RESUMO
BACKGROUND: Male infertility is a hot problem worldwide, but there are few treatments, especially male infertility caused by irradiation is difficult to treat. The aim of this study was to investigate and evaluate novel drugs for the treatment of male infertility caused by irradiation. METHODS: we randomly divided 18 male BALB/c mice into 3 groups: control, irradiated, and telmisartan. Both irradiated and telmisartan group completed whole-body 0.5 Gy five times irradiation, and the telmisartan group received intraperitoneal injection of telmisartan (1.2 mg/kg) daily on the next day after irradiation, and all groups were sampled on day 25 after irradiation. RESULTS: Sperm motility results show that total sperm motility of irradiated group was significantly lower compared with control group, and testicular HE results showed that testis in irradiated group were severely damaged. Compared with irradiated group, the total sperm motility, sperm concentration, testicular index, Johnsen score, and the seminiferous tubule layer numbers were higher in telmisartan group (P < 0.05). The immunohistochemical staining showed γ-H2AX expression is higher in telmisartan group compared with irradiated group. And the relative mRNA expression of PLZF, GFRA1, STRA8, DMRT1, SPO11, SYCP2, OVOL2, CCNA1, TJP3, RUNX2, TXNDC2 TNP1, and PRM3 in telmisartan group was all significantly higher than irradiated group (P < 0.05). CONCLUSION: In conclusion, in vivo experiments confirmed that telmisartan ameliorated the spermatogenic disorder in mice caused by fractionated low-dose irradiation via promoting spermatogenesis.
Assuntos
Infertilidade Masculina , Motilidade dos Espermatozoides , Masculino , Camundongos , Animais , Humanos , Telmisartan/metabolismo , Telmisartan/farmacologia , Sêmen , Espermatogênese , Testículo/metabolismo , Infertilidade Masculina/tratamento farmacológico , Infertilidade Masculina/etiologia , Proteínas de Membrana/metabolismo , Tiorredoxinas/metabolismo , Tiorredoxinas/farmacologia , Fatores de Transcrição/metabolismo , Fatores de Transcrição/farmacologia , Proteínas da Zônula de Oclusão/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/farmacologiaRESUMO
BACKGROUND: Adenomyosis is a benign uterine disease and affected patients present with symptoms such as menorrhagia, chronic pelvic pain, abnormal uterine bleeding, and infertility. However, the specific mechanisms by which adenomyosis occurs need to be further studied. OBJECTIVE: Dataset of adenomyosis from our hospital and a public database were analyzed using bioinformatics. Corresponding differentially expressed genes (DEGs) and gene enrichment were detected to explore potential genetic adenomyosis targets. METHODS: Clinical data on adenomyosis were accessed based on the pathological specimens of patients with adenomyosis obtained from the Shengjing Hospital. R software was used to screen for DEGs, and volcano and cluster maps were drawn. Adenomyosis datasets (GSE74373) were downloaded from the GEO database. GEO2R online tool was used to screen for DEGs between adenomyosis and normal controls. Genes with P < 0.01 and |logFC| >1 were selected as DEGs. DAVID software was used for functional and pathway enrichment analyses. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed on common DEGs to obtain descriptions of the genes. The online database STRING was used for interaction gene retrieval. Moreover, Cytoscape software was used to construct a protein-protein interaction (PPI) network map for common DEGs to visualize potential gene interactions and screen the hub genes. RESULTS: A total of 845 DEGs were identified in the dataset obtained from Shengjing Hospital. A total of 175 genes were downregulated, and 670 genes were upregulated. In the GSE74373 database, 1679 genes were differentially expressed, 916 genes were downregulated, and 763 genes were upregulated. A total of 40 downregulated and 148 upregulated common DEGs showed potential gene interactions. The top ten upregulated hub genes were CDH1, EPCAM, CLDN7, ESRP1, RAB25, SPINT1, PKP3, TJP3, GRHL2, and CDKN2A. CONCLUSION: Genes involved in tight junction may be key in the development of adenomyosis and may provide a potential treatment strategy for adenomyosis.
Assuntos
Adenomiose , Perfilação da Expressão Gênica , Feminino , Humanos , Mapeamento de Interação de Proteínas , Biomarcadores Tumorais/genética , Adenomiose/genética , Regulação Neoplásica da Expressão Gênica , Endométrio/metabolismo , Biologia Computacional , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas da Zônula de Oclusão/genética , Proteínas da Zônula de Oclusão/metabolismoRESUMO
Tight junctions (TJs) are cell-adhesion structures responsible for the epithelial barrier. We reported that accumulation of cholesterol at the apical junctions is required for TJ formation [K. Shigetomi, Y. Ono, T. Inai, J. Ikenouchi, J. Cell Biol. 217, 2373-2381 (2018)]. However, it is unclear how cholesterol accumulates and informs TJ formation-and whether cholesterol enrichment precedes or follows the assembly of claudins in the first place. Here, we established an epithelial cell line (claudin-null cells) that lacks TJs by knocking out claudins. Despite the lack of TJs, cholesterol normally accumulated in the vicinity of the apical junctions. Assembly of claudins at TJs is thought to require binding to zonula occludens (ZO) proteins; however, a claudin mutant that cannot bind to ZO proteins still formed TJ strands. ZO proteins were however necessary for cholesterol accumulation at the apical junctions through their effect on the junctional actomyosin cytoskeleton. We propose that ZO proteins not only function as scaffolds for claudins but also promote TJ formation of cholesterol-rich membrane domains at apical junctions.
Assuntos
Fosfoproteínas , Junções Íntimas , Proteínas da Zônula de Oclusão/metabolismo , Junções Íntimas/metabolismo , Fosfoproteínas/metabolismo , Proteína da Zônula de Oclusão-1/genética , Proteína da Zônula de Oclusão-1/metabolismo , Claudinas/genética , Claudinas/metabolismoRESUMO
BACKGROUND: The intestinal tract is considered the body's "engine" and the most impacted organ during sepsis. In this study, we explored toll-like receptor 4 (TLR4) functions in sepsis-induced intestinal barrier dysfunction. METHODS: Wild-type and TLR4-knockout (KO) mice were used to establish a sepsis-induced dysfunctional intestinal barrier model via the intraperitoneal injection of lipopolysaccharide (LPS, 10 mg/kg). Hematoxylin and eosin staining, Transmission electron microscope, enzyme linked immunosorbent assay, western blot, quantitative real-time polymerase chain reaction, TdT-mediated dUTP nick end labeling staining, 16 S rRNA gene sequencing were used to explore differences in inflammatory cytokines, apoptosis, tight junction (TJ) protein expression, and intestinal flora diversity between groups. RESULTS: TLR4-deficiency reduced procalcitonin and C-reactive protein to prevent sepsis, and also inhibited inflammatory response by decreasing interleukin (IL)- 1ß, IL-6 and tumor necrosis factor-α levels. Also, BAX/Bcl2 and cleaved-caspase 3 expressions were decreased in TLR4-KO mice to suppress the intestinal mucosal cell apoptosis. TJ proteins, including zonula occludens protein, Occludin and Claudin-5 were significantly increased and intestinal fatty acid binding protein, myosin light chain and myosin light chain kinase were reduced in TLR4-KO mice. Additionally, 16 S rRNA gene sequencing indicated that TLR4-deficiency improved flora diversity and altered normal and abnormal bacterial proportions. CONCLUSIONS: TLR4 deficiency alleviated LPS-induced intestinal barrier dysfunction by reducing inflammatory responses and apoptosis, impairing intestinal damage, and regulating intestinal flora disturbance.
Assuntos
Lipopolissacarídeos , Sepse , Camundongos , Animais , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismo , Quinase de Cadeia Leve de Miosina/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Caspase 3/metabolismo , Ocludina/metabolismo , Cadeias Leves de Miosina/metabolismo , Interleucina-6/metabolismo , Pró-Calcitonina/metabolismo , Proteína C-Reativa/metabolismo , Claudina-5/metabolismo , Hematoxilina , Amarelo de Eosina-(YS) , Proteína X Associada a bcl-2/metabolismo , Proteínas de Junções Íntimas/metabolismo , Citocinas/metabolismo , Sepse/induzido quimicamente , Proteínas de Ligação a Ácido Graxo , Proteínas da Zônula de Oclusão/metabolismoRESUMO
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
Phase separation underlies functional compartmentalization in living systems. Two recent studies (Beutel et al. and Schwayer et al.) show that zonula occludens (ZO) proteins of tight junctions (TJs) condense into compartments within the cytoplasm that display liquid properties. This ability to condense predicts normal TJ assembly and epithelial barrier function which are essential for vertebrate embryogenesis.
Assuntos
Transição de Fase , Animais , Células Epiteliais/metabolismo , Humanos , Modelos Biológicos , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo , Proteínas da Zônula de Oclusão/metabolismoRESUMO
O-GlcNAcylation, like phosphorylation, is a dynamic and rapid posttranslational modification which regulates many cellular processes. Phosphorylation on tyrosine and O-GlcNAcylation on nearby serine or threonine residues may modulate each other. Indeed, by using a microarray with a peptide model system based on the ZO-3 protein, extensive cross talk between O-GlcNAcylation by OGT and phosphorylation by kinases was observed. However, studying the effects of kinases and OGT without the reverse processes catalyzed by phosphatases and O-GlcNAcase (OGA) does not provide a complete picture of the cross talk. The study of the missing part showed that nearby phosphorylation affects the de-O-GlcNAcylation by OGA, but not to the same extent as it affects the O-GlcNAcylation by OGT. Both the phosphorylation and de-phosphorylation processes were only slightly affected by the presence of an O-GlcNAc residue on a nearby serine.
Assuntos
N-Acetilglucosaminiltransferases/metabolismo , Fragmentos de Peptídeos/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Processamento de Proteína Pós-Traducional , Tirosina/metabolismo , Proteínas da Zônula de Oclusão/metabolismo , Humanos , Fosforilação , Análise Serial de ProteínasRESUMO
Hereditary hearing loss affects about 1 per 1000 children. Mutations in GJB2, which encodes the connexin 26 protein (Cx26) involved in cochlear homeostasis, are found in about 50% of patients with autosomal recessive non-syndromic hearing loss. Deciphering the trafficking pathway of cochlear Cx26 in situ should represent an advance in understanding the pathogenic significance of many of these mutations. Connexins trafficking and delivery to lipid raft-associated gap junction plaques usually requires successively microtubule and actin networks. Here we show that cochlear Cx26 exhibits an unusual trafficking pathway. We observed that Cx26 assembly occurs in non-lipid raft membrane domains and that junctional plaques are devoid of actin and associated zonula occludens proteins. Using cytoskeleton-disrupting drugs in organotypic culture, we found that cochlear Cx26 gap junction assembly requires microtubules but not actin filaments. Altogether, our data provide an unexpected insight into Cx26 trafficking pathway and gap junction assembly in the cochlea.
Assuntos
Cóclea/metabolismo , Conexinas/metabolismo , Junções Comunicantes/metabolismo , Actinas/metabolismo , Animais , Animais Recém-Nascidos , Criança , Conexina 26 , Conexinas/genética , Surdez/genética , Surdez/metabolismo , Humanos , Técnicas In Vitro , Microdomínios da Membrana/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Modelos Biológicos , Mutação , Transporte Proteico , Proteínas da Zônula de Oclusão/metabolismoRESUMO
Th17 cells together with their hallmark cytokine interleukin (IL)-17 were identified as crucial contributing factors in the pathogenesis of thyroid autoimmunity. The cytokine-regulated tight junction (Tj) disruption is thought to be essential in the initiation and/or development of several diseases. Still, the role of IL-17 maintaining Tj integrity in autoimmune thyroid diseases (AITDs) has not yet been evaluated. We aimed to investigate integrity of the thyroid follicle by studying immunoexpression of cellular Tj - zonula occludens (ZO)-1 and claudin-1 proteins coupled to IL-17A and CD68 detection in AITD patients compared with controls.Thirty-five adult patients undergoing thyroidectomy and presenting 18 cases of Hashimoto thyroiditis (HT), 7 of Graves' disease (GD) as well as 10 subjects of colloid goiter without autoimmune component served as controls were enrolled in this study. An immunohistochemical analysis including IL-17A, ZO-1, claudin-1, and CD68 detection was performed in each case. The correlation of IL-17A with Tj and CD68 in patients with AITD was also analyzed.Apart from inflammatory cells, we evidenced a stronger expression level of IL17A in the thyroid follicular cells in HT patients when compared with GD or colloid goiter. A significant reduction of ZO-1 immunoreactivity was observed in the thyrocytes in HT patients, whereas no significant differences were found in claudin-1 expression in HT and GD compared with colloid goiter patients. A significantly higher number of thyroid follicles with CD68-positive cells was found in HT patients than that in patients with GD or colloid goiter. In HT patients, the expression of IL-17A in the follicular cells was positively correlated with CD68 immunopositivity, whereas no association with claudin-1 or ZO-1 expression was found. GD patients did not reveal any significant correlation of IL-17A with Tj and CD68.Strong overexpression of IL-17A observed in the thyroid epithelial cells is associated with the presence of intrafollicular CD68-positive cells in HT patients. We evidenced the changes in molecules of thyrocyte junctional complexes highlighting impairment of the thyroid follicle integrity in HT, but no association with IL-17A was found.
Assuntos
Doença de Graves/imunologia , Doença de Hashimoto/imunologia , Interleucina-17/metabolismo , Tireoidite Autoimune/imunologia , Proteínas de Junções Íntimas/metabolismo , Adulto , Idoso , Antígenos CD/imunologia , Antígenos de Diferenciação Mielomonocítica/imunologia , Claudinas/metabolismo , Citocinas/metabolismo , Feminino , Doença de Graves/patologia , Doença de Graves/cirurgia , Doença de Hashimoto/patologia , Doença de Hashimoto/cirurgia , Humanos , Imuno-Histoquímica/métodos , Masculino , Pessoa de Meia-Idade , Células Th17/imunologia , Glândula Tireoide/patologia , Glândula Tireoide/ultraestrutura , Proteínas da Zônula de Oclusão/metabolismoRESUMO
BACKGROUND: Epidermal Growth Factor (EGF) reduces necrotizing enterocolitis (NEC). However, its high cost virtually prohibits clinical use. To reduce cost, soybean expressing human EGF was developed. Here we report effectiveness of soybean-derived EGF in experimental NEC. METHODS: Newborn rats were subjected to the NEC-inducing regimen of formula feeding and hypoxia. Formula was supplemented with extract from EGF-expressing or empty soybeans. NEC pathology was determined microscopically. Localization of tight junction proteins JAM-A and ZO-1 was examined by immunofluorescence and levels of mucosal COX-2 and iNOS mRNAs by real time PCR. RESULTS: Soybean extract amounts corresponding to 150µg/kg/day EGF caused considerable mortality, whereas those corresponding to 75µg/kg/day EGF were well tolerated. There was no significant difference in NEC scores between animals fed plain formula and formula supplemented with empty soybean extract. Soybean-EGF-supplemented formula at 75µg/kg/day EGF significantly decreased NEC, attenuated dissociation of JAM-A and ZO-1 proteins from tight junctions, and reduced intestinal expression of COX-2 and iNOS mRNAs. CONCLUSION: Supplementation with soybean-expressed EGF significantly decreased NEC in the rat model. Soybean-expressed EGF may provide an economical solution for EGF administration and prophylaxis of clinical NEC.
Assuntos
Enterocolite Necrosante/prevenção & controle , Fator de Crescimento Epidérmico/uso terapêutico , Glycine max , Extratos Vegetais/uso terapêutico , Substâncias Protetoras/uso terapêutico , Animais , Animais Recém-Nascidos , Ciclo-Oxigenase 2/metabolismo , Modelos Animais de Doenças , Enterocolite Necrosante/patologia , Humanos , Fórmulas Infantis , Recém-Nascido , Recém-Nascido Prematuro , Doenças do Prematuro/patologia , Doenças do Prematuro/prevenção & controle , Mucosa Intestinal/metabolismo , Intestinos/patologia , Moléculas de Adesão Juncional/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , RNA Mensageiro/metabolismo , Ratos Sprague-Dawley , Proteínas Recombinantes/uso terapêutico , Proteínas da Zônula de Oclusão/metabolismoRESUMO
Microcystin (MC)-LR is a cyclic heptapeptide that acts as a potent reproductive system toxin. However, the underlying pathways of MCLR-induced reproductive system toxicity have not been well elucidated. The blood-testis barrier is mainly constituted by tight junctions (TJs) between adjacent Sertoli cells in the seminiferous epithelium near the basement membrane. The present study was designed to investigate changes in TJs and the underlying pathway in MC-LR-induced TJs toxicity in Sertoli cell. In our study, the transepithelial electrical resistance (TER) value was decreased in a dose dependent manner due to the markers of TJs occludin, claudin and zonula occludens-1 (ZO-1) expression decline. MC-LR is shown to induce cytotoxicity by inhibiting protein phosphatase 2A (PP2A) activity. Our results also showed that the PP2A activity presented a dose-dependent decline. Moreover, MC-LR stimulated protein expression of snail by Akt/GSK-3ß activation. The activated Akt/GSK-3ß and snail signaling pathway largely accounted for MC-LRinduced TJs toxicity, which could be partially reversed by snail siRNA interference or AKT chemical inhibitor in TM4 cells. These findings indicated that MC-LR inhibit the protein expression of TJs, and the activation of Akt/Snail signaling pathways due to PP2A inhibition is proposed to participate in this process.
Assuntos
Microcistinas/toxicidade , Proteínas Proto-Oncogênicas c-akt/metabolismo , Células de Sertoli/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição da Família Snail/metabolismo , Junções Íntimas/efeitos dos fármacos , Animais , Masculino , Camundongos , Ocludina/genética , Ocludina/metabolismo , Proteína Fosfatase 2/genética , Proteína Fosfatase 2/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Células de Sertoli/metabolismo , Fatores de Transcrição da Família Snail/genética , Junções Íntimas/metabolismo , Proteínas da Zônula de Oclusão/genética , Proteínas da Zônula de Oclusão/metabolismoRESUMO
Nuclear receptors (NRs) are a group of transcription factors emerging as players in normal and pathological CNS development. Clinically, an association between the constitutive androstane NR (CAR) and cognitive impairment was proposed, however never experimentally investigated. We wished to test the hypothesis that the impact of CAR on neurophysiology and behavior is underlined by cerebrovascular-neuronal modifications. We have used CAR(-/-) C57BL/6 and wild type mice and performed a battery of behavioral tests (recognition, memory, motor coordination, learning and anxiety) as well as longitudinal video-electroencephalographic recordings (EEG). Brain cell morphology was assessed using 2-photon or electron microscopy and fluorescent immunohistochemistry. We observed recognition memory impairment and increased anxiety-like behavior in CAR(-/-) mice, while locomotor activity was not affected. Concomitantly to memory deficits, EEG monitoring revealed a decrease in 3.5-7Hz waves during the awake/exploration and sleep periods. Behavioral and EEG abnormalities in CAR(-/-) mice mirrored structural changes, including tortuous fronto-parietal penetrating vessels. At the cellular level we found reduced ZO-1, but not CLDN5, tight junction protein expression in cortical and hippocampal isolated microvessel preparations. Interestingly, the neurotoxin kainic acid, when injected peripherally, provoked a rapid onset of generalized convulsions in CAR(-/-) as compared to WT mice, supporting the hypothesis of vascular permeability. The morphological phenotype of CAR(-/-) mice also included some modifications of GFAP/IBA1 glial cells in the parenchymal or adjacent to collagen-IV(+) or FITC(+) microvessels. Neuronal defects were also observed including increased cortical NEUN(+) cell density, hippocampal granule cell dispersion and increased NPY immunoreactivity in the CA1 region in CAR(-/-) mice. The latter may contribute to the in vivo phenotype. Our results indicate that behavioral and electroencephalographic changes in adult CAR(-/-) mice are concomitant to discrete developmental or structural brain defects. The latter could increase the vulnerability to neurotoxins. The possibility that interfering with nuclear receptors during development could contribute to adulthood brain changes is proposed.
Assuntos
Ansiedade/genética , Encéfalo/patologia , Transtornos da Memória/genética , Microvasos/patologia , Receptores Citoplasmáticos e Nucleares/deficiência , Animais , Benzazepinas/farmacologia , Encéfalo/ultraestrutura , Proteínas de Ligação ao Cálcio/metabolismo , Receptor Constitutivo de Androstano , Modelos Animais de Doenças , Comportamento Exploratório/fisiologia , Ácido Caínico/farmacologia , Locomoção/genética , Masculino , Transtornos da Memória/patologia , Transtornos da Memória/fisiopatologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas dos Microfilamentos/metabolismo , Microvasos/fisiopatologia , Microvasos/ultraestrutura , Neuroglia/patologia , Neuroglia/ultraestrutura , Neurônios/patologia , Neurônios/ultraestrutura , Receptores Citoplasmáticos e Nucleares/genética , Reconhecimento Psicológico/fisiologia , Junções Íntimas/patologia , Junções Íntimas/ultraestrutura , Proteínas da Zônula de Oclusão/metabolismoRESUMO
Morphogenesis requires dynamic coordination between cell-cell adhesion and the cytoskeleton to allow cells to change shape and move without losing tissue integrity. We used genetic tools and superresolution microscopy in a simple model epithelial cell line to define how the molecular architecture of cell-cell zonula adherens (ZA) is modified in response to elevated contractility, and how these cells maintain tissue integrity. We previously found that depleting zonula occludens 1 (ZO-1) family proteins in MDCK cells induces a highly organized contractile actomyosin array at the ZA. We find that ZO knockdown elevates contractility via a Shroom3/Rho-associated, coiled-coil containing protein kinase (ROCK) pathway. Our data suggest that each bicellular border is an independent contractile unit, with actin cables anchored end-on to cadherin complexes at tricellular junctions. Cells respond to elevated contractility by increasing junctional afadin. Although ZO/afadin knockdown did not prevent contractile array assembly, it dramatically altered cell shape and barrier function in response to elevated contractility. We propose that afadin acts as a robust protein scaffold that maintains ZA architecture at tricellular junctions.
Assuntos
Junções Aderentes/metabolismo , Proteínas dos Microfilamentos/fisiologia , Proteínas da Zônula de Oclusão/fisiologia , Citoesqueleto de Actina/metabolismo , Animais , Adesão Celular , Forma Celular , Citoesqueleto/metabolismo , Cães , Células Epiteliais/metabolismo , Células Epiteliais/ultraestrutura , Técnicas de Silenciamento de Genes , Células Madin Darby de Rim Canino , Proteínas dos Microfilamentos/metabolismo , Morfogênese , Proteínas da Zônula de Oclusão/genética , Proteínas da Zônula de Oclusão/metabolismoRESUMO
Tight junctions (TJs) are complex cell-cell junctions that form a barrier in the stratum granulosum of mammalian skin. Besides forming a barrier themselves, TJs influence other skin barriers, e.g. the stratum corneum barrier, and are influenced by other skin barriers, e.g. by the chemical, the microbiome, or the immunological barrier and likely by the basement membrane. This review summarizes the dynamic interaction of the TJ barrier with other barriers in the skin and the central role of TJs in skin barrier function.
Assuntos
Claudinas/metabolismo , Epiderme/fisiologia , Fenômenos Fisiológicos da Pele , Junções Íntimas/fisiologia , Proteínas da Zônula de Oclusão/metabolismo , Animais , Peptídeos Catiônicos Antimicrobianos/metabolismo , Epiderme/metabolismo , Humanos , Permeabilidade , Junções Íntimas/imunologia , Junções Íntimas/microbiologiaRESUMO
miR-18a represses angiogenesis and tumor evasion by weakening vascular endothelial growth factor and transforming growth factor-ß signaling to prolong the survival of glioma patients, although it is thought to be an oncogene. This study investigates the potential effects of miR-18a on the permeability of the blood-tumor barrier (BTB) and its possible molecular mechanisms. An in vitro BTB model was successfully established. The endogenous expression of miR-18a in glioma vascular endothelial cells (GECs) was significantly lower than that in normal vascular ECs, and the overexpression of miR-18a significantly increased the permeability of the BTB as well as downregulating the mRNA and protein expressions of tight junction-related proteins zonula occluden-1 (ZO-1), claudin-5, and occludin in GECs. Dual luciferase reporter assays revealed that miR-18a bound to the 3'-untranslated region (3'UTR) of myocyte enhancer factor 2D (MEF2D). The overexpression of both miR-18a and MEF2D with the 3'UTR significantly weakened the effect caused by miR-18a of decreasing the mRNA and protein expressions of ZO-1, claudin-5 and occludin and of increasing the permeability of the BTB. Chromatin immunoprecipitation showed that MEF2D could directly bind to KLF4 promoter. This study shows that miR-18a targets and negatively regulates MEF2D, which further regulates tight junction-related proteins ZO-1, claudin-5, and occludin through transactivation of KLF4 and, finally, changes the permeability of the BTB. MiR-18a should garner growing attention because it might serve as a potential target in opening the BTB and providing a new strategy for the treatment of gliomas.
Assuntos
Regulação para Baixo/fisiologia , Células Epiteliais/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Fatores de Transcrição MEF2/metabolismo , MicroRNAs/metabolismo , Proteínas da Zônula de Oclusão/metabolismo , Barreira Hematoencefálica/citologia , Permeabilidade Capilar/fisiologia , Linhagem Celular Transformada , Imunoprecipitação da Cromatina , Claudina-5/metabolismo , Glioma/patologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Peroxidase do Rábano Silvestre/metabolismo , Humanos , Fator 4 Semelhante a Kruppel , MicroRNAs/genética , Ocludina/metabolismo , Permeabilidade , RNA Mensageiro/metabolismo , Transfecção , Proteínas da Zônula de Oclusão/genética , Proteína da Zônula de Oclusão-1/metabolismoRESUMO
Coordinated cell proliferation and ability to form intercellular seals are essential features of epithelial tissue function. Tight junctions (TJs) classically act as paracellular diffusion barriers. More recently, their role in regulating epithelial cell proliferation in conjunction with scaffolding zonula occludens (ZO) proteins has come to light. The kidney collecting duct (CD) is a model of tight epithelium that displays intense proliferation during embryogenesis followed by very low cell turnover in the adult kidney. Here, we examined the influence of each ZO protein (ZO-1, -2 and -3) on CD cell proliferation. We show that all 3 ZO proteins are strongly expressed in native CD and are present at both intercellular junctions and nuclei of cultured CD principal cells (mCCDcl1). Suppression of either ZO-1 or ZO-2 resulted in increased G0/G1 retention in mCCDcl1 cells. ZO-2 suppression decreased cyclin D1 abundance while ZO-1 suppression was accompanied by increased nuclear p21 localization, the depletion of which restored cell cycle progression. Contrary to ZO-1 and ZO-2, ZO-3 expression at intercellular junctions dramatically increased with cell density and relied on the presence of ZO-1. ZO-3 depletion did not affect cell cycle progression but increased cell detachment. This latter event partly relied on increased nuclear cyclin D1 abundance and was associated with altered ß1-integrin subcellular distribution and decreased occludin expression at intercellular junctions. These data reveal diverging, but interconnected, roles for each ZO protein in mCCDcl1 proliferation. While ZO-1 and ZO-2 participate in cell cycle progression, ZO-3 is an important component of cell adhesion.
Assuntos
Túbulos Renais Coletores/citologia , Interferência de RNA , Proteínas da Zônula de Oclusão/metabolismo , Proteína da Zônula de Oclusão-1/metabolismo , Proteína da Zônula de Oclusão-2/metabolismo , Animais , Adesão Celular , Pontos de Checagem do Ciclo Celular , Proliferação de Células , Células Cultivadas , Ciclina D1/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Túbulos Renais Coletores/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Sprague-Dawley , Proteínas da Zônula de Oclusão/antagonistas & inibidores , Proteínas da Zônula de Oclusão/genética , Proteína da Zônula de Oclusão-1/antagonistas & inibidores , Proteína da Zônula de Oclusão-1/genética , Proteína da Zônula de Oclusão-2/antagonistas & inibidores , Proteína da Zônula de Oclusão-2/genéticaRESUMO
Tight junctions (TJ) regulate the paracellular passage of ions and molecules through the paracellular pathway and maintain plasma membrane polarity in epithelial and endothelial cells. Apart from these canonical functions, several proteins of the TJ have been found in recent years to regulate gene expression. This function is found in proteins that shuttle between the nucleus and TJs, and in integral TJ proteins. In this review, we will describe these proteins and their known mechanisms of gene regulation.
Assuntos
Adesão Celular/fisiologia , Membrana Celular/metabolismo , Regulação da Expressão Gênica/genética , Junções Íntimas/genética , Transporte Biológico/genética , Moléculas de Adesão Celular Neuronais/metabolismo , Claudinas/metabolismo , Células Endoteliais/metabolismo , Humanos , Proteínas com Domínio MARVEL/metabolismo , Fatores de Transcrição/metabolismo , Proteínas da Zônula de Oclusão/metabolismoRESUMO
The localization and activities of DbpA/ZONAB and YAP transcription factors are in part regulated by the density-dependent assembly of epithelial junctions. DbpA activity and cell proliferation are inhibited by exogenous overexpression of the tight junction (TJ) protein ZO-1, leading to a model whereby ZO-1 acts by sequestering DbpA at the TJ. However, mammary epithelial cells and mouse tissues knock-out for ZO-1 do not show increased proliferation, as predicted by this model. To address this discrepancy, we examined the localization and activity of DbpA and YAP in Madin-Darby canine kidney cells depleted either of ZO-1, or one of the related proteins ZO-2 and ZO-3 (ZO proteins), or all three together. Depletion of only one ZO protein had no effect on DbpA localization and activity, whereas depletion of ZO-1 and ZO-2, which is associated with reduced ZO-3 expression, resulted in increased DbpA localization in the cytoplasm. Only depletion of ZO-2 reduced the nuclear import of YAP. Mammary epithelial (Eph4) cells KO for ZO-1 showed junctional DbpA, demonstrating that ZO-1 is not required to sequester DbpA at junctions. However, further depletion of ZO-2 in Eph4 ZO-1KO cells, which do not express ZO-3, caused decreased junctional localization and expression of DbpA, which were rescued by the proteasome inhibitor MG132. In vitro binding assays showed that full-length ZO-1 does not interact with DbpA. These results show that ZO-2 is implicated in regulating the nuclear shuttling of YAP, whereas ZO proteins redundantly control the junctional retention and stability of DbpA, without affecting its shuttling to the nucleus.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Células CACO-2 , Diferenciação Celular , Linhagem Celular , Proteínas de Ligação a DNA/genética , Cães , Células Epiteliais/metabolismo , Feminino , Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Junções Intercelulares/metabolismo , Glândulas Mamárias Animais/metabolismo , Camundongos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Fatores de Transcrição/genética , Proteínas da Zônula de Oclusão/deficiência , Proteínas da Zônula de Oclusão/genética , Proteínas da Zônula de Oclusão/metabolismo , Proteína da Zônula de Oclusão-1/deficiência , Proteína da Zônula de Oclusão-1/genética , Proteína da Zônula de Oclusão-1/metabolismo , Proteína da Zônula de Oclusão-2/deficiência , Proteína da Zônula de Oclusão-2/genética , Proteína da Zônula de Oclusão-2/metabolismoRESUMO
Zonula Occludens (ZO) proteins are ubiquitous scaffolding proteins providing the structural basis for the assembly of multiprotein complexes at the cytoplasmic surface of the plasma membrane and linking transmembrane proteins to the filamentous cytoskeleton. They belong to the large family of membrane-associated guanylate kinase (MAGUK)-like proteins comprising a number of subfamilies based on domain content and sequence similarity. ZO proteins were originally described to localize specifically to tight junctions, or Zonulae Occludentes, but this notion was rapidly reconsidered since ZO proteins were found to associate with adherens junctions as well as with gap junctions, particularly with connexin-made intercellular channels, and also with a few other membrane channels. Accumulating evidence reveals that in addition to having passive scaffolding functions in organizing gap junction complexes, including connexins and cytoskeletals, ZO proteins (particularly ZO-1) also actively take part in the dynamic function as well as in the remodeling of junctional complexes in a number of cellular systems. This article is part of a Special Issue entitled: Reciprocal influences between cell cytoskeleton and membrane channels, receptors and transporters. Guest Editor: Jean Claude Hervé.