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Interaction of α Carboxyl Terminus 1 Peptide With the Connexin 43 Carboxyl Terminus Preserves Left Ventricular Function After Ischemia-Reperfusion Injury.
Jiang J, Hoagland D, Palatinus JA, He H, Iyyathurai J, Jourdan LJ, Bultynck G, Wang Z, Zhang Z, Schey K, Poelzing S, McGowan FX, Gourdie RG
(2019) J Am Heart Assoc 8: e012385
MeSH Terms: Animals, Computer Simulation, Connexin 43, Mice, Microscopy, Confocal, Myocardial Contraction, Myocardial Reperfusion Injury, Peptide Fragments, Phosphorylation, Surface Plasmon Resonance, Tandem Mass Spectrometry, Ventricular Function, Left, Zonula Occludens-1 Protein
Show Abstract · Added March 24, 2020
Background α Carboxyl terminus 1 (αCT1) is a 25-amino acid therapeutic peptide incorporating the zonula occludens-1 (ZO-1)-binding domain of connexin 43 (Cx43) that is currently in phase 3 clinical testing on chronic wounds. In mice, we reported that αCT1 reduced arrhythmias after cardiac injury, accompanied by increases in protein kinase Cε phosphorylation of Cx43 at serine 368. Herein, we characterize detailed molecular mode of action of αCT1 in mitigating cardiac ischemia-reperfusion injury. Methods and Results To study αCT1-mediated increases in phosphorylation of Cx43 at serine 368, we undertook mass spectrometry of protein kinase Cε phosphorylation assay reactants. This indicated potential interaction between negatively charged residues in the αCT1 Asp-Asp-Leu-Glu-Iso sequence and lysines (Lys345, Lys346) in an α-helical sequence (helix 2) within the Cx43-CT. In silico modeling provided further support for this interaction, indicating that αCT1 may interact with both Cx43 and ZO-1. Using surface plasmon resonance, thermal shift, and phosphorylation assays, we characterized a series of αCT1 variants, identifying peptides that interacted with either ZO-1-postsynaptic density-95/disks large/zonula occludens-1 2 or Cx43-CT, but with limited or no ability to bind both molecules. Only peptides competent to interact with Cx43-CT, but not ZO-1-postsynaptic density-95/disks large/zonula occludens-1 2 alone, prompted increased pS368 phosphorylation. Moreover, in an ex vivo mouse model of ischemia-reperfusion injury, preischemic infusion only with those peptides competent to bind Cx43 preserved ventricular function after ischemia-reperfusion. Interestingly, a short 9-amino acid variant of αCT1 (αCT11) demonstrated potent cardioprotective effects when infused either before or after ischemic injury. Conclusions Interaction of αCT1 with the Cx43, but not ZO-1, is correlated with cardioprotection. Pharmacophores targeting Cx43-CT could provide a translational approach to preserving heart function after ischemic injury.
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13 MeSH Terms
Metabolic consequences of inflammatory disruption of the blood-brain barrier in an organ-on-chip model of the human neurovascular unit.
Brown JA, Codreanu SG, Shi M, Sherrod SD, Markov DA, Neely MD, Britt CM, Hoilett OS, Reiserer RS, Samson PC, McCawley LJ, Webb DJ, Bowman AB, McLean JA, Wikswo JP
(2016) J Neuroinflammation 13: 306
MeSH Terms: Blood-Brain Barrier, Brain, Claudin-5, Cytokines, Dose-Response Relationship, Drug, Humans, Interleukin-1beta, Lab-On-A-Chip Devices, Lipopolysaccharides, Metabolic Networks and Pathways, Models, Biological, Protein Transport, Tight Junctions, Time Factors, Tumor Necrosis Factor-alpha, Zonula Occludens-1 Protein
Show Abstract · Added April 26, 2017
BACKGROUND - Understanding blood-brain barrier responses to inflammatory stimulation (such as lipopolysaccharide mimicking a systemic infection or a cytokine cocktail that could be the result of local or systemic inflammation) is essential to understanding the effect of inflammatory stimulation on the brain. It is through the filter of the blood-brain barrier that the brain responds to outside influences, and the blood-brain barrier is a critical point of failure in neuroinflammation. It is important to note that this interaction is not a static response, but one that evolves over time. While current models have provided invaluable information regarding the interaction between cytokine stimulation, the blood-brain barrier, and the brain, these approaches-whether in vivo or in vitro-have often been only snapshots of this complex web of interactions.
METHODS - We utilize new advances in microfluidics, organs-on-chips, and metabolomics to examine the complex relationship of inflammation and its effects on blood-brain barrier function ex vivo and the metabolic consequences of these responses and repair mechanisms. In this study, we pair a novel dual-chamber, organ-on-chip microfluidic device, the NeuroVascular Unit, with small-volume cytokine detection and mass spectrometry analysis to investigate how the blood-brain barrier responds to two different but overlapping drivers of neuroinflammation, lipopolysaccharide and a cytokine cocktail of IL-1β, TNF-α, and MCP1,2.
RESULTS - In this study, we show that (1) during initial exposure to lipopolysaccharide, the blood-brain barrier is compromised as expected, with increased diffusion and reduced presence of tight junctions, but that over time, the barrier is capable of at least partial recovery; (2) a cytokine cocktail also contributes to a loss of barrier function; (3) from this time-dependent cytokine activation, metabolic signature profiles can be obtained for both the brain and vascular sides of the blood-brain barrier model; and (4) collectively, we can use metabolite analysis to identify critical pathways in inflammatory response.
CONCLUSIONS - Taken together, these findings present new data that allow us to study the initial effects of inflammatory stimulation on blood-brain barrier disruption, cytokine activation, and metabolic pathway changes that drive the response and recovery of the barrier during continued inflammatory exposure.
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16 MeSH Terms
Diet-induced obesity in male C57BL/6 mice decreases fertility as a consequence of disrupted blood-testis barrier.
Fan Y, Liu Y, Xue K, Gu G, Fan W, Xu Y, Ding Z
(2015) PLoS One 10: e0120775
MeSH Terms: Animals, Blood-Testis Barrier, Body Weight, Clathrin, Diet, High-Fat, Estradiol, Female, Fertility, Lipids, Male, Mice, Mice, Inbred C57BL, Models, Animal, Obesity, Occludin, Receptors, Androgen, Sertoli Cells, Sperm Motility, Spermatozoa, Testis, Testosterone, Zonula Occludens-1 Protein
Show Abstract · Added December 4, 2020
Obesity is a complex metabolic disease that is a serious detriment to both children and adult health, which induces a variety of diseases, such as cardiovascular disease, type II diabetes, hypertension and cancer. Although adverse effects of obesity on female reproduction or oocyte development have been well recognized, its harmfulness to male fertility is still unclear because of reported conflicting results. The aim of this study was to determine whether diet-induced obesity impairs male fertility and furthermore to uncover its underlying mechanisms. Thus, male C57BL/6 mice fed a high-fat diet (HFD) for 10 weeks served as a model of diet-induced obesity. The results clearly show that the percentage of sperm motility and progressive motility significantly decreased, whereas the proportion of teratozoospermia dramatically increased in HFD mice compared to those in normal diet fed controls. Besides, the sperm acrosome reaction fell accompanied by a decline in testosterone level and an increase in estradiol level in the HFD group. This alteration of sperm function parameters strongly indicated that the fertility of HFD mice was indeed impaired, which was also validated by a low pregnancy rate in their mated normal female. Moreover, testicular morphological analyses revealed that seminiferous epithelia were severely atrophic, and cell adhesions between spermatogenic cells and Sertoli cells were loosely arranged in HFD mice. Meanwhile, the integrity of the blood-testis barrier was severely interrupted consistent with declines in the tight junction related proteins, occludin, ZO-1 and androgen receptor, but instead endocytic vesicle-associated protein, clathrin rose. Taken together, obesity can impair male fertility through declines in the sperm function parameters, sex hormone level, whereas during spermatogenesis damage to the blood-testis barrier (BTB) integrity may be one of the crucial underlying factors accounting for this change.
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MeSH Terms
Alveolar epithelial cells undergo epithelial-to-mesenchymal transition in response to endoplasmic reticulum stress.
Tanjore H, Cheng DS, Degryse AL, Zoz DF, Abdolrasulnia R, Lawson WE, Blackwell TS
(2011) J Biol Chem 286: 30972-80
MeSH Terms: Acetylcysteine, Animals, Cadherins, Endoplasmic Reticulum, Epithelium, Fibrosis, Humans, Membrane Proteins, Mesoderm, Mice, Microscopy, Phase-Contrast, Models, Biological, Mutation, Phosphoproteins, Pulmonary Alveoli, Pulmonary Surfactant-Associated Protein C, Rats, Tunicamycin, Zonula Occludens-1 Protein
Show Abstract · Added March 5, 2014
Expression of mutant surfactant protein C (SFTPC) results in endoplasmic reticulum (ER) stress in type II alveolar epithelial cells (AECs). AECs have been implicated as a source of lung fibroblasts via epithelial-to-mesenchymal transition (EMT); therefore, we investigated whether ER stress contributes to EMT as a possible mechanism for fibrotic remodeling. ER stress was induced by tunicamyin administration or stable expression of mutant (L188Q) SFTPC in type II AEC lines. Both tunicamycin treatment and mutant SFTPC expression induced ER stress and the unfolded protein response. With tunicamycin or mutant SFTPC expression, phase contrast imaging revealed a change to a fibroblast-like appearance. During ER stress, expression of epithelial markers E-cadherin and Zonula occludens-1 decreased while expression of mesenchymal markers S100A4 and α-smooth muscle actin increased. Following induction of ER stress, we found activation of a number of pathways, including MAPK, Smad, β-catenin, and Src kinase. Using specific inhibitors, the combination of a Smad2/3 inhibitor (SB431542) and a Src kinase inhibitor (PP2) blocked EMT with maintenance of epithelial appearance and epithelial marker expression. Similar results were noted with siRNA targeting Smad2 and Src kinase. Together, these studies reveal that induction of ER stress leads to EMT in lung epithelial cells, suggesting possible cross-talk between Smad and Src kinase pathways. Dissecting pathways involved in ER stress-induced EMT may lead to new treatment strategies to limit fibrosis.
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19 MeSH Terms
Raised intensity phonation compromises vocal fold epithelial barrier integrity.
Rousseau B, Suehiro A, Echemendia N, Sivasankar M
(2011) Laryngoscope 121: 346-51
MeSH Terms: Animals, Cadherins, Epithelium, Gene Expression, Membrane Proteins, Microscopy, Electron, Scanning, Microscopy, Electron, Transmission, Occludin, Phonation, Phosphoproteins, Polymerase Chain Reaction, Rabbits, Tight Junctions, Vibration, Vocal Cords, Zonula Occludens-1 Protein, beta Catenin
Show Abstract · Added May 29, 2014
OBJECTIVES/HYPOTHESIS - We investigated the hypothesis that 30 minutes of raised intensity phonation alters transcript levels of vocal fold intercellular tight junction proteins and disrupts the vocal fold epithelial barrier.
STUDY DESIGN - Prospective animal study.
METHODS - Eighteen New Zealand white breeder rabbits were randomly assigned to receive 30 minutes of raised intensity phonation or approximation of the vocal folds without phonation. Quantitative polymerase chain reaction (qPCR) was used to investigate transcript levels of the epithelial intercellular tight junction proteins, occludin and zonula occludin-1 (ZO-1), and the adherens junction proteins β-catenin and E-cadherin. Structural alterations to the vocal fold epithelium were further examined by scanning electron microscopy (SEM) and transmission electron microscopy (TEM).
RESULTS - Mann-Whitney U revealed significantly decreased occludin (P = .016) and β-catenin (P = .016) gene expression from rabbits undergoing raised intensity phonation compared with control. There were no significant differences in Z0-1 and E-cadherin gene expression between groups (P > .025). SEM revealed significant obliteration, desquamation, and evidence of microhole formation in rabbit vocal folds exposed to raised intensity phonation compared with control, whereas TEM revealed dilated intercellular morphology between groups.
CONCLUSIONS - Results provide support for the hypothesis that a transient episode of raised intensity phonation alters transcript levels of vocal fold intercellular tight junction proteins and disrupts integrity of the epithelial barrier. The loss of barrier integrity may have significant consequences on epithelial defenses and compromise protection of the underlying mucosa from damage secondary to prolonged vibration exposure.
Copyright © 2011 The American Laryngological, Rhinological, and Otological Society, Inc.
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17 MeSH Terms
Tight junction-associated signaling pathways modulate cell proliferation in uveal melanoma.
Jayagopal A, Yang JL, Haselton FR, Chang MS
(2011) Invest Ophthalmol Vis Sci 52: 588-93
MeSH Terms: Blotting, Western, CCAAT-Enhancer-Binding Proteins, Cell Adhesion Molecules, Cell Cycle, Cell Line, Tumor, Cell Proliferation, Flow Cytometry, Fluorescent Antibody Technique, Indirect, Genetic Vectors, Heat-Shock Proteins, Humans, Melanoma, Membrane Proteins, Muscle Proteins, Occludin, Phosphoproteins, Signal Transduction, Tight Junctions, Transcription Factors, Transfection, Uveal Neoplasms, Zonula Occludens-1 Protein
Show Abstract · Added May 27, 2014
PURPOSE - To investigate the role of tight junction (TJ)-associated signaling pathways in the proliferation of uveal melanoma.
METHODS - Human uveal melanoma cell lines overexpressing the TJ molecule blood vessel epicardial substance (Bves) were generated. The effects of Bves overexpression on TJ protein expression, cell proliferation, and cell cycle distribution were quantified. In addition, localization and transcription activity of the TJ-associated protein ZO-1-associated nucleic acid binding protein (ZONAB) were evaluated using immunofluorescence and bioluminescence reporter assays to study the involvement of Bves signaling in cell proliferation-associated pathways.
RESULTS - Bves overexpression in uveal melanoma cell lines resulted in increased expression of the TJ proteins occludin and ZO-1, reduced cell proliferation, and increased sequestration of ZONAB at TJs and reduced ZONAB transcriptional activity.
CONCLUSIONS - TJ proteins are present in uveal melanoma, and TJ-associated signaling pathways modulate cell signaling pathways relevant to proliferation in uveal melanoma.
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22 MeSH Terms
A microfluidic bioreactor with integrated transepithelial electrical resistance (TEER) measurement electrodes for evaluation of renal epithelial cells.
Ferrell N, Desai RR, Fleischman AJ, Roy S, Humes HD, Fissell WH
(2010) Biotechnol Bioeng 107: 707-16
MeSH Terms: Animals, Bioreactors, Cell Proliferation, Dogs, Electric Impedance, Electrodes, Epithelial Cells, Fluorescent Antibody Technique, Humans, Membrane Proteins, Microfluidic Analytical Techniques, Phosphoproteins, Staining and Labeling, Tight Junctions, Tubulin, Zonula Occludens-1 Protein
Show Abstract · Added August 21, 2013
We have developed a bilayer microfluidic system with integrated transepithelial electrical resistance (TEER) measurement electrodes to evaluate kidney epithelial cells under physiologically relevant fluid flow conditions. The bioreactor consists of apical and basolateral fluidic chambers connected via a transparent microporous membrane. The top chamber contains microfluidic channels to perfuse the apical surface of the cells. The bottom chamber acts as a reservoir for transport across the cell layer and provides support for the membrane. TEER electrodes were integrated into the device to monitor cell growth and evaluate cell-cell tight junction integrity. Immunofluorescence staining was performed within the microchannels for ZO-1 tight junction protein and acetylated α-tubulin (primary cilia) using human renal epithelial cells (HREC) and MDCK cells. HREC were stained for cytoskeletal F-actin and exhibited disassembly of cytosolic F-actin stress fibers when exposed to shear stress. TEER was monitored over time under normal culture conditions and after disruption of the tight junctions using low Ca(2+) medium. The transport rate of a fluorescently labeled tracer molecule (FITC-inulin) was measured before and after Ca(2+) switch and a decrease in TEER corresponded with a large increase in paracellular inulin transport. This bioreactor design provides an instrumented platform with physiologically meaningful flow conditions to study various epithelial cell transport processes.
© 2010 Wiley Periodicals, Inc.
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16 MeSH Terms
ZO-1 and ZONAB interact to regulate proximal tubular cell differentiation.
Pozzi A, Zent R
(2010) J Am Soc Nephrol 21: 388-90
MeSH Terms: Animals, Cell Differentiation, DNA-Binding Proteins, Kidney Tubules, Proximal, Membrane Proteins, Mice, Phosphoproteins, Transcription Factors, Zonula Occludens-1 Protein
Added February 24, 2014
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9 MeSH Terms
Inhibition of RhoA signaling with increased Bves in trabecular meshwork cells.
Russ PK, Kupperman AI, Presley SH, Haselton FR, Chang MS
(2010) Invest Ophthalmol Vis Sci 51: 223-30
MeSH Terms: Avian Proteins, Blotting, Western, Cell Adhesion Molecules, Cell Line, Electric Impedance, Fluorescein, Fluorescent Antibody Technique, Indirect, Humans, Membrane Proteins, Microfilament Proteins, Muscle Proteins, Myosin Light Chains, Occludin, Permeability, Phosphoproteins, Phosphorylation, Signal Transduction, Tight Junctions, Trabecular Meshwork, Transfection, Zonula Occludens-1 Protein, rhoA GTP-Binding Protein
Show Abstract · Added May 27, 2014
PURPOSE - Blood vessel epicardial substance (Bves) is a novel adhesion molecule that regulates tight junction (TJ) formation. TJs also modulate RhoA signaling, which has been implicated in outflow regulation. Given that Bves has been reported in multiple ocular tissues, the authors hypothesize that Bves plays a role in the regulation of RhoA signaling in trabecular meshwork (TM) cells.
METHODS - Human TM cell lines NTM-5 and NTM-5 transfected to overexpress Bves (NTM-w) were evaluated for TJ formation, and levels of occludin, cingulin, and ZO-1 protein were compared. Assays of TJ function were carried out using diffusion of sodium fluorescein and transcellular electrical resistance (TER). Levels of activated RhoA were measured using FRET probes, and phosphorylation of myosin light chain (MLC-p), a downstream target of RhoA, was assessed by Western blot analysis.
RESULTS - Overexpression of Bves led to increased TJ formation in NTM-5 cells. Increased TJ formation was confirmed by increased occludin, cingulin, and ZO-1 protein. Functionally, NTM-w cells showed decreased permeability and increased TER compared with NTM-5 cells, consistent with increased TJ formation. NTM-w cells also exhibited decreased levels of active RhoA and lower levels of MLC-p than did NTM-5 cells. These findings support a TJ role in RhoA signaling.
CONCLUSIONS - Increased Bves in TM cells leads to increased TJ formation with decreased RhoA activation and decreased MLC-p. This is the first report of a regulatory pathway upstream of RhoA in TM cells. In TM tissue, RhoA has been implicated in outflow regulation; thus, Bves may be a key regulatory molecule in aqueous outflow.
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22 MeSH Terms
Phosphorylation of Tyr-398 and Tyr-402 in occludin prevents its interaction with ZO-1 and destabilizes its assembly at the tight junctions.
Elias BC, Suzuki T, Seth A, Giorgianni F, Kale G, Shen L, Turner JR, Naren A, Desiderio DM, Rao R
(2009) J Biol Chem 284: 1559-69
MeSH Terms: Animals, CSK Tyrosine-Protein Kinase, Caco-2 Cells, Chickens, Dogs, Humans, Hydrogen Peroxide, Mass Spectrometry, Membrane Proteins, Mutation, Missense, Occludin, Oxidants, Phosphoproteins, Phosphorylation, Protein Binding, Protein Structure, Tertiary, Protein-Tyrosine Kinases, Proto-Oncogene Proteins, Rats, Tight Junctions, Tyrosine, Zonula Occludens-1 Protein, src-Family Kinases
Show Abstract · Added February 25, 2014
Occludin is phosphorylated on tyrosine residues during the oxidative stress-induced disruption of tight junction, and in vitro phosphorylation of occludin by c-Src attenuates its binding to ZO-1. In the present study mass spectrometric analyses of C-terminal domain of occludin identified Tyr-379 and Tyr-383 in chicken occludin as the phosphorylation sites, which are located in a highly conserved sequence of occludin, YETDYTT; Tyr-398 and Tyr-402 are the corresponding residues in human occludin. Deletion of YETDYTT motif abolished the c-Src-mediated phosphorylation of occludin and the regulation of ZO-1 binding. Y398A and Y402A mutations in human occludin also abolished the c-Src-mediated phosphorylation and regulation of ZO-1 binding. Y398D/Y402D mutation resulted in a dramatic reduction in ZO-1 binding even in the absence of c-Src. Similar to wild type occludin, its Y398A/Y402A mutant was localized at the plasma membrane and cell-cell contact sites in Rat-1 cells. However, Y398D/Y402D mutants of occludin failed to localize at the cell-cell contacts. Calcium-induced reassembly of Y398D/Y402D mutant occludin in Madin-Darby canine kidney cells was significantly delayed compared with that of wild type occludin or its T398A/T402A mutant. Furthermore, expression of Y398D/Y402D mutant of occludin sensitized MDCK cells for hydrogen peroxide-induced barrier disruption. This study reveals a unique motif in the occludin sequence that is involved in the regulation of ZO-1 binding by reversible phosphorylation of specific Tyr residues.
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23 MeSH Terms