RESUMO
Tight junctions act as a barrier between epithelial cells to limit the transport of the paracellular substance, which is a required function in various tissues to sequestrate diverse microenvironments and maintain a normal physiological state. Tight junctions are complexes that contain various proteins, like transmembrane proteins, scaffolding proteins, signaling proteins, etc. Defects in those tight junction- related proteins can lead to hearing loss in humans which is also recapitulated in many model organisms. The disruption of the barrier between the endolymph and perilymph caused by tight junction abnormalities will affect the microenvironment of hair cells; and this could be the reason for this type of hearing loss. Besides their functions as a typical barrier and channel, tight junctions are also involved in many signaling networks to regulate gene expression, cell proliferation, and differentiation. This review will summarize the structures, localization, and related signaling pathways of hearingrelated tight junction proteins and their potential contributions to the hearing disorder.
Assuntos
Perda Auditiva , Junções Íntimas , Humanos , Junções Íntimas/química , Junções Íntimas/genética , Junções Íntimas/metabolismo , Perda Auditiva/genética , Perda Auditiva/metabolismo , Proteínas de Junções Íntimas/genética , Proteínas de Junções Íntimas/metabolismo , Células Epiteliais/metabolismoRESUMO
BACKGROUND: Metastasis is the worst prognostic variable of patients with colorectal cancer (CRC). For the development of metastases, it is necessary that cancer cells detach from the primary tumor, migrate into the angiolymphatic system, and invade the tissue where they will develop. The breakdown of the tight junctions (TJs) plays an important role in colorectal metastatic tumors. Claudin-3 and occludin are the main component proteins of TJs. AIM: To analyze the expression and tissue content of claudin-3 and occludin in normal and neoplastic tissues of patients with metastatic CRC. METHODS: Fifty-seven consecutive patients with stage III and IV CRC were included. Fragments of neoplastic tissue were collected from the tumor margins, and samples of the normal tissue were collected from the same patient in a standardized distance of 10 cm from the cranial margin of the tumor. Immunohistochemistry technique was used to identify the tissue staining of claudin-3 and occludin. To measure the content of both proteins in cellular membranes of normal and cancer cells, a validated immunoscore was used. RESULTS: Claudin-3 and occludin in normal tissues are in the apical and lateral membranes of cells, while in the neoplastic, in cytoplasm. The mean of the tissue content of claudin-3 in the normal tissue was 2.57 ± 0.16, while in the neoplastic tissue was 1.03 ± 0.13. The contents of occludin were 2.77 ± 0.1 in normal tissue, while in the neoplastic were 1.08 ± 0.14. CONCLUSION: There is a reduction in the content of the claudin-3 and occludin in the cell membranes of the neoplastic tissue in patients with CRC.
Assuntos
Neoplasias Colorretais , Junções Íntimas , Humanos , Ocludina/análise , Ocludina/metabolismo , Claudina-3/análise , Claudina-3/metabolismo , Claudina-1/análise , Claudina-1/metabolismo , Junções Íntimas/química , Junções Íntimas/metabolismo , Junções Íntimas/patologia , Neoplasias Colorretais/patologiaRESUMO
Tight junctions (TJs) are essential components of epithelial tissues connecting neighboring cells to provide protective barriers. While their general function to seal compartments is well understood, their role in collective cell migration is largely unexplored. Here, the importance of the TJ zonula occludens (ZO) proteins ZO1 and ZO2 for epithelial migration is investigated employing video microscopy in conjunction with velocimetry, segmentation, cell tracking, and atomic force microscopy/spectroscopy. The results indicate that ZO proteins are necessary for fast and coherent migration. In particular, ZO1 and 2 loss (dKD) induces actomyosin remodeling away from the central cortex towards the periphery of individual cells, resulting in altered viscoelastic properties. A tug-of-war emerges between two subpopulations of cells with distinct morphological and mechanical properties: 1) smaller and highly contractile cells with an outward bulging apical membrane, and 2) larger, flattened cells, which, due to tensile stress, display a higher proliferation rate. In response, the cell density increases, leading to crowding-induced jamming and more small cells over time. Co-cultures comprising wildtype and dKD cells migrate inefficiently due to phase separation based on differences in contractility rather than differential adhesion. This study shows that ZO proteins are necessary for efficient collective cell migration by maintaining tissue fluidity and controlling proliferation.
Assuntos
Movimento Celular/fisiologia , Junções Íntimas/química , Junções Íntimas/metabolismo , Proteínas da Zônula de Oclusão/química , Proteínas da Zônula de Oclusão/metabolismo , Animais , Linhagem Celular , Cães , Células Epiteliais/química , Células Epiteliais/metabolismo , Epitélio/química , Epitélio/metabolismoRESUMO
Tight junctions (TJs) are intercellular junctions critical for building the epithelial barrier and maintaining epithelial polarity. The claudin family of membrane proteins play central roles in TJ structure and function. However, recent findings have uncovered claudin-independent aspects of TJ structure and function, and additional players including junctional adhesion molecules (JAMs), membrane lipids, phase separation of the zonula occludens (ZO) family of scaffolding proteins, and mechanical force have been shown to play important roles in TJ structure and function. In this review, we discuss how these new findings have the potential to transform our understanding of TJ structure and function, and how the intricate network of TJ proteins and membrane lipids dynamically interact to drive TJ assembly.
Assuntos
Junções Íntimas/química , Junções Íntimas/metabolismo , Animais , Polaridade Celular , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Humanos , Modelos BiológicosRESUMO
The Envelope (E) protein of SARS-CoV-2 is the most enigmatic protein among the four structural ones. Most of its current knowledge is based on the direct comparison to the SARS E protein, initially mistakenly undervalued and subsequently proved to be a key factor in the ER-Golgi localization and in tight junction disruption. We compared the genomic sequences of E protein of SARS-CoV-2, SARS-CoV and the closely related genomes of bats and pangolins obtained from the GISAID and GenBank databases. When compared to the known SARS E protein, we observed a significant difference in amino acid sequence in the C-terminal end of SARS-CoV-2 E protein. Subsequently, in silico modelling analyses of E proteins conformation and docking provide evidences of a strengthened binding of SARS-CoV-2 E protein with the tight junction-associated PALS1 protein. Based on our computational evidences and on data related to SARS-CoV, we believe that SARS-CoV-2 E protein interferes more stably with PALS1 leading to an enhanced epithelial barrier disruption, amplifying the inflammatory processes, and promoting tissue remodelling. These findings raise a warning on the underestimated role of the E protein in the pathogenic mechanism and open the route to detailed experimental investigations.
Assuntos
COVID-19/metabolismo , Proteínas de Membrana/química , Núcleosídeo-Fosfato Quinase/química , SARS-CoV-2/química , Junções Íntimas/química , Proteínas do Envelope Viral/química , Sequência de Aminoácidos , Animais , COVID-19/genética , Quirópteros/virologia , Bases de Dados Genéticas , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Simulação de Dinâmica Molecular , Núcleosídeo-Fosfato Quinase/genética , Núcleosídeo-Fosfato Quinase/metabolismo , Pangolins/virologia , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/química , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/genética , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/metabolismo , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , SARS-CoV-2/patogenicidade , Junções Íntimas/metabolismo , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismoRESUMO
Epithelial and endothelial monolayers are multicellular sheets that form barriers between the 'outside' and 'inside' of tissues. Cell-cell junctions, made by adherens junctions, tight junctions and desmosomes, hold together these monolayers. They form intercellular contacts by binding their receptor counterparts on neighboring cells and anchoring these structures intracellularly to the cytoskeleton. During tissue development, maintenance and pathogenesis, monolayers encounter a range of mechanical forces from the cells themselves and from external systemic forces, such as blood pressure or tissue stiffness. The molecular landscape of cell-cell junctions is diverse, containing transmembrane proteins that form intercellular bonds and a variety of cytoplasmic proteins that remodel the junctional connection to the cytoskeleton. Many junction-associated proteins participate in mechanotransduction cascades to confer mechanical cues into cellular responses that allow monolayers to maintain their structural integrity. We will discuss force-dependent junctional molecular events and their role in cell-cell contact organization and remodeling.
Assuntos
Junções Aderentes/química , Junções Intercelulares/química , Fenômenos Mecânicos , Proteínas de Membrana/química , Junções Aderentes/genética , Pressão Sanguínea/genética , Citoesqueleto/química , Desmossomos/química , Desmossomos/genética , Células Endoteliais/química , Células Epiteliais/química , Humanos , Junções Intercelulares/genética , Proteínas de Membrana/genética , Junções Íntimas/química , Junções Íntimas/genéticaRESUMO
Epithelial barrier function is regulated by a family of transmembrane proteins known as claudins. Functional tight junctions are formed when claudins interact with other transmembrane proteins, cytosolic scaffold proteins and the actin cytoskeleton. The predominant scaffold protein, zonula occludens-1 (ZO-1), directly binds to most claudin C-terminal domains, crosslinking them to the actin cytoskeleton. When imaged by immunofluorescence microscopy, tight junctions most frequently are linear structures that form between tricellular junctions. However, tight junctions also adapt non-linear architectures exhibiting either a ruffled or spiked morphology, which both are responses to changes in claudin engagement of actin filaments. Other terms for ruffled tight junctions include wavy, tortuous, undulating, serpentine or zig-zag junctions. Ruffling is under the control of hypoxia induced factor (HIF) and integrin-mediated signaling, as well as direct mechanical stimulation. Tight junction ruffling is specifically enhanced by claudin-2, antagonized by claudin-1 and requires claudin binding to ZO-1. Tight junction spikes are sites of active vesicle budding and fusion that appear as perpendicular projections oriented towards the nucleus. Spikes share molecular features with focal adherens junctions and tubulobulbar complexes found in Sertoli cells. Lung epithelial cells under stress form spikes due to an increase in claudin-5 expression that directly disrupts claudin-18/ZO-1 interactions. Together this suggests that claudins are not simply passive cargoes controlled by scaffold proteins. We propose a model where claudins specifically influence tight junction scaffold proteins to control interactions with the cytoskeleton as a mechanism that regulates tight junction assembly and function.
Assuntos
Moléculas de Adesão Celular/genética , Membrana Celular/genética , Claudinas/genética , Junções Íntimas/genética , Citoesqueleto de Actina/química , Citoesqueleto de Actina/genética , Moléculas de Adesão Celular/química , Membrana Celular/química , Permeabilidade da Membrana Celular/genética , Claudinas/química , Células Epiteliais/metabolismo , Humanos , Junções Íntimas/químicaRESUMO
Tight junctions regulate paracellular permeability size and charge selectively. Models have been proposed for the molecular architecture of tight junction strands and paracellular channels. However, they are not fully consistent with experimental and structural data. Here, we analysed the architecture of claudin-based tight junction strands and channels by cellular reconstitution of strands, structure-guided mutagenesis, in silico protein docking and oligomer modelling. Prototypic channel- (Cldn10b) and barrier-forming (Cldn3) claudins were analysed. Förster resonance energy transfer (FRET) assays indicated multistep claudin polymerisation, starting with cis-oligomerization specific to the claudin subtype, followed by trans-interaction-triggered cis-polymerisation. Alternative protomer interfaces were modelled in silico and tested by cysteine-mediated crosslinking, confocal- and freeze fracture EM-based analysis of strand formation. The analysed claudin mutants included also mutations causing the HELIX syndrome. The results indicated that protomers in Cldn10b and Cldn3 strands form similar antiparallel double rows, as has been suggested for Cldn15. Mutually stabilising -hydrophilic and hydrophobic - cis- and trans-interfaces were identified that contained novel key residues of extracellular segments ECS1 and ECS2. Hydrophobic clustering of the flexible ECS1 ß1ß2 loops together with ECS2-ECS2 trans-interaction is suggested to be the driving force for conjunction of tetrameric building blocks into claudin polymers. Cldn10b andâ¯Cldn3 are indicated to share this polymerisation mechanism. However, in the paracellular centre of tetramers, electrostatic repulsion may lead to formation of pores (Cldn10b) and electrostatic attraction to barriers (Cldn3). Combining in vitro data and in silico modelling, this study improves mechanistic understanding of paracellular permeability regulation by elucidating claudin assembly and its pathologic alteration as in HELIX syndrome.
Assuntos
Claudina-3/química , Claudinas/química , Multimerização Proteica , Junções Íntimas/química , Animais , Permeabilidade da Membrana Celular , Claudina-3/genética , Claudina-3/metabolismo , Claudinas/genética , Claudinas/metabolismo , Células HEK293 , Humanos , Canais Iônicos , Camundongos , Mutação , Conformação Proteica , Síndrome , Junções Íntimas/metabolismoRESUMO
Tricellular junctions are specialized cell-cell junctions formed at sites where three epithelial or endothelial cells make contact at their apical side. By holding three cells together, tricellular junctions contribute to the maintenance of epithelial barrier function and mechanical integrity. In addition, recent studies have uncovered new functions of tricellular junctions at both cellular and physiological levels. In this review, we describe the architecture and molecular components of tricellular junctions and discuss how tricellular junctions participate in various biological processes.
Assuntos
Junções Aderentes/metabolismo , Desmossomos/metabolismo , Junções Íntimas/metabolismo , Junções Aderentes/química , Animais , Desmossomos/química , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Células Epiteliais/fisiologia , Humanos , Proteínas de Junções Íntimas/metabolismo , Junções Íntimas/químicaRESUMO
Methodology to enhance the intestinal absorption of peptides is an important challenge due to their easily degradation and poor permeability across the intestinal epithelium. In this study, the fish-derived peptide (DGDDGEAGKIG)-loaded chitosan (CS) nanoparticles (CS/PEP-NPs) were prepared and investigated in Caco-2 monolayer model. The results indicated zeta potential of CS/PEP-NPs increased with the increase in molecular weight of CS (10-50 kDa). Transmission electron microscopy images revealed the CS/PEP-NPs were uniform spherical-shaped nanoparticles with a diameter of 50-200 nm (150 kDa). Compared to other CS/PEP-NPs, 150-kDa CS/PEP-NPs performed an outstanding apparent permeability coefficient (Papp, 2.29 × 10-5 cm s-1 ) and cumulative amount of peptide (120 min, 2,987 ng) in Caco-2 cells. CS/PEP-NPs could reduce the tight junction integrity of Caco-2 cells and enhance the intracellular fluorescence intensities of fluorescein isothiocyanate-labeled peptide. These findings suggest that chitosan nanoparticles are promising carriers to promote intestinal absorption of fish-derived peptide via paracellular pathway mediated by tight junctions. PRACTICAL APPLICATIONS: Chitosans are promising carriers to promote intestinal absorption of fish-derived peptide. The 150-kDa CS/PEP-NPs performed an outstanding apparent permeability coefficient (Papp, 2.29 × 10-5 cm s-1 ) and cumulative amount of peptide (120 min, 2,987 ng) in Caco-2 cells. CS/PEP-NPs could reduce the tight junction integrity of Caco-2 cells and enhance the peptide uptake by paracellular pathway. Chitosan nanoparticles can be developed as vehicles for enhancing the cellular uptake of peptide in food industry.
Assuntos
Quitosana/análogos & derivados , Peptídeos/química , Peptídeos/metabolismo , Animais , Disponibilidade Biológica , Células CACO-2 , Quitosana/química , Portadores de Fármacos , Proteínas de Peixes/química , Proteínas de Peixes/metabolismo , Peixes , Humanos , Nanopartículas , Permeabilidade , Junções Íntimas/química , Junções Íntimas/metabolismoRESUMO
Changes in permeability of retinal blood vessels contribute to macular edema and the pathophysiology of numerous ocular diseases, including diabetic retinopathy, retinal vein occlusions, and macular degeneration. Vascular endothelial growth factor (VEGF) induces retinal permeability and macular thickening in these diseases. However, inflammatory agents, such as tumor necrosis factor-α (TNF-α), also may drive vascular permeability, specifically in patients unresponsive to anti-VEGF therapy. Recent evidence suggests VEGF and TNF-α induce permeability through distinct mechanisms; however, both require the activation of atypical protein kinase C (aPKC). We provide evidence, using genetic mouse models and therapeutic intervention with small molecules, that inhibition of aPKC prevented or reduced vascular permeability in animal models of retinal inflammation. Expression of a kinase-dead aPKC transgene, driven by a vascular and hematopoietic restricted promoter, reduced retinal vascular permeability in an ischemia-reperfusion model of retinal injury. This effect was recapitulated with a small-molecule inhibitor of aPKC. Expression of the kinase-dead aPKC transgene dramatically reduced the expression of inflammatory factors and blocked the attraction of inflammatory monocytes and granulocytes after ischemic injury. Coinjection of VEGF with TNF-α was sufficient to induce permeability, edema, and retinal inflammation, and treatment with an aPKC inhibitor prevented VEGF/TNF-α-induced permeability. These data suggest that aPKC contributes to inflammation-driven retinal vascular pathology and may be an attractive target for therapeutic intervention.
Assuntos
Permeabilidade Capilar/fisiologia , Proteína Quinase C/antagonistas & inibidores , Vasos Retinianos/fisiologia , Animais , Permeabilidade Capilar/efeitos dos fármacos , Masculino , Camundongos Endogâmicos C57BL , Papiledema/induzido quimicamente , Papiledema/fisiopatologia , Ratos Long-Evans , Proteínas Recombinantes , Traumatismo por Reperfusão/fisiopatologia , Retinite/induzido quimicamente , Retinite/fisiopatologia , Junções Íntimas/química , Junções Íntimas/fisiologia , Fator de Necrose Tumoral alfa/farmacologia , Fator A de Crescimento do Endotélio Vascular/farmacologiaRESUMO
Calycosin, a bioactive component derived from Astragali Radix (AR; Huang Qi), has been shown to have an effect of anti-allergic dermatitis with unknown mechanism. This study aims to investigate the mechanism of calycosin related to tight junctions (TJs) and HIF-1α both in FITC-induced mice allergic contact dermatitis and in IL-1ß stimulated HaCaT keratinocytes. Th2 cytokines (IL-4, IL-5 and IL-13) were detected by ELISA. The epithelial TJ proteins (occludin, CLDN1 and ZO-1), initiative key cytokines (TSLP and IL-33) and HIF-1α were assessed by Western blot, real-time PCR, immunohistochemistry or immunofluorescence. Herein, we have demonstrated that allergic inflammation and the Th2 cytokines in ACD mice were reduced significantly by calycosin treatment. Meanwhile, calycosin obviously decreased the expression of HIF-1α and repaired TJs both in vivo and in vitro. In HaCaT keratinocytes, we noted that IL-1ß induced the deterioration of TJs, as well as the increased levels of TSLP and IL-33, which could be reversed by silencing HIF-1α. In addition, administration of 2-methoxyestradiolin (2-ME), a HIF-1α inhibitor,significantly repaired the TJs and alleviated the allergic inflammation in vivo. Furthermore, TJs were destroyed by DMOG or by overexpressing HIF-1α in HaCaT keratinocytes, and simultaneously, calycosin down-regulated the expression of HIF-1α and repaired the TJs in this process. These results revealed that calycosin may act as a potential anti-allergy and barrier-repair agent via regulating HIF-1α in AD and suggested that HIF-1α and TJs might be possible therapy targets for allergic dermatitis.
Assuntos
Dermatite Alérgica de Contato/tratamento farmacológico , Medicamentos de Ervas Chinesas/farmacologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Isoflavonas/farmacologia , Junções Íntimas/efeitos dos fármacos , 2-Metoxiestradiol/farmacologia , Animais , Astragalus propinquus , Claudina-1/genética , Claudina-1/imunologia , Citocinas/genética , Citocinas/imunologia , Dermatite Alérgica de Contato/etiologia , Dermatite Alérgica de Contato/genética , Dermatite Alérgica de Contato/imunologia , Medicamentos de Ervas Chinesas/química , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/imunologia , Células Epiteliais/patologia , Fluoresceína-5-Isotiocianato/administração & dosagem , Regulação da Expressão Gênica , Glicina/análogos & derivados , Glicina/farmacologia , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Subunidade alfa do Fator 1 Induzível por Hipóxia/imunologia , Interleucina-1beta/farmacologia , Interleucinas/genética , Interleucinas/imunologia , Queratinócitos/citologia , Queratinócitos/efeitos dos fármacos , Queratinócitos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Ocludina/genética , Ocludina/imunologia , Transdução de Sinais , Pele/efeitos dos fármacos , Pele/imunologia , Pele/patologia , Junções Íntimas/química , Junções Íntimas/imunologia , Proteína da Zônula de Oclusão-1/genética , Proteína da Zônula de Oclusão-1/imunologia , Linfopoietina do Estroma do TimoRESUMO
Tight junction (TJ) protein assembly controls permeability across epithelial and endothelial cells; thus, biochemical interactions that control the TJ assembly have physiological and biomedical significance. In this work, we employed multiscale simulations to probe the TJ self-assembly of five classic claudins (-1, -2, -4, -15, and -19). Claudin proteins assembled into dimeric and occasionally trimeric interfaces that subsequently formed larger polymeric strands. Using orientation-angle analysis to decompose polymeric strands, we found that individual claudins prefer certain dimer interfaces to others. Despite variations in the exact dimer populations observed in individual claudins, there appears to be an overall conformational uniformity in the type of dimeric interactions formed by the claudin family of proteins. A detailed structural characterization of the trimeric assemblies revealed that they could be putative receptors for trimeric Clostridium perfringens enterotoxin. Full characterization of the claudin-2 dimer interface revealed a cysteine cross-linkable interaction, which could be assembled into a symmetric pore of 7.4 Å average diameter. We extended the analysis of pore structure to other classic claudins and found that the distribution of polar residues lining the pore volume varied considerably between the barrier- and pore-forming claudins, potentially delineating the functionality in classic claudins.
Assuntos
Claudinas/química , Claudinas/metabolismo , Clostridium perfringens/metabolismo , Cisteína/química , Dimerização , Enterotoxinas/química , Enterotoxinas/metabolismo , Simulação de Dinâmica Molecular , Ligação Proteica , Multimerização Proteica , Estrutura Terciária de Proteína , Junções Íntimas/química , Junções Íntimas/metabolismoRESUMO
The apical junctional complex (AJC), which includes tight junctions (TJs) and adherens junctions (AJs), determines the epithelial polarity, cell-cell adhesion and permeability barrier. An intriguing characteristic of a TJ is the dynamic nature of its multiprotein complex. Occludin is the most mobile TJ protein, but its significance in TJ dynamics is poorly understood. On the basis of phosphorylation sites, we distinguished a sequence in the C-terminal domain of occludin as a regulatory motif (ORM). Deletion of ORM and expression of a deletion mutant of occludin in renal and intestinal epithelia reduced the mobility of occludin at the TJs. ORM deletion attenuated Ca2+ depletion, osmotic stress and hydrogen peroxide-induced disruption of TJs, AJs and the cytoskeleton. The double point mutations T403A/T404A, but not T403D/T404D, in occludin mimicked the effects of ORM deletion on occludin mobility and AJC disruption by Ca2+ depletion. Both Y398A/Y402A and Y398D/Y402D double point mutations partially blocked AJC disruption. Expression of a deletion mutant of occludin attenuated collective cell migration in the renal and intestinal epithelia. Overall, this study reveals the role of ORM and its phosphorylation in occludin mobility, AJC dynamics and epithelial cell migration.
Assuntos
Junções Aderentes/química , Ocludina/química , Fosfoproteínas/química , Junções Íntimas/química , Junções Aderentes/genética , Animais , Cálcio/metabolismo , Movimento Celular/genética , Polaridade Celular/genética , Citoesqueleto/química , Citoesqueleto/genética , Cães , Células Epiteliais/química , Recuperação de Fluorescência Após Fotodegradação , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Humanos , Junções Intercelulares/química , Junções Intercelulares/genética , Células Madin Darby de Rim Canino , Ocludina/genética , Fosfoproteínas/genética , Fosforilação/genética , Mutação Puntual/genética , Domínios Proteicos/genética , Junções Íntimas/genéticaRESUMO
Herein, thioglycolic acid modified N-octyl-O, N'-glycol chitosan (N-mercapto acetyl-N'-octyl-O, Nâ³-glycol chitosan, abbreviated as SH-OGC) was synthesized to improve the oral bioavailability of paclitaxel (PTX). PTX was readily solubilized into the hydrophobic inner core of SH-OGC. Pharmacokinetic studies demonstrated that the bioavailability of PTX was greatly enhanced when delivered by SH-OGC compared to Taxol® or non-sulfhydrylated OGC micelles. Caco-2 cell experiments confirmed PTX or rhodamine-123-loaded SH-OGC demonstrated effective cellular accumulation via caveola-mediated endocytosis along with the inhibition of P-gp efflux. Furthermore, Caco-2 transport studies demonstrated that the mechanistic basis of SH-OGC efficacy was attributed to P-gp inhibition, enhanced permeability of tight junctions and clathrin-mediated transcytosis across the endothelium. In addition, SH-OGC exhibited increased intestinal retention through thiol-mediated mucoadhesion compared with OGC according to results of mucoadhesion evaluation on freshly excised rat intestine. In summary, SH-OGC micelles may present as a promising delivery vehicle for enhancing the oral bioavailability of P-gp substrates.
Assuntos
Antineoplásicos/farmacologia , Quitosana/análogos & derivados , Quitosana/farmacocinética , Portadores de Fármacos/farmacocinética , Paclitaxel/administração & dosagem , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/antagonistas & inibidores , Administração Oral , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/metabolismo , Disponibilidade Biológica , Células CACO-2 , Quitosana/administração & dosagem , Quitosana/síntese química , Portadores de Fármacos/administração & dosagem , Portadores de Fármacos/síntese química , Portadores de Fármacos/metabolismo , Endocitose , Humanos , Mucosa Intestinal/química , Micelas , Paclitaxel/metabolismo , Tamanho da Partícula , Ratos Sprague-Dawley , Junções Íntimas/química , Verapamil/farmacologiaRESUMO
Free-living amoebae of the genus Acanthamoeba are protozoa ubiquitously found in nature. Some species of the genus are potentially pathogenic for humans provoking keratitis in healthy individuals, often in contact lens wearers and opportunistic infections such as pneumonitis, fatal granulomatous encephalitis and skin infections, particularly in immunocompromised individuals. The pathogenic mechanisms of these amoebae are poorly understood, however it had been suggested that contact dependent mechanisms are important during invasion, regardless of the epithelia type, since amoebae penetrate epithelia separating tight junction (TJ). This study was undertaken to determine whether Acanthamoeba sp. (T4) damages the barrier function of the TJ in MDCK epithelial monolayers. Actin cytoskeleton staining and electron microscopy analyses were performed; paracellular permeability and TJ sealing were evaluated by apicobasolateral diffusion of ruthenium red and transepithelial resistance (TER) measurements; immunofluorescence and Western blot assays were performed to locate and estimate expression of TJ protein claudins 2 (Cldn2) and 4 (Cldn4). The results show that Acanthamoeba sp. crosses the MDCK monolayer without altering the actin cytoskeleton or the morphology of the cells. When trophozoites or conditioned medium interact with the monolayer, paracellular diffusion of ruthenium red increases. After 6 h, the amoebae, but not their conditioned medium, increase the TER, and Cldn2 is removed from the TJ, and its overall content in the cells diminishes, while Cldn4 is targeted to the TJ without changing its expression level. In conclusion Acanthamoeba (T4) crosses MDCK monolayer without damaging the cells, increasing permeability and TER through Cldn2 degradation, and redirecting Cldn4 to TJ. These results strongly suggest that contact-dependent mechanisms are relevant during amoebae invasion.
Assuntos
Acanthamoeba/fisiologia , Células Madin Darby de Rim Canino/parasitologia , Junções Íntimas/parasitologia , Acanthamoeba/patogenicidade , Acanthamoeba/ultraestrutura , Animais , Western Blotting , Claudina-2/metabolismo , Claudina-4/metabolismo , Meios de Cultivo Condicionados , Cães , Impedância Elétrica , Imunofluorescência , Indicadores e Reagentes/metabolismo , Células Madin Darby de Rim Canino/ultraestrutura , Microscopia Eletrônica de Transmissão , Permeabilidade , Rutênio Vermelho/metabolismo , Junções Íntimas/química , Junções Íntimas/metabolismo , Trofozoítos/fisiologia , Trofozoítos/ultraestruturaRESUMO
Tight-junctions between epithelial cells of biological barriers are specialized molecular structures that regulate the flux of solutes across the barrier, parallel to cell walls. The tight-junction backbone is made of strands of transmembrane proteins from the claudin family, but the molecular mechanism of its function is still not completely understood. Recently, the crystal structure of a mammalian claudin-15 was reported, displaying for the first time the detailed features of transmembrane and extracellular domains. Successively, a structural model of claudin-15-based paracellular channels has been proposed, suggesting a putative assembly that illustrates how claudins associate in the same cell (via cis interactions) and across adjacent cells (via trans interactions). Although very promising, the model offers only a static conformation, with residues missing in the most important extracellular regions and potential steric clashes. Here we present detailed atomic models of paracellular single and double pore architectures, obtained from the putative assembly and refined via structural modeling and all-atom molecular dynamics simulations in double membrane bilayer and water environment. Our results show an overall stable configuration of the complex with a fluctuating pore size. Extracellular residue loops in trans interaction are able to form stable contacts and regulate the size of the pore, which displays a stationary radius of 2.5-3.0 Å at the narrowest region. The side-by-side interactions of the cis configuration are preserved via stable hydrogen bonds, already predicted by cysteine crosslinking experiments. Overall, this work introduces an improved version of the claudin-15-based paracellular channel model that strengthens its validity and that can be used in further computational studies to understand the structural features of tight-junctions regulation.
Assuntos
Claudinas/química , Simulação de Dinâmica Molecular , Junções Íntimas/química , Simulação por Computador , Cristalografia por Raios X , Humanos , Ligação de Hidrogênio , Estrutura Molecular , Conformação Proteica , Domínios Proteicos , Multimerização Proteica , Software , Solventes/químicaRESUMO
Porcine epidemic diarrhea virus (PEDV), the causative agent of porcine epidemic diarrhea, has caused huge economic losses in pig-producing countries. Although PEDV was long believed to replicate in the intestinal epithelium by using aminopeptidase N as a receptor, the mechanisms of PEDV infection are not fully characterized. In this study, we found that PEDV infection of epithelial cells results in disruption of the tight junctional distribution of occludin to its intracellular location. Overexpression of occludin in target cells makes them more susceptible to PEDV infection, whereas ablation of occludin expression by use of small interfering RNA (siRNA) in target cells significantly reduces their susceptibility to virus infection. However, the results observed with occludin siRNA indicate that occludin is not required for virus attachment. We conclude that occludin plays an essential role in PEDV infection at the postbinding stages. Furthermore, we observed that macropinocytosis inhibitors blocked occludin internalization and virus entry, indicating that virus entry and occludin internalization are closely coupled. However, the macropinocytosis inhibitors could not impede virus replication once the virus had entered host cells. This suggests that occludin internalization by macropinocytosis or a macropinocytosis-like process is involved in the virus entry events. Immunofluorescence confocal microscopy showed that PEDV was trapped at cellular junctional regions upon macropinocytosis inhibitor treatment, indicating that occludin may serve as a scaffold in the vicinity of virus entry. Collectively, these data show that occludin plays an essential role in PEDV infection during late entry events. Our observation may provide novel insights into PEDV infection and related pathogenesis.IMPORTANCE Tight junctions are highly specialized membrane domains whose main function is to attach adjacent cells to each other, thereby forming intercellular seals. Here we investigate, for the first time, the role of the tight junction protein occludin in PEDV infection. We observed that PEDV infection induced the internalization of occludin. By using genetic modification methods, we demonstrate that occludin plays an essential role in PEDV infection. Moreover, PEDV entry and occludin internalization seem to be closely coupled. Our findings reveal a new mechanism of PEDV infection.
Assuntos
Ocludina/metabolismo , Vírus da Diarreia Epidêmica Suína/fisiologia , Junções Íntimas/química , Ligação Viral , Internalização do Vírus , Animais , Linhagem Celular , Chlorocebus aethiops , Citoplasma/metabolismo , Células Epiteliais/virologia , Ocludina/deficiência , Ocludina/genética , Vírus da Diarreia Epidêmica Suína/efeitos dos fármacos , Vírus da Diarreia Epidêmica Suína/patogenicidade , RNA Interferente Pequeno , Suínos , Junções Íntimas/patologia , Junções Íntimas/virologia , Células Vero , Internalização do Vírus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacosRESUMO
Monolayered epithelia are composed of tight cell assemblies that ensure polarized exchanges. EpCAM, an unconventional epithelial-specific cell adhesion molecule, is assumed to modulate epithelial morphogenesis in animal models, but little is known regarding its cellular functions. Inspired by the characterization of cellular defects in a rare EpCAM-related human intestinal disease, we find that the absence of EpCAM in enterocytes results in an aberrant apical domain. In the course of this pathological state, apical translocation towards tricellular contacts (TCs) occurs with striking tight junction belt displacement. These unusual cell organization and intestinal tissue defects are driven by the loss of actomyosin network homoeostasis and contractile activity clustering at TCs, yet is reversed by myosin-II inhibitor treatment. This study reveals that adequate distribution of cortical tension is crucial for individual cell organization, but also for epithelial monolayer maintenance. Our data suggest that EpCAM modulation protects against epithelial dysplasia and stabilizes human tissue architecture.
Assuntos
Células Epiteliais/química , Epitélio/química , Actomiosina/química , Actomiosina/genética , Actomiosina/metabolismo , Adolescente , Fenômenos Biomecânicos , Células CACO-2 , Polaridade Celular , Criança , Pré-Escolar , Diarreia Infantil/genética , Diarreia Infantil/metabolismo , Enterócitos/química , Enterócitos/metabolismo , Molécula de Adesão da Célula Epitelial/química , Molécula de Adesão da Célula Epitelial/genética , Molécula de Adesão da Célula Epitelial/metabolismo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Epitélio/metabolismo , Feminino , Humanos , Lactente , Síndromes de Malabsorção/genética , Síndromes de Malabsorção/metabolismo , Masculino , Junções Íntimas/química , Junções Íntimas/genética , Junções Íntimas/metabolismoRESUMO
Previous reports showed that Maresin 1 (MaR1) possessed organ protection effects and could attenuate acute lung injury. Here, we aim to figure out whether MaR1 can maintain the permeability of lung epithelial cells by regulating the expression of tight junction protein during lung injury. Monolayer of murine lung epithelial cells was stimulated by lipopolysaccharide (LPS) with or without MaR1 and the permeability was evaluated. The expression of Claudin-1 and ZO-1 in lung epithelial cells was analyzed by immunofluorescence staining and western blotting. MaR1 was given to the mice after LPS induced acute lung injury. The permeability of lung was assessed by Evans Blue extravasation, lung wet/dry ratio and protein concentration in bronchoalveolar lavage fluid. Lung injury score was also evaluated. The expression of Claudin-1 and ZO-1 in the lung was analyzed by immunofluorescence staining. Results showed that MaR1 maintained the permeability of lung epithelial cells and upregulated the expression of Claudin-1 and ZO-1 after LPS stimulation. In acute lung injury mice, MaR1 upregulated the expression of Claudin-1 and ZO-1, decreased lung permeability, and reduced lung injury. In summary, this study suggests that MaR1 can maintain the permeability of lung epithelial cells by upregulating the expression of Claudin-1 and ZO-1 in acute lung injury.