restricted to the periphery of GJ plaques, S365A, S365E, S368A, S368E, and S373A mutants bound ZO-1 throughout the GJ plaques, while the S373E mutant did not bind ZO-1 at all. Inability to disengage from ZO-1 correlated with increased GJ plaque size and increased connexin protein half-life, while maintaining GJ channels in an open, functional state. Quantitative clathrin-binding analyses revealed no significant alterations in clathrin-binding efficiency, suggesting that the inability to disengage from ZO-1 prevented maturation of functional into nonfunctional/endocytic channels, rather than ZO-1 interfering with GJ endocytosis directly. Collectively, our results indicate that ZO-1 binding regulates channel accrual, while disengagement from ZO-1 is critical for GJ channel closure and transitioning GJ channels for endocytosis. Intriguingly, these transitional ZO-1 binding/release and channel-aging steps are mediated by a series of hierarchical phosphorylation/dephosphorylation events at S373, S365, and S368, well-known Cx43 Akt, protein kinase A, and protein kinase C phosphorylation sites located in the vicinity of the ZO-1 binding site.
Asunto(s)
Conexina 43/metabolismo , Uniones Comunicantes/fisiología , Proteína de la Zonula Occludens-1/metabolismo , Animales , Sitios de Unión , Conexina 43/genética , Conexina 43/fisiología , Conexinas/metabolismo , Perros , Endocitosis , Células HeLa , Humanos , Células de Riñón Canino Madin Darby , Fosforilación/fisiología , Unión Proteica , Proteolisis , Proteína de la Zonula Occludens-1/fisiologíaRESUMEN
Fundamental processes in cell adhesion, motility, and rigidity adaptation are regulated by integrin-mediated adhesion to the extracellular matrix (ECM). The link between the ECM component fibronectin (fn) and integrin α5ß1 forms a complex with ZO-1 in cells at the edge of migrating monolayers, regulating cell migration. However, how this complex affects the α5ß1-fn link is unknown. Here we show that the α5ß1/ZO-1 complex decreases the resistance to force of α5ß1-fn adhesions located at the edge of migrating cell monolayers while also increasing α5ß1 recruitment. Consistently with a molecular clutch model of adhesion, this effect of ZO-1 leads to a decrease in the density and intensity of adhesions in cells at the edge of migrating monolayers. Taken together, our results unveil a new mode of integrin regulation through modification of the mechanical properties of integrin-ECM links, which may be harnessed by cells to control adhesion and migration.
Asunto(s)
Integrina alfa5beta1/metabolismo , Proteína de la Zonula Occludens-1/metabolismo , Animales , Células CHO , Adhesión Celular/fisiología , Movimiento Celular , Cricetulus , Matriz Extracelular/metabolismo , Fibronectinas/metabolismo , Fibronectinas/fisiología , Humanos , Integrina alfa5beta1/fisiología , Integrinas/metabolismo , Mecanotransducción Celular/fisiología , Unión Proteica , Proteína de la Zonula Occludens-1/fisiologíaRESUMEN
OCTN2 (SLC22A5) is an organic cation/carnitine transporter belonging to the solute carrier transporters (SLC) family. OCTN2 is ubiquitously expressed and its presence was shown in various brain cells, including the endothelial cells forming blood-brain barrier, where it was mainly detected at abluminal membrane and in proximity of tight junctions (TJ). Since OCTN2 contains a PDZ-binding domain, the present study was focused on a possible role of transporter interaction with a TJ-associated protein ZO-1, containing PDZ domains and detected in rat Octn2 proteome. We showed previously that activation of protein kinase C (PKC) in rat astrocytes regulates Octn2 surface presence and activity. Regulation of a wild type Octn2 and its deletion mutant without a PDZ binding motif were studied in heterologous expression system in HEK293 cells. Plasma membrane presence of overexpressed Octn2 did not depend on either PKC activation or presence of PDZ-binding motif, anyhow, as assayed in proximity ligation assay, the truncation of PDZ binding motif resulted in a strongly diminished Octn2/ZO-1 interaction and in a decreased transporter activity. The same effects on Octn2 activity were detected upon PKC activation, what correlated with ZO-1 phosphorylation. It is postulated that ZO-1, when not phosphorylated by PKC, keeps Octn2 in an active state, while elimination of this binding in ΔPDZ mutant or after ZO-1 phosphorylation leads to diminution of Octn2 activity.
Asunto(s)
Proteínas de Transporte de Catión Orgánico/metabolismo , Proteína Quinasa C/metabolismo , Proteína de la Zonula Occludens-1/fisiología , Animales , Perros , Células HEK293 , Humanos , Células de Riñón Canino Madin Darby , Fosforilación , Unión Proteica , Transducción de Señal , Miembro 5 de la Familia 22 de Transportadores de SolutosRESUMEN
The organization and integrity of epithelial tight junctions depend on interactions between claudins, ZO scaffolding proteins, and the cytoskeleton. However, although binding between claudins and ZO-1/2/3 and between ZO-1/2/3 and numerous cytoskeletal proteins has been demonstrated in vitro, fluorescence recovery after photobleaching analysis suggests interactions in vivo are likely highly dynamic. Here we use superresolution live-cell imaging in a model fibroblast system to examine relationships between claudins, ZO-1, occludin, and actin. We find that GFP claudins make easily visualized dynamic strand patches between two fibroblasts; strand dynamics is constrained by ZO-1 binding. Claudin association with actin is also dependent on ZO-1, but colocalization demonstrates intermittent rather than continuous association between claudin, ZO-1, and actin. Independent of interaction with ZO-1 or actin, claudin strands break and reanneal; pulse-chase-pulse analysis using SNAP-tagged claudins showed preferential incorporation of newly synthesized claudins into break sites. Although claudin strand behavior in fibroblasts may not fully recapitulate that of epithelial tight junction strands, this is the first direct demonstration of the ability of ZO-1 to stabilize claudin strands. We speculate that intermittent tethering of claudins to actin may allow for accommodation of the paracellular seal to physiological or pathological alterations in cell shape or movement.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Proteína de la Zonula Occludens-1/metabolismo , Proteína de la Zonula Occludens-1/fisiología , Actinas/metabolismo , Animales , Línea Celular , Claudina-1/metabolismo , Claudinas/metabolismo , Proteínas del Citoesqueleto/metabolismo , Citoesqueleto/fisiología , Células HEK293 , Humanos , Proteínas de la Membrana/metabolismo , Imagen Molecular/métodos , Ocludina/metabolismo , Fosfoproteínas/metabolismo , Ratas , Uniones Estrechas/metabolismoRESUMEN
In S334ter-line-3 rat model of Retinitis Pigmentosa (RP), rod cell death induces the rearrangement of cones into mosaics of rings while the fibrotic processes of Müller cells remodel to fill the center of the rings. In contrast, previous work established that DL-alpha-aminoadipic-acid (AAA), a compound that transiently blocks Müller cell metabolism, abolishes these highly structured cone rings. Simultaneously, adherens-junction associated protein, Zonula occludens-1 (ZO-1) expression forms in a network between the photoreceptor segments and Müller cells processes. Thus, we hypothesized that AAA treatment alters the cone mosaic rings by disrupting the distal sealing formed by these fibrotic processes, either directly or indirectly, by down regulating the expression of ZO-1. Therefore, we examined these processes and ZO-1 expression at the outer retina after intravitreal injection of AAA and observed that AAA treatment transiently disrupts the distal glial sealing in RP retina, plus induces cones in rings to become more homogeneous. Moreover, ZO-1 expression is actively suppressed after 3 days of AAA treatment, which coincided with cone ring disruption. Similar modifications of glial sealing and cone distribution were observed after injection of siRNA to inhibit ZO-1 expression. These findings support our hypothesis and provide additional information about the critical role played by ZO-1 in glial sealing and shaping the ring mosaic in RP retina. These studies represent important advancements in the understanding of retinal degeneration's etiology and pathophysiology.
Asunto(s)
Ácido 2-Aminoadípico/farmacología , Células Ependimogliales/fisiología , Células Fotorreceptoras Retinianas Conos/patología , Retinitis Pigmentosa/fisiopatología , Proteína de la Zonula Occludens-1/fisiología , Ácido 2-Aminoadípico/administración & dosificación , Animales , Muerte Celular , Modelos Animales de Enfermedad , Regulación hacia Abajo/efectos de los fármacos , Células Ependimogliales/efectos de los fármacos , Femenino , Fibrosis , Filamentos Intermedios/metabolismo , Inyecciones Intravítreas , Masculino , Opsinas/deficiencia , Opsinas/genética , Interferencia de ARN , ARN Interferente Pequeño/genética , Ratas , Ratas Mutantes , Ratas Sprague-Dawley , Retinitis Pigmentosa/patología , Transgenes , Proteína de la Zonula Occludens-1/antagonistas & inhibidoresRESUMEN
The tight junction-based paracellular pathway plays an important role in saliva secretion. Zonula occludens (ZO) proteins are submembranous proteins of tight junction complex; however, their function in salivary epithelium is poorly understood. Here, we found that activation of transient receptor potential vanilloid subtype 1 (TRPV1) by capsaicin increased rat saliva secretion both in vivo and ex vivo. Meanwhile, TRPV1 activation enlarged the width of tight junctions between neighboring acinar cells, increased the paracellular ï¬ux of 4-kDa fluorescein isothiocyanate (FITC)-dextran in submandibular gland (SMG) tissues, and decreased transepithelial electric resistance (TER) in SMG-C6 cells. ZO-1, -2, and -3 were distributed principally to the apical lateral region of acinar cells in SMG tissues and continuously encircled the peripheries of SMG-C6 cells in the untreated condition. TRPV1 activation obviously diminished ZO-1 and -2 staining, but not ZO-3 or ß-catenin, at the cell-cell contacts ex vivo and in vitro. Moreover, in untreated SMG-C6 cells, ZO-1 and -2 single or double knockdown by small interfering RNA (siRNA) increased the paracellular ï¬ux of 4-kDa FITC-dextran. In capsaicin-treated cells, ZO-1 and -2 single or double knockdown abolished, whereas their re-expression restored, the capsaicin-induced increase in paracellular permeability. Furthermore, TRPV1 activation increased RhoA activity, and inhibition of either RhoA or Rho kinase (ROCK) abolished the capsaicin-induced TER decrease as well as ZO-1 and -2 redistribution. These results indicate that ZO-1 and -2 play crucial roles in both basal salivary epithelial barrier function and TRPV1-modulated paracellular transport. RhoA-ROCK signaling pathway is responsible for TRPV1-modulated paracellular permeability as well as ZO-1 and -2 redistribution.
Asunto(s)
Glándula Submandibular/fisiología , Canales Catiónicos TRPV/fisiología , Uniones Estrechas/fisiología , Proteína de la Zonula Occludens-1/fisiología , Proteína de la Zonula Occludens-2/fisiología , Animales , Western Blotting , Permeabilidad de la Membrana Celular/fisiología , Epitelio , Técnicas de Silenciamiento del Gen , Masculino , Microscopía Electrónica de Transmisión , Reacción en Cadena de la Polimerasa , Ratas , Ratas Sprague-Dawley , Salivación/fisiologíaRESUMEN
Intercellular junctions are crucial for mechanotransduction, but whether tight junctions contribute to the regulation of cell-cell tension and adherens junctions is unknown. Here, we demonstrate that the tight junction protein ZO-1 regulates tension acting on VE-cadherin-based adherens junctions, cell migration, and barrier formation of primary endothelial cells, as well as angiogenesis in vitro and in vivo. ZO-1 depletion led to tight junction disruption, redistribution of active myosin II from junctions to stress fibers, reduced tension on VE-cadherin and loss of junctional mechanotransducers such as vinculin and PAK2, and induced vinculin dissociation from the α-catenin-VE-cadherin complex. Claudin-5 depletion only mimicked ZO-1 effects on barrier formation, whereas the effects on mechanotransducers were rescued by inhibition of ROCK and phenocopied by JAM-A, JACOP, or p114RhoGEF down-regulation. ZO-1 was required for junctional recruitment of JACOP, which, in turn, recruited p114RhoGEF. ZO-1 is thus a central regulator of VE-cadherin-dependent endothelial junctions that orchestrates the spatial actomyosin organization, tuning cell-cell tension, migration, angiogenesis, and barrier formation.
Asunto(s)
Uniones Adherentes/metabolismo , Permeabilidad Capilar , Células Endoteliales/fisiología , Neovascularización Fisiológica , Proteína de la Zonula Occludens-1/fisiología , Actomiosina/metabolismo , Animales , Antígenos CD/metabolismo , Cadherinas/metabolismo , Moléculas de Adhesión Celular/metabolismo , Movimiento Celular , Células Cultivadas , Claudina-5/metabolismo , Proteínas del Citoesqueleto/metabolismo , Citoesqueleto/metabolismo , Humanos , Mecanotransducción Celular , Ratones Endogámicos C57BL , Miosinas/metabolismo , Transporte de Proteínas , Receptores de Superficie Celular/metabolismo , Uniones Estrechas/metabolismoAsunto(s)
Moléculas de Adhesión Celular/fisiología , Emparejamiento Cromosómico/fisiología , Neuritas/fisiología , Animales , Cadherinas/fisiología , Humanos , Ratones , Nectinas , Enfermedades del Sistema Nervioso/fisiopatología , Seudópodos/fisiología , Proteína de la Zonula Occludens-1/fisiologíaRESUMEN
The discovery that PSD-95/Discs large/ZO-1 (PDZ) domains can function as lipid-binding modules, in particular interacting with phosphoinositides (PIs), was made more than 10 years ago (Mol Cell 9(6): 1215-1225, 2002). Confirmatory studies and a series of functional follow-ups established PDZ domains as dual specificity modules displaying both peptide and lipid binding, and prompted a rethinking of the mode of action of PDZ domains in the control of cell signaling. In this chapter, after introducing PDZ domains, PIs and methods for studying protein-lipid interactions, we focus on (i) the prevalence and the specificity of PDZ-PIs interactions, (ii) the molecular determinants of PDZ-PIs interactions, (iii) the integration of lipid and peptide binding by PDZ domains, (iv) the common features of PIs interacting PDZ domains and (v) the regulation and functional significance of PDZ-PIs interactions.
Asunto(s)
Dominios PDZ/fisiología , Fosfatidilinositoles/química , Proteínas Adaptadoras Transductoras de Señales , Animales , Proteínas de Unión al Calcio/química , Proteínas de Unión al Calcio/fisiología , Proteínas Portadoras/química , Proteínas Portadoras/fisiología , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/fisiología , Humanos , Proteínas de la Membrana/química , Proteínas de la Membrana/fisiología , Proteínas Musculares/química , Proteínas Musculares/fisiología , Proteínas Nucleares/química , Proteínas Nucleares/fisiología , Fosfatidilinositoles/fisiología , Sinteninas/química , Sinteninas/fisiología , Proteína de la Zonula Occludens-1/química , Proteína de la Zonula Occludens-1/fisiologíaRESUMEN
The tight junction is a multi-protein complex and is the apical most junctional complex in certain epithelial and endothelial cells. A great deal of attention has been devoted to the understanding of these proteins in contributing to the barrier function - that is, regulating the paracellular flux or permeability between adjacent cells. However, tight junction proteins are now recognized as having functions beyond the barrier. The focus of this review is to discuss the barrier function of the tight junction and to summarize the literature with a focus on the role of tight junction proteins in proliferation, transformation, and metastasis.
Asunto(s)
Transformación Celular Neoplásica , Neoplasias/etiología , Proteínas de Uniones Estrechas/fisiología , Animales , Claudinas/fisiología , Humanos , Ocludina/fisiología , Uniones Estrechas/fisiología , Proteína de la Zonula Occludens-1/fisiologíaRESUMEN
Tight junctions (TJs) form a selective barrier for ions, water, and macromolecules in simple epithelia. In keratinocytes and epidermis, TJs were shown to be involved in individual barrier functions. The absence of the TJ protein claudin-1 (Cldn1) in mice results in a skin-barrier defect characterized by lethal water loss. However, detailed molecular analyses of the various TJ barriers in keratinocytes and the contribution of distinct TJ proteins are missing. Herein, we discriminate TJ-dependent paracellular resistance from transcellular resistance in cultured keratinocytes using the two-path impedance spectroscopy. We demonstrate that keratinocyte TJs form a barrier for Na(+), Cl(-), and Ca(2+), and contribute to barrier function for water and larger molecules of different size. In addition, knockdown of Cldn1, Cldn4, occludin, and zonula occludens-1 increased paracellular permeabilities for ions and larger molecules, demonstrating that all of these TJ proteins contribute to barrier formation. Remarkably, Cldn1 and Cldn4 are not critical for TJ barrier function for water in submerged keratinocyte cultures. However, Cldn1 influences stratum corneum (SC) proteins important for SC water barrier function, and is crucial for TJ barrier formation for allergen-sized macromolecules.
Asunto(s)
Permeabilidad de la Membrana Celular/fisiología , Iones/metabolismo , Queratinocitos/metabolismo , Sustancias Macromoleculares/metabolismo , Proteínas de Uniones Estrechas/fisiología , Uniones Estrechas/fisiología , Agua/metabolismo , Animales , Células Cultivadas , Claudina-1/deficiencia , Claudina-1/genética , Claudina-1/fisiología , Claudina-4/deficiencia , Claudina-4/genética , Claudina-4/fisiología , Queratinocitos/citología , Masculino , Ratones , Ratones Noqueados , Modelos Animales , Ocludina/deficiencia , Ocludina/genética , Ocludina/fisiología , Proteínas de Uniones Estrechas/deficiencia , Proteínas de Uniones Estrechas/genética , Proteína de la Zonula Occludens-1/deficiencia , Proteína de la Zonula Occludens-1/genética , Proteína de la Zonula Occludens-1/fisiologíaRESUMEN
In many gut chronic inflammatory conditions, intestinal epithelium (IE) is deprived of the protection of the mucus secreted by IE-specialized cells. In these events, bleeding and subsequent lysis of erythrocytes are common. This may lead to the release of high amounts of heme in the intestinal lumen, which interacts with IE. Previous works from our group have shown that heme itself is a proinflammatory molecule, activating a number of phlogistic signaling events in a nicotinamide adenine dinucleotide phosphate oxidase (NADPHox)-dependent manner. In this study, we aim to evaluate the effects of heme upon a well-established nontransformed small intestine epithelial cell lineage (IEC 6). Our results show that free heme evokes intracellular reactive oxygen species (ROS) production by IEC 6 cells, which is inhibited both by pharmacological inhibition with diphenyleneiodonium (10 µM), a NADPHox inhibitor, and small interfering RNA-mediated suppression of NOX1, a constitutive NADPHox isoform present in intestinal epithelial cells. Focal adhesion kinase phosphorylation and actin cytoskeleton polymerization are also induced by heme in a NADPHox-dependent manner. Heme increases monolayer permeability and redistributes key modulators of cell-cell adhesion as zona occludens-1 and E-cadherin proteins via NADPHox signaling. Heme promotes IEC 6 cell migration and proliferation, phenomena also regulated by NADPHox-derived ROS. Heme, in NADPHox-activating concentrations, is able to induce mRNA expression of IL-6, a cytokine implicated in inflammatory and tumorigenic responses. These data indicate a prominent role for heme-derived signaling in the pathophysiology of intestinal mucosa dysfunction and address an important role of NADPHox activity on the pathogenesis of intestinal inflammatory conditions.