Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
J Cell Biol
1997 Jan 27;1362:399-409. doi: 10.1083/jcb.136.2.399.
Show Gene links
Show Anatomy links
A synthetic peptide corresponding to the extracellular domain of occludin perturbs the tight junction permeability barrier.
Wong V
,
Gumbiner BM
.
???displayArticle.abstract??? Occludin, the putative tight junction integral membrane protein, is an attractive candidate for a protein that forms the actual sealing element of the tight junction. To study the role of occludin in the formation of the tight junction seal, synthetic peptides (OCC1 and OCC2) corresponding to the two putative extracellular domains of occludin were assayed for their ability to alter tight junctions in Xenopus kidneyepithelial cell line A6. Transepithelial electrical resistance and paracellular tracer flux measurements indicated that the second extracellular domain peptide (OCC2) reversibly disrupted the transepithelial permeability barrier at concentrations of < 5 microM. Despite the increased paracellular permeability, there were no changes in gross epithelial cell morphology as determined by scanning EM. The OCC2 peptide decreased the amount of occludin present at the tight junction, as assessed by indirect immunofluorescence, as well as decreased total cellular content of occludin, as assessed by Western blot analysis. Pulse-labeling and metabolic chase analysis suggested that this decrease in occludin level could be attributed to an increase in turnover of cellular occludin rather than a decrease in occludin synthesis. The effect on occludin was specific because other tight junction components, ZO-1, ZO-2, cingulin, and the adherens junction protein E-cadherin, were unaltered by OCC2 treatment. Therefore, the peptide corresponding to the second extracellular domain of occludin perturbs the tight junction permeability barrier in a very specific manner. The correlation between a decrease in occludin levels and the perturbation of the tight junction permeability barrier provides evidence for a role of occludin in the formation of the tight junction seal.
Figure 2. A synthetic peptide (OCC2) corresponding to the entire second extracellular domain of chick occludin decreased TER of A6 cell monolayers. (a) Effect of various synthetic peptides on TER. OCC1 (corresponding to the entire first extracellular domain of chick occludin), OCC2 (corresponding to the entire second extracellular domain of chick occludin), OCC2(S) (corresponding to the scrambled sequence of the entire second extracellular domain of chick occludin), and DMSO solvent control were used. Newly confluent A6 cell monolayers (starting TER ∼1,000 Ωcm2) that were still developing TER were used. Cell monolayers were treated with a final concentration of 5 μM OCC1, 5 μM OCC2, 5 μM OCC2(S), or DMSO (0.05%) for 66 h, and peptides were replenished every 24 h. At the end of the 66-h peptide incubation, TER for control monolayers reached ∼5,000–6,000 Ωcm2. n = 6 for each condition. (b) Time course of effect of OCC2 peptide on TER of A6 cells that were still developing TER. Cell monolayers that had attained TER ∼750 Ωcm2 were treated with a final concentration of 5 μM OCC1 (n = 4) or 5 μM OCC2 (n = 5) at t = 0. Peptides were replenished at 30 h. (c) Dose dependency of OCC2 peptide on TER in A6 cell monolayers that were still developing TER. A6.2 cells were allowed to grow to confluency in normal medium and were subsequently changed to low calcium medium for 18 h. The low calcium medium was replaced with normal calcium medium containing a final concentration of 0.2, 0.5, 2, and 5 μM OCC2. TER were measured after 4 d when control cell monolayers developed TER of ∼3,000 Ωcm2. n = 3 for all concentrates tested. (d) Time course of OCC2 peptide on TER of steady-state A6 cell monolayers that were confluent for ∼2 wk (TER ∼8,000 Ωcm2). Cells were treated with a final concentration of 5 μM OCC2 at t = 0. Untreated monolayers were done in parallel as control. Peptides were replenished at 22 and 76 h. n = 3 for all conditions. (e) Dose dependency of OCC2 peptide on TER in steady-state A6 monolayers (TER ∼6,000 Ωcm2). Cell monolayers were treated with a final concentration of 0.5, 1, 2, 5, and 10 μM OCC2. TER was measured at 40 h after peptide addition. The TER of each individual monolayer is plotted. Each concentration of OCC2 was done on duplicate monolayers. All error bars represent standard error.
Figure 3. OCC2 increased the paracellular flux of membraneimpermeant tracer molecules. (a) Effects of OCC2 on the flux of [3H]mannitol, [14C]inulin, Texas red–conjugated neutral dextran (mol wt 3,000), and Texas red–conjugated neutral dextran (mol wt 40,000). OCC1 was used as control peptide. A6 cell monolayers were allowed to grow until TER reached ∼1,200 Ωcm2. Cell monolayers were then treated with a final concentration of 5 μl OCC1 or OCC2 for 36 h. TER was measured and tracers flux assays were performed as described in Materials and Methods. For all four tracers, n = 8 and error bars represent SEM. (b–e) The relationship between tracer flux and TER changes induced by OCC2 treatment. Absolute flux values for individual A6 cell monolayers were plotted against TER of the same monolayer. (b) [3H]mannitol, (c) [14C]inulin, (d) neutral dextran (mol wt 3,000) conjugated with Texas red, and (e) neutral dextran (mol wt 40,000) conjugated with Texas red.
Figure 4. OCC2 reduced junctional stainings of occludin but not ZO-1, cingulin, ZO-2, and E-cadherin. A6 cell monolayers from the paracellular tracer flux assays described in Fig. 3 were processed for indirect immunofluorescence microscopy at the end of the flux assays. OCC1-treated monolayers had TER of ∼2,500 Ωcm2, and OCC2-treated monolayers had TER of ∼250 Ωcm2. OCC1-treated (a, c, e, g, and i) and OCC2-treated (b, d, f, h, and j) monolayers were immunostained in parallel for occludin (a and b), ZO-1 (c and d), cingulin (e and f), ZO-2 (g and h), and E-cadherin (i and j).
Figure 5. OCC2 specifically decreased total cellular occludin levels. (a) Western blots of occludin, cingulin, ZO-1, ZO-2, and E-cadherin of total cell lysates from monolayers that were treated with OCC1, OCC2, or DMSO solvent control. A6 cells were allowed to grow until TER reached ∼1,000 Ωcm2, and monolayers were treated with 10 μM of OCC1, 10 μM OCC2, or 0.1% DMSO for 24 h. (b) Only the peptide that decreased TER also caused a decrease in occludin levels. Western blot of occludin in A6 total cell lysates of monolayers that were treated with OCC1, OCC2(U) (unmodified), OCC2, or OCC2(S) (scrambled). A6 cells were allowed to grow to confluency in normal medium and were subsequently changed to low calcium medium for 18 h. A6 cells were then replenished with normal calcium media containing peptides at a final concentration of 5 μM. OCC2(U), unmodified OCC2, and OCC2(S), scrambled sequence of OCC2. Peptides were replenished every 24 h, and cells were extracted for analysis at 4 d after initial peptide treatment. (c) Occludin synthesis was not reduced by OCC2 treatment. A6 cells that were either untreated or treated for either 2 or 22 h with a final concentration of 5 μM OCC2 were subsequently labeled for 2.5 h with [35S]methionine followed by immunoprecipitation (IP) of occludin. (d) Turnover of occludin was enhanced by OCC2 treatment. A6 cells were metabolically labeled 20 h with [35S]methionine. At the end of the labeling period (t = 0), fresh media (without [35S]methionine) containing 10 μM OCC2 was added for 12 h followed by immunoprecipitation (IP) of occludin. Untreated A6 cells were used in parallel as a control.
Figure 6. The effects of OCC2 on TER and occludin accumulation were reversible. (a) Reversibility of TER after OCC2 removal. A6 cell monolayers that had TER of ∼1,700 Ωcm2 were treated at t = 0 with a final concentration of 5 μM OCC1 or OCC2. At t = 24 h, peptides were either replenished (OCC1 and OCC2) or removed (OCC2 Recovery) from the cells. OCC1 (n = 6), OCC2 (n = 6), and OCC2 recovery (n = 3). (b) Recovery of junctional stainings of occludin after OCC2 removal. A6 cell monolayers that had TER ∼1,000 Ωcm2 were treated at t = 0 with a final concentration of 5 μM OCC1 or OCC2. At t = 24 h, peptides were either replenished (OCC1 and OCC2) or removed (OCC2 recovery) from the cells. At t = 60 h, cells were processed for indirect immunofluorescence microscopy of occludin. OCC1 (TER ∼2,200 Ωcm2), OCC2 (TER ∼250 Ωcm2), and OCC2 recovery (TER ∼2,300 Ωcm2).
Figure 7. OCC2 did not cause morphological changes in A6 cell monolayers as observed by scanning EM. Confluent A6 cells grown on polylysine-coated coverslips were treated 24 h with a final concentration of 10 μM OCC1 (a and d), 10 μM OCC2 (b and e), and 0.1% DMSO (c and f). Cells were then processed for scanning EM. A6 cells were ∼7–10 μM in diam.
Anderson,
Characterization of ZO-1, a protein component of the tight junction from mouse liver and Madin-Darby canine kidney cells.
1988, Pubmed
Anderson,
Characterization of ZO-1, a protein component of the tight junction from mouse liver and Madin-Darby canine kidney cells.
1988,
Pubmed
Anderson,
ZO-1 mRNA and protein expression during tight junction assembly in Caco-2 cells.
1989,
Pubmed
Ando-Akatsuka,
Interspecies diversity of the occludin sequence: cDNA cloning of human, mouse, dog, and rat-kangaroo homologues.
1996,
Pubmed
Atisook,
Effects of phlorizin and sodium on glucose-elicited alterations of cell junctions in intestinal epithelia.
1990,
Pubmed
Baker,
Epithelial cells retain junctions during mitosis.
1993,
Pubmed
Ballard,
Regulation of tight-junction permeability during nutrient absorption across the intestinal epithelium.
1995,
Pubmed
Baron,
Extrusion of colonic epithelial cells in vitro.
1990,
Pubmed
Buse,
Transforming growth factor-alpha abrogates glucocorticoid-stimulated tight junction formation and growth suppression in rat mammary epithelial tumor cells.
1995,
Pubmed
Choi,
Expression of cell adhesion molecule E-cadherin in Xenopus embryos begins at gastrulation and predominates in the ectoderm.
1989,
Pubmed
,
Xenbase
Citi,
Cingulin, a new peripheral component of tight junctions.
1988,
Pubmed
Citi,
Cingulin: characterization and localization.
1989,
Pubmed
Claude,
Fracture faces of zonulae occludentes from "tight" and "leaky" epithelia.
1973,
Pubmed
Fujimoto,
Freeze-fracture replica electron microscopy combined with SDS digestion for cytochemical labeling of integral membrane proteins. Application to the immunogold labeling of intercellular junctional complexes.
1995,
Pubmed
Furuse,
Occludin: a novel integral membrane protein localizing at tight junctions.
1993,
Pubmed
Furuse,
Direct association of occludin with ZO-1 and its possible involvement in the localization of occludin at tight junctions.
1994,
Pubmed
Gonzalez-Mariscal,
Tight junction formation in cultured epithelial cells (MDCK).
1985,
Pubmed
Gumbiner,
Identification of a 160-kDa polypeptide that binds to the tight junction protein ZO-1.
1991,
Pubmed
Gumbiner,
A functional assay for proteins involved in establishing an epithelial occluding barrier: identification of a uvomorulin-like polypeptide.
1986,
Pubmed
Gumbiner,
Structure, biochemistry, and assembly of epithelial tight junctions.
1987,
Pubmed
Hollander,
Crohn's disease--a permeability disorder of the tight junction?
1988,
Pubmed
Jesaitis,
Molecular characterization and tissue distribution of ZO-2, a tight junction protein homologous to ZO-1 and the Drosophila discs-large tumor suppressor protein.
1994,
Pubmed
Madara,
Structural abnormalities of jejunal epithelial cell membranes in celiac sprue.
1980,
Pubmed
Madara,
Occluding junction structure-function relationships in a cultured epithelial monolayer.
1985,
Pubmed
Meldolesi,
Ca++-dependent disassembly and reassembly of occluding junctions in guinea pig pancreatic acinar cells. Effect of drugs.
1978,
Pubmed
Milks,
The effect of neutrophil migration on epithelial permeability.
1986,
Pubmed
Perkins,
Transport properties of toad kidney epithelia in culture.
1981,
Pubmed
,
Xenbase
Posalaky,
The gastric mucosal barrier: tight junction structure in gastritis and ulcer biopsies.
1989,
Pubmed
Powell,
Barrier function of epithelia.
1981,
Pubmed
Sagaties,
The structural basis of the inner blood-retina barrier in the eye of Macaca mulatta.
1987,
Pubmed
Schulzke,
Tight junction regulation during impaired ion transport in blind loops of rat jejunum.
1990,
Pubmed
Stevenson,
Identification of ZO-1: a high molecular weight polypeptide associated with the tight junction (zonula occludens) in a variety of epithelia.
1986,
Pubmed
Swift,
Intercellular junctions in hepatocellular carcinoma.
1983,
Pubmed
Wolburg,
Modulation of tight junction structure in blood-brain barrier endothelial cells. Effects of tissue culture, second messengers and cocultured astrocytes.
1994,
Pubmed
Woo,
Antagonistic regulation of tight junction dynamics by glucocorticoids and transforming growth factor-beta in mouse mammary epithelial cells.
1996,
Pubmed
Zahraoui,
A small rab GTPase is distributed in cytoplasmic vesicles in non polarized cells but colocalizes with the tight junction marker ZO-1 in polarized epithelial cells.
1994,
Pubmed
Zettl,
Glucocorticoid-induced formation of tight junctions in mouse mammary epithelial cells in vitro.
1992,
Pubmed
Zhong,
Monoclonal antibody 7H6 reacts with a novel tight junction-associated protein distinct from ZO-1, cingulin and ZO-2.
1993,
Pubmed
Zhong,
Localization of the 7H6 antigen at tight junctions correlates with the paracellular barrier function of MDCK cells.
1994,
Pubmed