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Interaction of phosphorylated Rab11-FIP2 with Eps15 regulates apical junction composition.
Lapierre LA, Manning EH, Mitchell KM, Caldwell CM, Goldenring JR
(2017) Mol Biol Cell 28: 1088-1100
MeSH Terms: Adaptor Proteins, Signal Transducing, Animals, Cadherins, Carrier Proteins, Cell Polarity, Dogs, Endosomes, Epithelial Cells, Gene Knockout Techniques, HEK293 Cells, Humans, Intercellular Junctions, Madin Darby Canine Kidney Cells, Membrane Proteins, Occludin, Phosphorylation, Protein Binding, Protein Transport, rab GTP-Binding Proteins
Show Abstract · Added April 18, 2017
MARK2 regulates the establishment of polarity in Madin-Darby canine kidney (MDCK) cells in part through phosphorylation of serine 227 of Rab11-FIP2. We identified Eps15 as an interacting partner of phospho-S227-Rab11-FIP2 (pS227-FIP2). During recovery from low calcium, Eps15 localized to the lateral membrane before pS227-FIP2 arrival. Later in recovery, Eps15 and pS227-FIP2 colocalized at the lateral membrane. In MDCK cells expressing the pseudophosphorylated FIP2 mutant FIP2(S227E), during recovery from low calcium, Eps15 was trapped and never localized to the lateral membrane. Mutation of any of the three NPF domains within GFP-FIP2(S227E) rescued Eps15 localization at the lateral membrane and reestablished single-lumen cyst formation in GFP-FIP2(S227E)-expressing cells in three-dimensional (3D) culture. Whereas expression of GFP-FIP2(S227E) induced the loss of E-cadherin and occludin, mutation of any of the NPF domains of GFP-FIP2(S227E) reestablished both proteins at the apical junctions. Knockdown of Eps15 altered the spatial and temporal localization of pS227-FIP2 and also elicited formation of multiple lumens in MDCK 3D cysts. Thus an interaction of Eps15 and pS227-FIP2 at the appropriate time and location in polarizing cells is necessary for proper establishment of epithelial polarity.
© 2017 Lapierre et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).
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19 MeSH Terms
Effects of phonation time and magnitude dose on vocal fold epithelial genes, barrier integrity, and function.
Kojima T, Valenzuela CV, Novaleski CK, Van Deusen M, Mitchell JR, Garrett CG, Sivasankar MP, Rousseau B
(2014) Laryngoscope 124: 2770-8
MeSH Terms: Animals, Cadherins, Cyclooxygenase 2, Disease Models, Animal, Follow-Up Studies, Gene Expression Regulation, Interleukin-1beta, Microscopy, Electron, Scanning, Occludin, Phonation, RNA, Messenger, Rabbits, Real-Time Polymerase Chain Reaction, Time Factors, Transforming Growth Factor beta1, Vocal Cords, beta Catenin
Show Abstract · Added February 12, 2015
OBJECTIVES/HYPOTHESIS - To investigate the effects of increasing time and magnitude doses of vibration exposure on transcription of the vocal fold's junctional proteins, structural alterations, and functional tissue outcomes.
STUDY DESIGN - Animal study.
METHODS - 100 New Zealand White breeder rabbits were studied. Dependent variables were measured in response to increasing time doses (30, 60, or 120 minutes) and magnitude doses (control, modal intensity, and raised intensity) of vibration exposure. Messenger RNA expression of occludin, zonula occluden-1 (ZO-1), E-cadherin, β-catenin, interleukin 1β, cyclooxygenase-2, transforming growth factor β-1, and fibronectin were measured. Tissue structural alterations were assessed using transmission electron microscopy (TEM). Transepithelial resistance was used to measure functional tissue outcomes.
RESULTS - Occludin gene expression was downregulated in vocal folds exposed to 120-minute time doses of raised-intensity phonation, relative to control, and modal-intensity phonation. ZO-1 gene expression was upregulated following a 120-minute time dose of modal-intensity phonation, compared to control, and downregulated after a 120-minute time dose of raised-intensity phonation, compared to modal-intensity phonation. E-cadherin gene expression was downregulated after a 120-minute time dose of raised-intensity phonation, compared to control and modal-intensity phonation. TEM revealed extensive desquamation of the stratified squamous epithelial cells with increasing time and magnitude doses of vibration exposure. A general observation of lower transepithelial resistance measures was made in tissues exposed to raised-intensity phonation compared to all other groups.
CONCLUSIONS - This study provides evidence of vocal fold tissue responses to varying time and magnitude doses of vibration exposure.
LEVEL OF EVIDENCE - NA.
© 2014 The American Laryngological, Rhinological and Otological Society, Inc.
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17 MeSH Terms
Small intestinal intraepithelial TCRγδ+ T lymphocytes are present in the premature intestine but selectively reduced in surgical necrotizing enterocolitis.
Weitkamp JH, Rosen MJ, Zhao Z, Koyama T, Geem D, Denning TL, Rock MT, Moore DJ, Halpern MD, Matta P, Denning PW
(2014) PLoS One 9: e99042
MeSH Terms: Animals, Cells, Cultured, Enterocolitis, Necrotizing, Female, Gene Expression Regulation, Humans, Infant, Newborn, Infant, Premature, Interleukin-17, Intestine, Small, Male, Mice, Mice, Inbred C57BL, Occludin, Receptors, Antigen, T-Cell, gamma-delta, T-Lymphocyte Subsets
Show Abstract · Added June 10, 2014
BACKGROUND - Gastrointestinal barrier immaturity predisposes preterm infants to necrotizing enterocolitis (NEC). Intraepithelial lymphocytes (IEL) bearing the unconventional T cell receptor (TCR) γδ (γδ IEL) maintain intestinal integrity and prevent bacterial translocation in part through production of interleukin (IL) 17.
OBJECTIVE - We sought to study the development of γδ IEL in the ileum of human infants and examine their role in NEC pathogenesis. We defined the ontogeny of γδ IEL proportions in murine and human intestine and subjected tcrδ-/- mice to experimental gut injury. In addition, we used polychromatic flow cytometry to calculate percentages of viable IEL (defined as CD3+ CD8+ CD103+ lymphocytes) and the fraction of γδ IEL in surgically resected tissue from infants with NEC and gestational age matched non-NEC surgical controls.
RESULTS - In human preterm infants, the proportion of IEL was reduced by 66% in 11 NEC ileum resections compared to 30 non-NEC controls (p<0.001). While γδ IEL dominated over conventional αβ IEL early in gestation in mice and in humans, γδ IEL were preferential decreased in the ileum of surgical NEC patients compared to non-NEC controls (50% reduction, p<0.05). Loss of IEL in human NEC was associated with downregulation of the Th17 transcription factor retinoic acid-related orphan nuclear hormone receptor C (RORC, p<0.001). TCRδ-deficient mice showed increased severity of experimental gut injury (p<0.05) with higher TNFα expression but downregulation of IL17A.
CONCLUSION - Complimentary mouse and human data suggest a role of γδ IEL in IL17 production and intestinal barrier production early in life. Specific loss of the γδ IEL fraction may contribute to NEC pathogenesis. Nutritional or pharmacological interventions to support γδ IEL maintenance in the developing small intestine could serve as novel strategies for NEC prevention.
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16 MeSH Terms
Phosphorylation of Rab11-FIP2 regulates polarity in MDCK cells.
Lapierre LA, Avant KM, Caldwell CM, Oztan A, Apodaca G, Knowles BC, Roland JT, Ducharme NA, Goldenring JR
(2012) Mol Biol Cell 23: 2302-18
MeSH Terms: Adherens Junctions, Animals, Blotting, Western, Cadherins, Catenins, Cell Line, Cell Polarity, Claudins, Dogs, Epithelial Cells, Green Fluorescent Proteins, HEK293 Cells, Humans, Kidney, Membrane Proteins, Microscopy, Confocal, Mutation, Occludin, Phosphorylation, Reverse Transcriptase Polymerase Chain Reaction, Serine, Tight Junctions, Vesicular Transport Proteins
Show Abstract · Added October 7, 2013
The Rab11 effector Rab11-family interacting protein 2 (Rab11-FIP2) regulates transcytosis through its interactions with Rab11a and myosin Vb. Previous studies implicated Rab11-FIP2 in the establishment of polarity in Madin-Darby canine kidney (MDCK) cells through phosphorylation of Ser-227 by MARK2. Here we examine the dynamic role of Rab11-FIP2 phosphorylation on MDCK cell polarity. Endogenous Rab11-FIP2 phosphorylated on Ser-227 coalesces on vesicular plaques during the reestablishment of polarity after either monolayer wounding or calcium switch. Whereas expression of the nonphosphorylatable Rab11-FIP2(S227A) elicits a loss in lumen formation in MDCK cell cysts grown in Matrigel, the putative pseudophosphorylated Rab11-FIP2(S227E) mutant induces the formation of cysts with multiple lumens. On permeable filters, Rab11-FIP2(S227E)-expressing cells exhibit alterations in the composition of both the adherens and tight junctions. At the adherens junction, p120 catenin and K-cadherin are retained, whereas the majority of the E-cadherin is lost. Although ZO-1 is retained at the tight junction, occludin is lost and the claudin composition is altered. Of interest, the effects of Rab11-FIP2 on cellular polarity did not involve myosin Vb or Rab11a. These results indicate that Ser-227 phosphorylation of Rab11-FIP2 regulates the composition of both adherens and tight junctions and is intimately involved in the regulation of polarity in epithelial cells.
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23 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 Cycle, Cell Line, Tumor, Cell Proliferation, Flow Cytometry, Fluorescent Antibody Technique, Indirect, Genetic Vectors, Heat-Shock Proteins, Humans, Melanoma, Membrane 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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20 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
PKC eta regulates occludin phosphorylation and epithelial tight junction integrity.
Suzuki T, Elias BC, Seth A, Shen L, Turner JR, Giorgianni F, Desiderio D, Guntaka R, Rao R
(2009) Proc Natl Acad Sci U S A 106: 61-6
MeSH Terms: Animals, Binding Sites, Cell Line, Dogs, Epithelium, Humans, Membrane Proteins, Mutation, Occludin, Phosphorylation, Protein Kinase C, Tight Junctions
Show Abstract · Added February 25, 2014
PKC eta is expressed predominantly in the epithelial tissues; however, its role in the regulation of epithelial tight junctions (TJs) is unknown. We present evidence that PKC eta phosphorylates occludin on threonine residues (T403 and T404) and plays a crucial role in the assembly and/or maintenance of TJs in Caco-2 and MDCK cell monolayers. Inhibition of PKC eta by specific pseudo substrate inhibitor or knockdown of PKC eta by specific shRNA disrupts the junctional distribution of occludin and ZO-1 and compromises the epithelial barrier function. Expression of dominant negative, PKC eta(K394R) disrupts the TJ and barrier function, whereas wild-type PKC eta and constitutively active PKC eta(A161E) enhance the TJ integrity. Inhibition and knockdown of PKC eta or expression of PKC eta(K394R) induce dephosphorylation of occludin on threonine residues, whereas active PKC eta elevates occludin phosphorylation. PKC eta directly interacts with the C-terminal domain of occludin and phosphorylates it on highly conserved T403 and T404. T403/404A mutations result in the loss of occludin's ability to localize at the TJs, whereas T403/404D mutations attenuates the PKC eta inhibitor-mediated redistribution of occludin from the intercellular junctions. These results reveal an important mechanism of epithelial TJ regulation by PKC eta.
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12 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, 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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22 MeSH Terms
Helicobacter pylori dysregulation of gastric epithelial tight junctions by urease-mediated myosin II activation.
Wroblewski LE, Shen L, Ogden S, Romero-Gallo J, Lapierre LA, Israel DA, Turner JR, Peek RM
(2009) Gastroenterology 136: 236-46
MeSH Terms: Animals, Cells, Cultured, Electric Impedance, Gastric Mucosa, Helicobacter pylori, Membrane Proteins, Mice, Myosin Light Chains, Myosin Type II, Myosin-Light-Chain Kinase, Occludin, Phosphorylation, Tight Junctions, Urease, rho-Associated Kinases
Show Abstract · Added March 5, 2014
BACKGROUND & AIMS - Helicobacter pylori-induced gastritis predisposes to the development of gastric cancer. Increased epithelial tight junction permeability and alterations in apical-junctional complexes are also associated with an increased risk of carcinogenesis. Phosphorylation of myosin regulatory light chain (MLC) by MLC kinase (MLCK) regulates tight junction function. We determined whether MLCK was activated by H pylori and defined the mechanisms through which such activation dysregulates gastric epithelial barrier function.
METHODS - MKN28 gastric epithelial cells were cocultured with the H pylori cag(+) strain 60190 or cagA(-), cagE(-), ureB(-), or vacA(-) mutants. MLC phosphorylation and barrier integrity were determined by immunoblot analysis and transepithelial electrical resistance measurements, respectively. Localization of the tight junction protein occludin was determined by immunocytochemistry in MKN28 cells and INS-GAS mice.
RESULTS - H pylori induced a progressive loss of barrier function that was attenuated by inactivation of ureB, but not cagA, cagE, or vacA. Reductions in transepithelial electrical resistance were also dependent on functional urease activity. H pylori increased MLC phosphorylation in epithelial monolayers; this was significantly decreased by inhibition of MLCK or Rho kinase or by loss of UreB. H pylori infection of either cultured monolayers or hypergastrinemic INS-GAS mice induced occludin endocytosis, reflecting cytoskeletally mediated disruption of tight junctions.
CONCLUSIONS - H pylori increases MLC phosphorylation, occludin internalization and barrier dysfunction in gastric epithelial cells. This process requires functional urease activity and is independent of the cag pathogenicity island or VacA. These data provide new insights into the mechanisms by which H pylori disrupts gastric barrier function.
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15 MeSH Terms