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tight junction [keywords]
- Farrerol regulates occludin expression in hydrogen peroxide-induced EA.hy926 cells by modulating ERK1/2 activity. [JOURNAL ARTICLE]
- Eur J Pharmacol 2014 Apr 12.
Endothelial tight junction is a crucial intracellular junctional structure that controls paracellular permeability across vascular endothelium. Oxidative stress-mediated elevation in endothelial permeability is associated with pathogenesis of several cardiovascular diseases. In the present research, the regulation of farrerol on occludin, a transmembrane proteins associated with endothelial tight junction, was investigated in hydrogen peroxide-induced human endothelium-derived EA.hy926 cells. Western blot analysis demonstrated that H2O2 exposure caused a significant decrease in occludin expression, but had little effect on ZO-1 expression, and the decrease of occludin expression was significantly attenuated by farrerol in a dose-dependent manner. Meanwhile, immunofluorescent staining assay also demonstrated that the loss of occludin expression induced by H2O2 exposure was restored by farrerol pretreatment. Further investigations showed that farrerol prevented H2O2-induced activation of extracellular signal-regulated kinase (ERK) 1/2 in a dose-dependent manner. The use of U0126, a specific inhibitor of MEK1/2, proved that H2O2-induced decrease of occludin in EA.hy926 cells was likely associated with activation of ERK1/2, which indicated that the regulation of farrerol on occludin expression in H2O2-induced EA.hy926 cells was likely related to the modulation of ERK1/2 activation. In conclusion, the present study demonstrates for the first time that farrerol has potential effects on oxidative stress-induced endothelial tight junction disruption and suggests that farrerol is a potential candidate for the intervention of endothelial permeability-associated cardiovascular diseases.
- Molecular insights into NF2/Merlin tumor suppressor function. [REVIEW]
- FEBS Lett 2014 Apr 9.
The FERM domain protein Merlin, encoded by the NF2 tumor suppressor gene, regulates cell proliferation in response to adhesive signaling. The growth inhibitory function of Merlin is induced by intercellular adhesion and inactivated by joint integrin/receptor tyrosine kinase signaling. Merlin contributes to the formation of cell junctions in polarized tissues, activates anti-mitogenic signaling at tight-junctions, and inhibits oncogenic gene expression. Thus, inactivation of Merlin causes uncontrolled mitogenic signaling and tumorigenesis. Merlin's predominant tumor suppressive functions are attributable to its control of oncogenic gene expression through regulation of Hippo signaling. Notably, Merlin translocates to the nucleus where it directly inhibits the CRL4(DCAF1) E3 ubiquitin ligase, thereby suppressing inhibition of the Lats kinases. A dichotomy in NF2 function has emerged whereby Merlin acts at the cell cortex to organize cell junctions and propagate anti-mitogenic signaling, whereas it inhibits oncogenic gene expression through the inhibition of CRL4(DCAF1) and activation of Hippo signaling. The biochemical events underlying Merlin's normal function and tumor suppressive activity will be discussed in this Review, with emphasis on recent discoveries that have greatly influenced our understanding of Merlin biology.
- Cyclic Stretch Disrupts Apical Junctional Complexes in Caco-2 Cell Monolayers by a JNK-2, c-Src and MLCK-Dependent Mechanism. [JOURNAL ARTICLE]
- Am J Physiol Gastrointest Liver Physiol 2014 Apr 10.
The intestinal epithelium is subjected to various types of mechanical stress. In this study, we investigated the impact of cyclic stretch on tight junction and adherens junction integrity in Caco-2 cell monolayers. Stretch for 2 hours resulted in dramatic modulation of tight junction protein distribution from linear organization into wavy structure. Continuation of cyclic stretch for 6 hours led to redistribution of tight junction proteins from the intercellular junctions into intracellular compartment. Disruption of tight junctions was associated with redistribution of adherens junction proteins, E-cadherin and β-catenin, and dissociation of the actin cytoskeleton at the actomyosin belt. Stretch activates JNK2, c-Src and MLCK. Inhibition of JNK, Src kinase or MLCK activity and knockdown of JNK2 or c-Src attenuated stretch-induced disruption of tight junctions, adherens junctions and actin cytoskeleton. Stretch increased tyrosine phosphorylation of occludin, ZO-1, E-cadherin and β-catenin. Inhibition of JNK or Src kinase attenuated stretch-induced occludin phosphorylation. Immunofluorescence localization indicated that phospho-MLC co-localizes with the vesicle-like actin structure at the actomyosin belt in stretched cells. On the other hand, phospho-c-Src co-localizes with the actin at the apical region of cells. This study demonstrates that cyclic stretch disrupts tight junctions and adherens junctions by a JNK2, c-Src and MLCK-dependent mechanism.
- The Altered Tight Junctions: An Important Gateway of Bacterial Translocation in Cachexia Patients with Advanced Gastric Cancer. [JOURNAL ARTICLE]
- J Interferon Cytokine Res 2014 Apr 10.
Tight junctions (TJs) are the structural basis for the intestinal epithelium barrier. Increased intestinal permeability caused by variations in TJ proteins may result in bacterial translocation (BT). There is increasing evidence that BT might contribute to the occurrence and development of cancer cachexia, but the details are not known. Aims, we undertook further investigations into the pathway of BT in cancer cachexia. Results: BT-positive patients had a higher level of claudins-2 (CL-2, P=0.035) and a lower level of occludin (P=0.038) and Zonula occluden-1 (P=0.01) than BT-negative patients. Moreover, the levels of IL-6, TNF-α, and IFN-γ in BT-positive cachexia patients were higher compared with BT-negative cachexia patients (P<0.001, P=0.01, P<0.001) and BT-positive noncachexia patients (P<0.001, P=0.025, P<0.001). In the BT-positive cachexia patients, the local concentration of IL-6, TNF-α, and IFN-γ, in the middle colic vein, was higher than in the peripheral venous (P=0.04, P=0.03, P=0.038). In addition, endotoxin was detected within the small intestinal wall, and the concentration of endotoxin decreased from the mucosal side to the serosal side gradually in BT-positive patients. This study suggests that the altered TJs could be an important gateway of BT in gastric cancer cachexia and local cytokines could play a more important role than systemic cytokines in the process.
- Immunological and molecular targets of atopic dermatitis treatment. [JOURNAL ARTICLE]
- Br J Dermatol 2014 Apr 11.
Atopic dermatitis (AD) is a common, chronic inflammatory skin disease with a highly variable clinical phenotype and heterogeneous pathophysiology. Its pathogenesis is associated with alterations to both the skin barrier and the immune system, which may in turn be influenced by genetic mutations and the patient's environment. Basic and translational research, as well as clinical trials, has helped broaden our knowledge of the molecular mechanisms underlying the development of AD and to identify potential treatment targets and approaches. These include new ways of reducing transepidermal water loss and the shedding of corneocytes, new ways of interacting with established molecular targets (such as histamine receptors and interleukins and other T-cell cytokines), and the identification of new molecular targets (such as toll-like receptors and tight junction proteins). Well-established treatment options such as emollients, corticosteroids and topical calcineurin inhibitors will clearly continue to have a role in treating AD. Among the new agents that could be joining them in the near future are sphinganin (a precursor of ceramides 1 and 3), cannabinoids, highly targeted monoclonal antibodies and subcutaneous immunotherapy. This article is protected by copyright. All rights reserved.
- A multiple-channel, multiple-assay platform for characterization of full-range shear stress effects on vascular endothelial cells. [JOURNAL ARTICLE]
- Lab Chip 2014 Apr 9.
Vascular endothelial cells (VECs), which line blood vessels and are key to understanding pathologies and treatments of various diseases, experience highly variable wall shear stress (WSS) in vivo (1-60 dyn cm(-2)), imposing numerous effects on physiological and morphological functions. Previous flow-based systems for studying these effects have been limited in range, and comprehensive information on VEC functions at the full spectrum of WSS has not been available yet. To allow rapid characterization of WSS effects, we developed the first multiple channel microfluidic platform that enables a wide range (~15×) of homogeneous WSS conditions while simultaneously allowing trans-monolayer assays, such as permeability and trans-endothelial electrical resistance (TEER) assays, as well as cell morphometry and protein expression assays. Flow velocity/WSS distributions between channels were predicted with COMSOL simulations and verified by measurement using an integrated microflow sensor array. Biomechanical responses of the brain microvascular endothelial cell line bEnd.3 to the full natural spectrum of WSS were investigated with the platform. Under increasing WSS conditions ranging from 0 to 86 dyn cm(-2), (1) permeabilities of FITC-conjugated dextran and propidium iodide decreased, respectively, at rates of 4.06 × 10(-8) and 6.04 × 10(-8) cm s(-1) per dyn cm(-2); (2) TEER increased at a rate of 0.8 Ω cm(2) per dyn cm(-2); (3) increased alignment of cells along the flow direction under increasing WSS conditions; and finally (4) increased protein expression of both the tight junction component ZO-1 (~5×) and the efflux transporter P-gp (~6×) was observed at 86 dyn cm(-2) compared to static controls via western blot. We conclude that the presented microfluidic platform is a valid approach for comprehensively assaying cell responses to fluidic WSS.
- Modulation of cell polarization by the Na+/K+ ATPase associated protein FXYD5 (dysadherin). [JOURNAL ARTICLE]
- Am J Physiol Cell Physiol 2014 Apr 9.
FXYD5 (dysadherin or RIC) is a transmembrane auxiliary subunit of the Na(+)/K(+) ATPase shown to increase its Vmax. FXYD5 has also been identified as a cancer associated protein whose expression in tumor derived cell lines impairs cytoskeletal organization and increases cell motility. Previously, we have demonstrated that the expression of FXYD5 in M1 cells derived from mouse kidney collecting duct, impairs the formation of tight and adherence junctions. The current study aimed to further explore effects of FXYD5 at a single cell level. It was found that in M1 as well as three other cell lines, FXYD5 inhibits transformation of adhered single cells from the initial radial shape to a flattened, elongated shape, in the first stage of monolayer formation. This is also correlated to less ordered actin cables and fewer focal points. Structure-function analysis has demonstrated that the transmembrane domain of FXYD5, and not its unique extracellular segment, mediates the inhibition of change in cell shape. This domain has been shown before to be involved in the association of FXYD5 with the Na(+)/K(+) ATPase which leads to the increase in Vmax. Furthermore, specific transmembrane point mutations in FXYD5 that either increase or decrease its effect on cell elongation had a corresponding effect on the co-immunoprecipitation of FXYD5 with αNa(+)/K(+) ATPase . These findings lend support to the possibility that FXYD5 affects cell polarization through its transmembrane domain interaction with the Na(+)/K(+) ATPase. Yet interaction of FXYD5 with other proteins can not be excluded.
- Inhibition of JNK in HaCaT cells induced tight junction formation with decreased expression of cytokeratin 5, cytokeratin 17 and desmoglein 3. [JOURNAL ARTICLE]
- Histochem Cell Biol 2014 Apr 9.
Epidermal keratinocytes proliferate in the basal layer, differentiate, migrate through the spinous layer, granular layer and cornified layer, and finally are peeled off from the surface of skin with layer-specific expression of differentiation markers, including cytokeratins and cell-cell junction proteins such as desmogleins. Basal cells express CK5, CK14 and Ki67. In contrast, the suprabasal cells in the spinous and granular layers express CK1 and CK10 without Ki67. Inhibition of c-Jun NH2-terminal protein kinase (JNK) in HaCaT cells, a human epidermal keratinocyte cell line, induced the formation of tight junctions, which occurs in the granular layer in vivo. These cells lost their expression of CK5 and CK17, exhibited decreased expression of desmoglein 3 and had no Ki67 labeling in the nucleus. These results suggest that inhibition of JNK causes HaCaT cells to differentiate from basal- and spinous-like cells to granular-like cells. The inhibition of JNK in HaCaT cells provides a useful in vitro model system to study the differentiation of epidermal keratinocytes.
- Blood-testis barrier and spermatogenesis: lessons from genetically-modified mice. [JOURNAL ARTICLE]
- Asian J Androl 2014 Mar 28.
The blood-testis barrier (BTB) is found between adjacent Sertoli cells in the testis where it creates a unique microenvironment for the development and maturation of meiotic and postmeiotic germ cells in seminiferous tubes. It is a compound proteinous structure, composed of several types of cell junctions including tight junctions (TJs), adhesion junctions and gap junctions (GJs). Some of the junctional proteins function as structural proteins of BTB and some have regulatory roles. The deletion or functional silencing of genes encoding these proteins may disrupt the BTB, which may cause immunological or other damages to meiotic and postmeiotic cells and ultimately lead to spermatogenic arrest and infertility. In this review, we will summarize the findings on the BTB structure and function from genetically-modified mouse models and discuss the future perspectives.
- In Vivo Two-photon Fluorescence Microscopy Reveals Disturbed Cerebral Capillary Blood Flow and Increased Susceptibility to Ischemic Insults in Diabetic Mice. [JOURNAL ARTICLE]
- CNS Neurosci Ther 2014 Apr 8.
Diabetes mellitus increases the risk of stroke, but the mechanisms are unclear. The present study tested the hypothesis that diabetes mellitus disturbs the brain microcirculation and increases the susceptibility to cerebral damage in a middle cerebral artery occlusion (MCAO) model of ischemia.Diabetes was induced by streptozocin in mice expressing green fluorescent protein in endothelial cells (Tie2-GFP mice). Four weeks later, they were subjected to transient (20 min) MCAO. In vivo blood flow was measured by two-photon laser-scanning microscopy (TPLSM) in cerebral arteries, veins, and capillaries.There was a significant decrease in red blood cell (RBC) velocity in capillaries in diabetic mice as assessed by TPLSM, yet the regional cerebral blood flow, as assessed by laser Doppler flowmetry, was maintained. Brain capillary flow developed turbulence after MCAO only in diabetic mice. These mice sustained increased neurological deficits after MCAO which were accompanied by an exaggerated degradation of tight junction proteins and blunted CaMKII phosphorylation in cerebral tissues indicating disruption of the blood-brain barrier and disturbed cognitive potential.Diabetic mice are more susceptible to disturbances of cerebral capillary blood flow which may predispose them to neurovascular defects following ischemia.