RESUMO
Epithelial barrier function is commonly analyzed using transepithelial electrical resistance, which measures ion flux across a monolayer, or by adding traceable macromolecules and monitoring their passage across the monolayer. Although these methods measure changes in global barrier function, they lack the sensitivity needed to detect local or transient barrier breaches, and they do not reveal the location of barrier leaks. Therefore, we previously developed a method that we named the zinc-based ultrasensitive microscopic barrier assay (ZnUMBA), which overcomes these limitations, allowing for detection of local tight junction leaks with high spatiotemporal resolution. Here, we present expanded applications for ZnUMBA. ZnUMBA can be used in Xenopus embryos to measure the dynamics of barrier restoration and actin accumulation following laser injury. ZnUMBA can also be effectively utilized in developing zebrafish embryos as well as cultured monolayers of Madin-Darby canine kidney (MDCK) II epithelial cells. ZnUMBA is a powerful and flexible method that, with minimal optimization, can be applied to multiple systems to measure dynamic changes in barrier function with spatiotemporal precision.
Assuntos
Células Epiteliais , Zinco , Animais , Cães , Peixe-Zebra , Células Madin Darby de Rim Canino , Junções Íntimas , ActinasRESUMO
Podocytes serve as part of the renal filtration unit with slit diaphragms. Although the structure of slit diaphragms between two cells is well characterized, how the tricellular contact of podocytes is organized and how it changes in injured podocytes remains unknown. This study focused on a tricellular junction protein, angulin-3, and its localization in healthy podocytes, in developmental stages, and in pathologic conditions, using a newly established monoclonal antibody. Angulin-3 was confined at tricellular junctions of primordial podocytes, then transiently localized at bicellular junctions as foot process interdigitation developed and the intercellular junctions rearranged into slit diaphragm, and eventually distributed in a sparse punctate pattern on the foot processes of adult podocytes. In the rodent podocyte injury models, angulin-3 showed bicellular localization between the foot processes, and the localization turned from punctate to dashed linear pattern along the effaced foot processes with the progression of podocyte injury. Angulin-3 also accumulated between foot processes in a linear pattern in kidney biopsy samples of human nephrotic syndrome. Additionally, the line length of angulin-3 staining signal correlated with risk of relapse under glucocorticoid therapy in patients with minimal change nephrotic syndrome. This study proposes an image program to score the linearity of the accumulation pattern of angulin-3 to evaluate the relapse risk of patients with minimal change nephrotic syndrome.
Assuntos
Nefrose Lipoide , Podócitos , Adulto , Humanos , Podócitos/metabolismo , Junções Íntimas/patologia , Nefrose Lipoide/metabolismo , Nefrose Lipoide/patologia , Junções Intercelulares/metabolismo , RecidivaRESUMO
BACKGROUND: Cell adhesion is indispensable for appropriate tissue architecture and function in multicellular organisms. Besides maintaining tissue integrity, cell adhesion molecules, including tight-junction proteins claudins (CLDNs), exhibit the signaling abilities to control a variety of physiological and pathological processes. However, it is still fragmentary how cell adhesion signaling accesses the nucleus and regulates gene expression. METHODS: By generating a number of knockout and rescued human breast cell lines and comparing their phenotypes, we determined whether and how CLDN4 affected breast cancer progression in vitro and in vivo. We also identified by RNA sequencing downstream genes whose expression was altered by CLDN4-adhesion signaling. Additionally, we analyzed by RT-qPCR the CLDN4-regulating genes by using a series of knockout and add-back cell lines. Moreover, by immunohistochemistry and semi-quantification, we verified the clinicopathological significance of CLDN4 and the nuclear receptor LXRß (liver X receptor ß) expression in breast cancer tissues from 187 patients. RESULTS: We uncovered that the CLDN4-adhesion signaling accelerated breast cancer metabolism and progression via LXRß. The second extracellular domain and the carboxy-terminal Y197 of CLDN4 were required to activate Src-family kinases (SFKs) and the downstream AKT in breast cancer cells to promote their proliferation. Knockout and rescue experiments revealed that the CLDN4 signaling targets the AKT phosphorylation site S432 in LXRß, leading to enhanced cell proliferation, migration, and tumor growth, as well as cholesterol homeostasis and fatty acid metabolism, in breast cancer cells. In addition, RT-qPCR analysis showed the CLDN4-regulated genes are classified into at least six groups according to distinct LXRß- and LXRßS432-dependence. Furthermore, among triple-negative breast cancer subjects, the "CLDN4-high/LXRß-high" and "CLDN4-low and/or LXRß-low" groups appeared to exhibit poor outcomes and relatively favorable prognoses, respectively. CONCLUSIONS: The identification of this machinery highlights a link between cell adhesion and transcription factor signalings to promote metabolic and progressive processes of malignant tumors and possibly to coordinate diverse physiological and pathological events.
Assuntos
Proteínas Proto-Oncogênicas c-akt , Neoplasias de Mama Triplo Negativas , Humanos , Claudina-4/genética , Claudina-4/metabolismo , Receptores X do Fígado/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Claudinas/genética , Claudinas/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Linhagem Celular TumoralRESUMO
Recent studies have revealed that aberrant expression of tight junction (TJ) proteins is a hallmark of various solid tumors and it is recognized as a useful therapeutic target. Claudin-6 (CLDN6), a member of the family of TJ transmembrane proteins, is an ideal therapeutic target because it is not expressed in human adult normal tissues. In this study, we found that CLDN6 is highly expressed in uterine cervical adenocarcinoma (ADC) and that high CLDN6 expression was correlated with lymph node metastasis and lymphovascular infiltration and was an independent prognostic factor. Shotgun proteome analysis revealed that cell-cell adhesion-related proteins and drug metabolism-associated proteins (aldo-keto reductase [AKR] family proteins) were significantly increased in CLDN6-overexpressing cells. Furthermore, overexpression of CLDN6 enhanced cell-cell adhesion properties and attenuated sensitivity to anticancer drugs including doxorubicin, daunorubicin, and cisplatin. Taken together, the results indicate that aberrant expression of CLDN6 enhances malignant potentials and drug resistance of cervical ADC, possibly due to increased cell-cell adhesion properties and drug metabolism. Our findings provide an insight into a new therapeutic strategy, a CLDN6-targeting therapy, against cervical ADC.
Assuntos
Adenocarcinoma , Neoplasias do Colo do Útero , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/genética , Adulto , Claudinas/genética , Resistência a Medicamentos , Feminino , Humanos , Neoplasias do Colo do Útero/tratamento farmacológico , Neoplasias do Colo do Útero/genéticaRESUMO
Cell adhesion is essential for proper tissue architecture and function in multicellular organisms. Cell adhesion molecules not only maintain tissue integrity but also possess signaling properties that contribute to diverse cellular events such as cell growth, survival, differentiation, polarity, and migration; however, the underlying molecular basis remains poorly defined. Here we identify that the cell adhesion signal initiated by the tight-junction protein claudin-6 (CLDN6) regulates nuclear receptor activity. We show that CLDN6 recruits and activates Src-family kinases (SFKs) in second extracellular domain-dependent and Y196/200-dependent manners, and SFKs in turn phosphorylate CLDN6 at Y196/200. We demonstrate that the CLDN6/SFK/PI3K/AKT axis targets the AKT phosphorylation sites in the retinoic acid receptor γ (RARγ) and the estrogen receptor α (ERα) and stimulates their activities. Interestingly, these phosphorylation motifs are conserved in 14 of 48 members of human nuclear receptors. We propose that a similar link between diverse cell adhesion and nuclear receptor signalings coordinates a wide variety of physiological and pathological processes.
Assuntos
Adesão Celular/fisiologia , Claudinas/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Animais , Linhagem Celular , Claudinas/genética , Receptor alfa de Estrogênio/metabolismo , Regulação da Expressão Gênica , Humanos , Camundongos , Mutação , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Domínios Proteicos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores do Ácido Retinoico/genética , Receptores do Ácido Retinoico/metabolismo , Transdução de Sinais , Tirosina/genética , Quinases da Família src/metabolismo , Receptor gama de Ácido RetinoicoRESUMO
The neurovascular unit (NVU) consists of neurons, glial cells, microvascular cells, and extracellular matrix, and is involved in a variety of physiological and pathological processes in the central nervous system (CNS). Within the NVU, the microvascular endothelial cells and pericytes principally contribute to maintaining the integrity of the blood-brain barrier (BBB). Various types of cells are connected to each other in the NVU by diverse cell adhesion molecules, of which claudin-5 (CLDN5) is by far the most abundantly expressed tight-junction protein in brain microvascular endothelial cells and absolutely required for the maintenance of the BBB. This review highlights recent progress in understanding the region-specific regulation and dysregulation of CLDN5 expression in CNS health and disorders. We also discuss how CLDN5 expression is regionally disrupted within the NVU. In addition, we focus on the link between cell adhesion and transcription factor signalings and describe the possible involvement of CLDN5-adhesion signaling in brain health and disorders.
Assuntos
Barreira Hematoencefálica/metabolismo , Encefalopatias/metabolismo , Claudina-5/metabolismo , Células Endoteliais/metabolismo , Transdução de Sinais , Junções Íntimas/metabolismo , Animais , Barreira Hematoencefálica/patologia , Encefalopatias/genética , Encefalopatias/patologia , Claudina-5/genética , Células Endoteliais/patologia , Regulação da Expressão Gênica , Humanos , Junções Íntimas/genética , Junções Íntimas/patologiaRESUMO
BACKGROUND: Within the claudin (CLDN) family, CLDN12 mRNA expression is altered in various types of cancer, but its clinicopathological relevance has yet to be established due to the absence of specific antibodies (Abs) with broad applications. METHODS: We generated a monoclonal Ab (mAb) against human/mouse CLDN12 and verified its specificity. By performing immunohistochemical staining and semiquantification, we evaluated the relationship between CLDN12 expression and clinicopathological parameters in tissues from 138 cases of cervical cancer. RESULTS: Western blot and immunohistochemical analyses revealed that the established mAb selectively recognized the CLDN12 protein. Twenty six of the 138 cases (18.8%) showed low CLDN12 expression, and the disease-specific survival (DSS) and recurrence-free survival rates were significantly decreased compared with those in the high CLDN12 expression group. We also demonstrated, via univariable and multivariable analyses, that the low CLDN12 expression represents a significant prognostic factor for the DSS of cervical cancer patients (HR 3.412, p = 0.002 and HR 2.615, p = 0.029, respectively). CONCLUSIONS: It can be concluded that a reduced CLDN12 expression predicts a poor outcome for cervical cancer. The novel anti-CLDN12 mAb could be a valuable tool to evaluate the biological relevance of the CLDN12 expression in diverse cancer types and other diseases.
Assuntos
Claudinas/biossíntese , Regulação Neoplásica da Expressão Gênica , Proteínas de Neoplasias/biossíntese , Neoplasias do Colo do Útero/metabolismo , Neoplasias do Colo do Útero/mortalidade , Adulto , Idoso , Intervalo Livre de Doença , Feminino , Células HEK293 , Humanos , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Taxa de Sobrevida , Neoplasias do Colo do Útero/patologiaRESUMO
We previously reported that site-selective claudin-5 (CLDN5) breakdown and protein kinase A (PKA) activation are observed in brain microvessels of schizophrenia, but the underlying molecular basis remains unknown. The 5-HT1 receptors decline the intracellular cAMP levels and inactivate the major downstream PKA, and the 5-HT1A receptor is a promising target for schizophrenia. Therefore, we elucidated the involvement of serotonin/5-HT1A signaling in the endothelial CLDN5 expression. We demonstrate, by immunohistochemistry using post-mortem human brain tissue, that the 5-HT1A receptor is expressed in brain microvascular endothelial cells (BMVECs) and mural cells of the normal prefrontal cortex (PFC) gray matter. We also show that PKA is aberrantly activated not only in BMVECs but also in mural cells of the schizophrenic PFC. We subsequently revealed that the endothelial cell-pericyte tube-like structure was formed in a novel two-dimensional co-culture of human primary BMVECs and a human brain-derived pericyte cell line, in both of which the 5-HT1A receptor was expressed. Furthermore, we disclose that the serotonin/5-HT1A signaling enhances endothelial CLDN5 expression in BMVECs under two-dimensional co-culture conditions. Our findings provide novel insights into the physiological and pathological significance of serotonin/5-HT1A signaling in the region-specific regulation of the blood-brain barrier.
Assuntos
Claudina-5/genética , Células Endoteliais/metabolismo , Regulação da Expressão Gênica , Acoplamento Neurovascular , Receptor 5-HT1A de Serotonina/metabolismo , Serotonina/metabolismo , Transdução de Sinais , Biomarcadores , Encéfalo/metabolismo , Comunicação Celular , Claudina-5/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Imunofluorescência , Humanos , Pericitos/patologiaRESUMO
Neural progenitors acquire GFAP expression during the perinatal period and continue to generate granule cells (GCs) in the hippocampal dentate gyrus throughout adulthood. Cellular characteristics of GFAP+ progenitor-derived late-born GCs in comparison with early-born GCs remain unknown. Using genetic fate mapping in mice, we show that early- and late-born GCs are concentrated in the outer and inner side of the GC layer, respectively. We then identify that a nuclear orphan receptor Nr4A2 is preferentially expressed by early-born GCs. Nr4a2 expression is dynamically regulated in response to restraint stress and glucocorticoid levels, indicating that Nr4a2 is a stress-regulated gene in GCs. Acute stress suppresses but chronic stress conversely induces Nr4a2 expression in GCs. The survival of newly generated GCs is impaired by chronic restraint stress and long-term stress after middle age decreases the proportion of late-born GCs in aged mice. Thus, early- and late-born GCs exhibit characteristic anatomical distribution, differential gene expression, and distinct response to environmental stress.
Assuntos
Giro Denteado/metabolismo , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares/genética , Envelhecimento/genética , Envelhecimento/metabolismo , Animais , Giro Denteado/citologia , Giro Denteado/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento , Glucocorticoides/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Neurogênese/genética , Neurogênese/fisiologia , Restrição Física , Estresse FisiológicoRESUMO
Extracellular vesicles (EVs) are membrane-bound intercellular communication vehicles that transport proteins, lipids and nucleic acids with regulatory capacity between cells. RNA profiling using microarrays and sequencing technologies has revolutionized the discovery of EV-RNA content, which is crucial to understand the molecular mechanism of EV function. Recent studies have indicated that EVs are enriched with specific RNAs compared to the originating cells suggestive of an active sorting mechanism. Here, we present the comparative transcriptome analysis of human osteoblasts and their corresponding EVs using next-generation sequencing. We demonstrate that osteoblast-EVs are specifically depleted of cellular mRNAs that encode proteins involved in basic cellular activities, such as cytoskeletal functions, cell survival and apoptosis. In contrast, EVs are significantly enriched with 254 mRNAs that are associated with protein translation and RNA processing. Moreover, mRNAs enriched in EVs encode proteins important for communication with the neighboring cells, in particular with osteoclasts, adipocytes and hematopoietic stem cells. These findings provide the foundation for understanding the molecular mechanism and function of EV-mediated interactions between osteoblasts and the surrounding bone microenvironment.
Assuntos
Vesículas Extracelulares/genética , Osteoblastos/metabolismo , Transcriptoma , Linhagem Celular , Vesículas Extracelulares/metabolismo , Humanos , RNA Mensageiro/química , RNA Mensageiro/genéticaRESUMO
Beyond forming bone, osteoblasts play pivotal roles in various biologic processes, including hematopoiesis and bone metastasis. Extracellular vesicles (EVs) have been implicated in intercellular communication via transfer of proteins and nucleic acids between cells. We focused on the proteomic characterization of nonmineralizing (NMOBs) and mineralizing (MOBs) human osteoblast (SV-HFOs) EVs and investigated their effect on human prostate cancer (PC3) cells by microscopic, proteomic, and gene expression analyses. Proteomic analysis showed that 97% of the proteins were shared among NMOB and MOB EVs, and 30% were novel osteoblast-specific EV proteins. Label-free quantification demonstrated mineralization stage-dependent 5-fold enrichment of 59 and 451 EV proteins in NMOBs and MOBs, respectively. Interestingly, bioinformatic analyses of the osteoblast EV proteomes and EV-regulated prostate cancer gene expression profiles showed that they converged on pathways involved in cell survival and growth. This was verified by in vitro proliferation assays where osteoblast EV uptake led to 2-fold increase in PC3 cell growth compared to cell-free culture medium-derived vesicle controls. Our findings elucidate the mineralization stage-specific protein content of osteoblast-secreted EVs, show a novel way by which osteoblasts communicate with prostate cancer, and open up innovative avenues for therapeutic intervention.
Assuntos
Calcificação Fisiológica/fisiologia , Osteoblastos/metabolismo , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Calcificação Fisiológica/genética , Comunicação Celular/genética , Comunicação Celular/fisiologia , Diferenciação Celular , Linhagem Celular Tumoral , Proliferação de Células , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Perfilação da Expressão Gênica , Humanos , Masculino , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Osteoblastos/patologia , Neoplasias da Próstata/genética , Proteômica , Microambiente TumoralRESUMO
PURPOSE: LPA is one of several physiologically active lipid mediators that promote cell proliferation and invasion, and are present in serum, ascites and urine. LPA receptor is a G-protein coupled receptor that is considered a potential therapeutic target for some malignant cancers. We evaluated the expression of LPA receptors in bladder cancer and the effect of LPA in bladder cancer invasion. MATERIALS AND METHODS: Using real-time polymerase chain reaction and immunohistochemical staining we determined LPA receptor expression in bladder cancer specimens from patients with bladder cancer, including 12 with Ta or T1 and 15 with T2-T4 disease. ROCK expression, myosin light chain phosphorylation and Matrigel™ invasion assays were done and morphological observations were made to assess LPA effects in T24 cells, which were derived from bladder cancer. RESULTS: Notably LPA1 mRNA expression was significantly higher in muscle invasive bladder cancer specimens than in nonmuscle invasive specimens. Strong LPA1 expression was evident on cell membranes in muscle invasive specimens. T24 cell invasion was increased by LPA treatment and invasiveness was decreased by LPA1 siRNA or LPA1 inhibitor. LPA treatment increased ROCK1 expression and myosin light chain phosphorylation, and induced morphological changes, including lamellipodia formation and cell rounding. CONCLUSIONS: Results indicate that LPA signaling via LPA1 activation promoted bladder cancer invasion. LPA1 might be useful to detect bladder cancer with highly invasive potential and become a new therapeutic target for invasive bladder cancer treatment.
Assuntos
Receptores de Ácidos Lisofosfatídicos/biossíntese , Receptores de Ácidos Lisofosfatídicos/fisiologia , Neoplasias da Bexiga Urinária/metabolismo , Neoplasias da Bexiga Urinária/patologia , Idoso , Feminino , Humanos , Masculino , Invasividade Neoplásica , Células Tumorais Cultivadas , Neoplasias da Bexiga Urinária/cirurgiaRESUMO
BACKGROUND: S100 family proteins have recently been identified as biomarkers in various cancers. Of this protein family, S100A14 and S100A16 are also believed to play an important role in tumor progression. The aim of the present study was to clarify the clinical significance and functional role of these molecules in breast cancer. METHODS: In a clinical study, an immunohistochemical analysis of S100A14 and S100A16 expression in archival specimens of primary tumors of 167 breast cancer patients was performed. The relationship of S100A14 and S100A16 expression to patient survival and clinicopathological variables was statistically analyzed. In an experimental study, the subcellular localization and function of these molecules was examined by using the human breast cancer cell lines MCF7 and SK-BR-3, both of which highly express S100A14 and S100A16 proteins. Cells transfected with expression vectors and siRNA for these genes were characterized using in vitro assays for cancer invasion and metastasis. RESULTS: Immunohistochemical analysis of 167 breast cancer cases showed strong cell membrane staining of S100A14 (53% of cases) and S100A16 (31% of cases) with a significant number of cases with co-expression (p < 0.001). Higher expression levels of these proteins were significantly associated with a younger age (<60 years), ER-negative status, HER2-positive status and a poorer prognosis. Co-expression of the two proteins showed more aggressive features with poorer prognosis. In the human breast cancer cell lines MCF7 and SK-BR-3, both proteins were colocalized on the cell membrane mainly at cell-cell attachment sites. Immunoprecipitation and immunofluorescence analyses demonstrated that the 100A14 protein can bind to actin localized on the cell membrane in a calcium-independent manner. A Boyden chamber assay showed that S100A14 and S100A16 knockdown substantially suppressed the invasive activity of both cell lines. Cell motility was also inhibited by S100A14 knockdown in a modified dual color wound-healing assay. CONCLUSIONS: To our knowledge, this is the first report showing the correlation of expression of S100A14, S100A16, and co-expression of these proteins with poor prognosis of breast cancer patients. In addition, our findings indicate that S100A14 and S100A16 can promote invasive activity of breast cancer cells via an interaction with cytoskeletal dynamics. S100A14 and S100A16 might be prognostic biomarkers and potential therapeutic targets for breast cancer.
Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/mortalidade , Proteínas de Ligação ao Cálcio/genética , Expressão Gênica , Proteínas S100/genética , Actinas/metabolismo , Adulto , Idoso , Biomarcadores Tumorais , Neoplasias da Mama/patologia , Neoplasias da Mama/cirurgia , Proteínas de Ligação ao Cálcio/metabolismo , Linhagem Celular Tumoral , Movimento Celular/genética , Feminino , Técnicas de Silenciamento de Genes , Humanos , Imuno-Histoquímica , Pessoa de Meia-Idade , Gradação de Tumores , Estadiamento de Neoplasias , Prognóstico , Ligação Proteica , Transporte Proteico , Proteínas S100/metabolismoAssuntos
Neoplasias Cardíacas/secundário , Ruptura/etiologia , Neoplasias da Bexiga Urinária/patologia , Idoso de 80 Anos ou mais , Tamponamento Cardíaco/diagnóstico por imagem , Tamponamento Cardíaco/etiologia , Neoplasias Cardíacas/complicações , Neoplasias Cardíacas/diagnóstico por imagem , Humanos , Masculino , Metástase Neoplásica , Ruptura/diagnóstico por imagem , Tomografia Computadorizada por Raios X , Neoplasias da Bexiga Urinária/complicações , Urotélio/patologiaRESUMO
Genome editing is a powerful tool for establishing gene knockout or mutant cell lines. Here, we present a protocol for establishing knockout cell clones by deletion of large gene fragments using CRISPR-Cas9 with multiple guide RNAs. We describe steps for designing guide RNAs, cloning them into CRISPR-Cas9 vectors, cell seeding, transfection into cultured cells, clonal selection, and screening assays. This protocol can delete gene regions over 100 kbp, including GC-rich domains, and is applicable to various cell lines. For complete details on the use and execution of this protocol, please refer to Saito et al.,1 Saito and Endo et al.,2 and Higashi et al.3.
RESUMO
Epithelial tissues cover the surfaces and lumens of the internal organs of multicellular animals and crucially contribute to internal environment homeostasis by delineating distinct compartments within the body. This vital role is known as epithelial barrier function. Epithelial cells are arranged like cobblestones and intricately bind together to form an epithelial sheet that upholds this barrier function. Central to the restriction of solute and fluid diffusion through intercellular spaces are occluding junctions, tight junctions in vertebrates and septate junctions in invertebrates. As part of epithelial tissues, cells undergo constant renewal, with older cells being replaced by new ones. Simultaneously, the epithelial tissue undergoes relative rearrangement, elongating, and shifting directionally as a whole. The movement or shape changes within the epithelial sheet necessitate significant deformation and reconnection of occluding junctions. Recent advancements have shed light on the intricate mechanisms through which epithelial cells sustain their barrier function in dynamic environments. This review aims to introduce these noteworthy findings and discuss some of the questions that remain unanswered.
Assuntos
Células Epiteliais , Junções Íntimas , Animais , Humanos , Células Epiteliais/metabolismo , Células Epiteliais/citologia , Junções Íntimas/metabolismo , Junções Íntimas/fisiologia , Epitélio/metabolismo , Epitélio/fisiologiaRESUMO
Tight junctions (TJs) are cell-cell junction structures critical for controlling paracellular permeability. On freeze-fracture replica electron microscopy, they appear as a continuous network of fibrils (TJ strands). TJ strands function as zippers that create a physical barrier against paracellular diffusion of molecules. The morphology of the TJ strand network varies greatly between tissues, and in recent years, studies have highlighted the mechanisms regulating the morphology of TJ strand networks and on their relevance to barrier function. In this review, we discuss evidence regarding the components of the TJ strand and the mechanisms for creating the TJ strand network. Furthermore, we discuss and hypothesize how its morphology contributes to the establishment of the epithelial barrier.
Assuntos
Epitélio , Junções Íntimas , Junções Íntimas/química , Junções Íntimas/fisiologiaRESUMO
Cell adhesion molecules, including integrins, cadherins, and claudins (CLDNs), are known to activate Src-family kinases (SFKs) that organize a variety of physiological and pathological processes; however, the underlying molecular basis remains unclear. Here, we identify the SFK members that are coupled with the CLDN6-adhesion signaling. Among SFK subtypes, BLK, FGR, HCK, and SRC were highly expressed in F9 cells and concentrated with CLDN6 along cell borders during epithelial differentiation. Immunoprecipitation assay showed that BLK and SRC, but not FGR or HCK, form a complex with CLDN6 via the C-terminal cytoplasmic domain. We also demonstrated, by pull-down assay, that recombinant BLK and SRC proteins directly bind to the C-terminal cytoplasmic domain of CLDN6 (CLDN6C). Unexpectedly, both recombinant SFK proteins recognized the CLDN6C peptide in a phosphotyrosine-independent manner. Furthermore, by comparing phenotypes of F9:Cldn6:Blk-/- and F9:Cldn6:Src-/- cells with those of wild-type F9 and F9:Cldn6 cells, we revealed that BLK and SRC are essential for CLDN6-triggered cellular events, namely epithelial differentiation and the expression of retinoid acid receptor target genes. These results indicate that selective SFK members appear to participate in the CLDN-adhesion signaling.
Assuntos
Transdução de Sinais , Quinases da Família src , Quinases da Família src/metabolismo , Moléculas de Adesão Celular , Integrinas , Receptores do Ácido Retinoico , Claudinas/genética , Claudinas/metabolismoRESUMO
TJs maintain the epithelial barrier by regulating paracellular permeability. Since TJs are under dynamically fluctuating intercellular tension, cells must continuously survey and repair any damage. However, the underlying mechanisms allowing cells to sense TJ damage and repair the barrier are not yet fully understood. Here, we showed that proteinases play an important role in the maintenance of the epithelial barrier. At TJ break sites, EpCAM-claudin-7 complexes on the basolateral membrane become accessible to apical membrane-anchored serine proteinases (MASPs) and the MASPs cleave EpCAM. Biochemical data and imaging analysis suggest that claudin-7 released from EpCAM contributes to the rapid repair of damaged TJs. Knockout (KO) of MASPs drastically reduced barrier function and live-imaging of TJ permeability showed that MASPs-KO cells exhibited increased size, duration, and frequency of leaks. Together, our results reveal a novel mechanism of TJ maintenance through the localized proteolysis of EpCAM at TJ leaks, and provide a better understanding of the dynamic regulation of epithelial permeability.
Assuntos
Claudinas , Molécula de Adesão da Célula Epitelial , Serina Proteases Associadas a Proteína de Ligação a Manose , Junções Íntimas , Claudinas/genética , Claudinas/metabolismo , Molécula de Adesão da Célula Epitelial/genética , Molécula de Adesão da Célula Epitelial/metabolismo , Serina Proteases Associadas a Proteína de Ligação a Manose/metabolismo , Proteólise , Junções Íntimas/metabolismo , Técnicas de Inativação de GenesRESUMO
BACKGROUND: Ovarian cancer has the worst outcome among gynecological malignancies; therefore, biomarkers that could contribute to the early diagnosis and/or prognosis prediction are urgently required. In the present study, we focused on the secreted protein spondin-1 (SPON1) and clarified the prognostic relevance in ovarian cancer. METHODS: We developed a monoclonal antibody (mAb) that selectively recognizes SPON1. Using this specific mAb, we determined the expression of SPON1 protein in the normal ovary, serous tubal intraepithelial carcinoma (STIC), and ovarian cancer tissues, as well as in various normal adult tissues by immunohistochemistry, and verified its clinicopathological significance in ovarian cancer. RESULTS: The normal ovarian tissue was barely positive for SPON1, and no immunoreactive signals were detected in other healthy tissues examined, which was in good agreement with data obtained from gene expression databases. By contrast, upon semi-quantification, 22 of 242 ovarian cancer cases (9.1%) exhibited high SPON1 expression, whereas 64 (26.4%), 87 (36.0%), and 69 (28.5%) cases, which were designated as SPON1-low, possessed the moderate, weak, and negative SPON1 expression, respectively. The STIC tissues also possessed SPON1-positive signals. The 5-year recurrence-free survival (RFS) rate in the SPON1-high group (13.6%) was significantly lower than that in the SPON1-low group (51.2%). In addition, high SPON1 expression was significantly associated with several clinicopathological variables. Multivariable analysis revealed that high SPON1 was an independent prognostic factor for RFS of ovarian cancer. CONCLUSIONS: SPON1 represents a prognostic biomarker for ovarian cancer, and the anti-SPON1 mAb could be valuable as an outcome predictor.