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Cystic Fibrosis/Pulmonary Research and Treatment Center (R.S.E., M.K.V., L.G.J.), and the Departments of Medicine and Pharmacology, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina (L.G.J.); and University of Queensland, Brisbane, Australia (N.B., I.T.)
Received July 27, 2005; accepted November 15, 2005
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Abstract |
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; Gumbiner et al., 1991). Tight junctions comprise transmembrane proteins such as occludin (Furuse et al., 1998), claudins (Furuse et al., 1998; Morita et al., 1999), junctional adhesion molecule (Martin-Padura et al., 1998), and coxsackie virus B and Ad2/5 receptor (Cohen et al., 2001), as well as cytoplasmic molecules such as ZO-1 (Stevenson and Goodenough, 1984), ZO-2 (Itoh et al., 1999), ZO-3 (Haskins et al., 1998), cingulin (Citi and Cordenonsi, 1998), and 7H6 (Zhong et al., 1993). Regulatory molecules, including tyrosine kinases, proteases, and GTPases, also colocalize near the tight junction. Interactions between the transmembrane components and cytoplasmic molecules, along with the cytoskeleton and regulatory molecules, are thought to modulate the gate and fence functions of tight junctions.
Several groups have investigated the modulation of tight junction gate function as a method to enhance drug uptake in intestinal epithelia (van Hoogdalem et al., 1990; Swenson et al., 1994; Yamamoto et al., 1996). Like intestinal epithelia, the airway epithelium is also resistant to the uptake of apically delivered macromolecules. Agents that modulate tight junctions, such as EGTA, sodium caprate, the sodium salt of the saturated medium-chain fatty acid capric acid (C10) or lauric acid (C12), polidocanol, and lysophosphatidyl choline have been shown to increase the permeability of airway tight junction and also enhance gene transfer (Duan et al., 1998; Parsons et al., 1998; Coyne et al., 2000, 2003; Wang et al., 2000; Chu et al., 2001; Limberis et al., 2002). However, delivery of these agents that alter multiple proteins in the tight junction has been linked to inflammation in airways in vitro and in vivo. Alternative strategies that target specific tight junction proteins could prove to be less toxic to airway epithelia. One such strategy directly targeted occludin to enhance tight junction permeability to molecular tracers in a Xenopus laevis kidney epithelial cell line A6 by basolateral delivery of synthetic peptides corresponding to the second extracellular loop of occludin (Wong and Gumbiner, 1997). A similar modulation in tight junction permeability of solutes in intestinal epithelia by apical delivery of a synthetic occludin peptide has also been reported by Tavelin et al. (2003), who showed that the conjugation of a lipoamino acid to the occludin peptide inhibited enzymatic degradation of the peptide by apical peptidases. Suppression of occludin by stable expression of short interfering RNA with associated changes in the gate functions of tight junctions in MDCK cells (Yu et al., 2005) provides further evidence for the role of occludin in tight junction functions.
Thus, occludin is a potentially good target for modulating tight junction barrier function. Occludin is a 
60-kDa integral membrane protein of tight junction fibrils that spans the membrane four times with three cytoplasmic domains and two extracellular loops (ECLs). The first ECL has a high tyrosine and glycine composition, whereas the second loop is rich in tyrosine residues. Both extracellular loops of occludin consist solely of uncharged residues with the exception of one or two charged residues adjacent to the membrane. Occludin localizes to tight junctions, and its overexpression is known to increase transepithelial resistance in mammalian epithelial cells (McCarthy et al., 1996). Alteration of occludin expression has been shown to increase epithelial permeability, and the absence of occludin from tight junctions has been shown to have no significant affect on tight junction morphology (Balda et al., 1996; Saitou et al., 2000).
We hypothesized that apically applied occludin peptide could specifically modulate tight junction permeability in airway epithelia and enhance the paracellular flux of molecular tracers and viral vectors. To test this hypothesis, we addressed the following issues: 1) whether specific targeting of occludin in airway epithelial tight junctions by apical application of occludin peptide could enhance paracellular permeability to macromolecules such as dextrans and gene transfer vectors, 2) whether the specificity of occludin peptide would affect the expression and distribution of other tight junction proteins, and 3) whether specific targeting of occludin could reduce the toxicity typically observed with other tight junction modulating agents.
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Materials and Methods |
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). Because a previous study showed that the conjugation of a lipoamino acid to the occludin peptide inhibited enzymatic degradation of the peptide (Tavelin et al., 2003) by apical peptidases, OP90–103 was conjugated to a lipophilic amino acid moiety at the N terminus of OP90–103, resulting in C14-OP90–103. A scrambled peptide conjugated to the same lipophilic amino acid moiety (C14-SGLSGGTRDYGTGL-NH2) was used as a control in all experiments.
Cell Culture. Primary airway cells from human subjects were isolated in accordance with guidelines approved by the Committee on the Protection of the Rights of Human Subjects. Well differentiated human airway epithelial (WD HAE) cells were isolated from surgical specimens, plated at a density of 2 x 105 cells/12 mm on TranswellCol inserts (0.4-µm pore size; Corning Inc., Acton, MA), and maintained in a 50:50 mixture of light-harvesting complex basal medium (Biofluids, Rockville, MD) and Dulbecco's modified Eagle's medium with 4.5 g/l glucose supplemented with growth factors, retinoic acid, and bovine serum albumin as described previously (Fulcher et al., 2005). Upon reaching confluence, culture medium was aspirated from the apical surface, and cells maintained at an air-liquid interface for 3 to 4 weeks. Cultures with >10% cilia, as determined by microscopy, and a transepithelial electrical resistance (RT) of 500 
/cm2 were selected for experiments.
Electrophysiological Measurements. The RT of primary HAE cells was monitored with an ohmmeter (EVOM; World Precision Instruments, Sarasota, FL). Culture medium or HEPES-Ringer solution was added to the apical and basolateral surfaces of WD HAE cells in Transwell-Col inserts and incubated for 20 min at 37°C, after which RT was measured.
Measurement of Permeability. To determine the optimal concentration of occludin peptide for increased tight junction permeability, a dose-response curve was performed. Primary HAE cells were grown on Transwell-Col inserts under air liquid interface conditions as described previously (Fulcher et al., 2005). Occludin peptides were applied to the apical surface of 25- to 28-day-old WD HAE cultures in the following concentrations: 10, 30, 100, 300, and 1000 µM. Control cultures were treated with vehicle (no peptide) or 1000 µM scrambled peptide. Transepithelial resistance (RT) was monitored with an ohmmeter at 10-min intervals. When RT decreased, occludin peptide was removed from the culture medium and the recovery of RT was monitored at 6, 24, and 48 h. The optimal concentrations of occludin peptide (300 and 1000 µM) that reduced RT rapidly (<30 min) and allowed for full recovery of RT after occludin peptide removal were selected for subsequent experiments. Once RT had decreased, the maximal effect of occludin peptide on the permeation of dextrans in WD HAE cells was measured. FITC-labeled dextrans of 70 or 2000 kDa were applied at a concentration of 5 mg/ml to the lumen (source) after removal of the occludin peptide from the culture medium. The appearance of dextrans in the basolateral bath (sink) was measured in 10-µl samples obtained from the sink every 10 min for 60 min and in 10 µl of source samples at time 0 and at 60 min. Fluorescence was measured in samples at 496 nm. The paracellular permeability (Papp) coefficients were calculated as described previously (Stutts et al., 1981).
Measurement of Transduction Efficiency. A recombinant, first generation, E1, E3-deleted adenovirus serotype 5 vector encoding a LacZ transgene (AdlacZ) and an adeno-associated vector encoding a green fluorescent protein (GFP) transgene (AAV2 U1a GFP) were prepared by the University of North Carolina at Chapel Hill Gene Therapy Vector Core. Cultures of WD HAE epithelia were apically exposed to 1000 µM scrambled peptide and 300 or 1000 µM occludin peptide for 20 min. AdlacZ at a multiplicity of infection of 300 or AAV2 U1a GFP at 500 transducing units/cell was applied to the lumen after removal of the occludin peptide from the culture medium. After infection for2hat 37°C, cells were washed with PBS and incubated for an additional 48 h for Ad-mediated LacZ detection. Adeno-associated vector-mediated GFP detection was performed by fluorescent microscopy at 4 weeks post-transduction, which is the optimal time required for synthesis of the complementary strand in AAV expression. LacZ expression was detected by X-Gal histochemistry. The cultures were stained in 5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside (X-Gal) for 4 h at 37°C. To minimize background staining, the pH of all solutions was adjusted to 8.0 with Tris buffer (20 mM final concentration). LacZ protein levels were quantitated by 
-galactosidase enzymatic analysis (Galactostar Light assay; Tropix, Bedford, MA) according to the manufacturer's instructions. GFP expression was detected by fluorescent microscopy. Vehicle-treated cultures were not exposed to scrambled peptide or occludin peptide but received the same concentration of viral vectors as the other treatment groups.
Measurement of Cellular Toxicity. Occludin peptide-induced cellular toxicity was assessed by the amount of lactate dehydrogenase (LDH) leakage into the culture medium because an increase in the number of cell membrane-damaged cells results in increased LDH levels in the culture supernatant. WD HAE cells were apically exposed to 1000 µM scrambled peptide or 300 and 1000 µM occludin peptide. At 0, 6, and 24 h after treatment, culture medium was collected, and LDH levels were measured using a commercial kit [LDH Release Detection Kit (LDH); Roche] and analyzed according to the manufacturer's instructions. In brief, 100 µl of cell-free supernatant was added in duplicate to wells in a 96-well microtiter plate, followed by the addition of 100 µl of LDH assay reaction mixture. After a 90-min incubation at room temperature, the absorbance was read on an enzyme-linked immunosorbent assay microplate reader at 492 nm. Background values were subtracted from each reading, and the average absorbance for each sample was calculated. The mean percent occludin peptide-induced LDH release for each sample was calculated as % occludin peptide-induced LDH release = [(ABSexpt–ABSlow)/(ABShigh–ABSlow)] x 100, where ABSexpt i s the mean absorbance of treated cells, ABSlow is the mean absorbance of culture medium, and ABShigh is the mean absorbance of Triton X-100-treated cells.
Transepithelial Permeability. For visualization of paracellular permeability in live WD HAE cells, cultures were treated with 300 or 1000 µM occludin peptide in HEPES-Ringer solution containing 2 mg/ml Texas Red-labeled 70-kDa dextran, and XZ-axis scans were recorded by confocal microscopy at 1, 5, 10, 15, 20, and 30 min after apical application.
Western Blotting. WD HAE cultures were apically exposed to 1000 µM scrambled peptide or 300 and 1000 µM occludin peptide. At 6 and 24 h after treatment, whole cell lysates from occludin peptidetreated and control cultures were prepared with 0.1% Triton X-100 extraction buffer containing phenylmethanesulfonyl fluoride and dithiothreitol. Equal amounts of protein (50 µg) were loaded onto 12% Tris-glycine gels (Novex, San Diego, CA). After electrophoresis for 1.5 h at 150 V, protein was transferred to polyvinylidine difluoride membrane at 33 V and blocked in 5% fat-free milk. Membranes were probed with antihuman occludin (1:500), ZO-1 (1:500), claudin-1 (1:500), or claudin-4 (1:500) antibodies (Zymed Laboratories, South San Francisco, CA) in phosphate-buffered saline-Tween 20. Proteins were visualized with a peroxidase-conjugated secondary antibody (1:10,000) by ECL.
Immunofluorescence and Confocal Microscopy. To determine the effects of scrambled peptide and occludin peptide on the localization of occludin, ZO-1, claudin-1, and claudin-4, indirect immunofluorescence was performed on vehicle-treated, scrambled peptide-treated, and occludin peptide-treated cultures at 24 h after peptide treatment. Cells were permeabilized with methanol at –20°C for 10 min and rehydrated 3 x 10 min with PBS. Cells were then blocked with 1x PBS containing 5% BSA (+0.5% Triton X-100 for claudin-1 staining) for 30 min at room temperature. After three washes in 1x PBS, antibodies to occludin, ZO-1, claudin-1, and claudin-4 (Zymed) at a dilution of 1:1000 were added to the apical surface for 1 h. Cells were washed with PBS and Alexa-labeled secondary antibodies (GE Healthcare, Little Chalfont, Buckinghamshire, UK), diluted to 10 µg/ml in 10% goat serum/PBS, were added to the apical surface and incubated for 1 h at room temperature. Cells were postfixed in 4% paraformaldehyde and images captured with a Zeiss 510 laser-scanning microscope (Carl Zeiss Inc., Thornwood, NY).
Measurement of Fence Function. To determine the effect of occludin peptide on membrane integrity, fence function was evaluated by assessing intramembrane diffusion of BODIPY-sphingomyelin. The apical domains of filter-grown WD HAE cells were labeled with BODIPY-sphingomyelin/BSA complexes that were prepared in P-buffer (10 mM HEPES, pH 7.4, 145 mM NaCl, 1 mM sodium pyruvate, 10 mM glucose, and 3 mM CaCl2) by slowly mixing 10 ml of 0.8 mg/ml defatted BSA (Sigma-Aldrich, St. Louis, MO) with 200 µl of BODIPY-FL-C5-sphingomyelin (Invitrogen, Carlsbad, CA) stock solution (1 mM in dimethyl sulfoxide) under vigorous vortexing. Cells were subsequently labeled with 1:2 diluted BODIPY-sphingomyelin/BSA complexes for 10 min on ice. After the cells were washed four times with P-buffer, they were mounted on an optical chamber and images were captured in an XZ plane by confocal microscopy. Occludin peptide at 1000 µM was applied to the lumen and serial images captured at 30 s and 1, 2, 5, 10, 20, and 30 min after occludin peptide application.
Statistical Analysis. Data are presented as means ± S.E.M. A one-way analysis of variance and Holm-Sidak's method for all pairwise multiple comparison procedures were used to determine statistical significance of observed differences (P < 0.05).
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Effect of Occludin Peptide on Gene Transfer Efficiency. To determine whether the occludin peptide could increase tight junction permeability to biologically relevant macromolecules such as gene transfer vectors, the transduction efficiencies of an adenoviral vector encoding a LacZ transgene (AdLacZ) and an adeno-associated viral vector encoding a GFP transgene (AAV2 U1a GFP) were evaluated in WD HAE cells apically exposed to 300 and 1000 µM occludin peptide, the two highest concentrations that rapidly reduced transepithelial resistance. The effect of 1000 µM scrambled peptide on AdLacZ gene transfer efficiency was also evaluated. Similar to our results with molecular tracers, pretreatment of WD HAE cells with 1000 µM occludin peptide significantly enhanced the transduction efficiencies of both AdLacZ (multiplicity of infection 300) and AAV2 U1a GFP (multiplicity of infection 500) vectors compared with vehicle control cultures or 1000 µM scrambled peptidetreated cells, as assessed by X-Gal staining (Fig. 4A) and GFP fluorescent imaging (Fig. 4B), respectively. Enzymatic analysis of the level of Ad-mediated -galactosidase expression showed significantly higher levels of this protein in cultures pretreated apically with 1000 µM occludin peptide compared with that of vehicle-treated or scrambled peptidetreated control cultures transduced with the AdLacZ vector alone (Fig. 4C). Vehicle control cultures and scrambled peptide-treated cells had -galactosidase activity levels of 208 ± 24 mU and 218 ± 35 mU of -galactosidase/mg of protein, respectively, whereas 300 µM occludin peptide-treated cultures had an activity of 419 ± 110 mU -galactosidase/mg of protein that exhibited a trend but were not significantly different from vehicle- or scrambled peptide-treated cultures. However, 1000 µM occludin peptide-treated cultures exhibited a significantly enhanced mean activity of 7215 ± 912 mU -galactosidase/mg of protein (P < 0.001). Thus, apical pretreatment of WD HAE cells with 1000 µM occludin peptide significantly enhanced tight junction permeability, allowing for increased penetration of both Ad and AAV vectors to the basolateral membrane where the viral receptors are localized (Walters et al., 1999), resulting in greater viral binding and internalization.
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Effect of Occludin Peptide on Cellular Permeability. To determine whether the increase in permeability to dextrans and viral vectors was associated with increased cellular rather than paracellular permeability, we assessed solute permeability in live occludin peptide-treated WD HAE cultures with Texas Red-labeled 70-kDa dextrans and XZ confocal microscopy scans. Although 70-kDA dextrans are fairly large molecules, it has previously been demonstrated that even larger molecular tracers such as 2000-kDa dextrans can enter into permeabilized HAE cells (Coyne et al., 2003). Therefore, we assessed the cellular uptake of fluorescently labeled 70-kDa dextrans in occludin peptide-treated cultures as a measure of transcellular permeability. Our results showed that 1000 µM occludin peptide application did not induce cellular uptake of the fluorescently labeled dextran, because no fluorescence was detected within the epithelial cells for up to 30 min after occludin peptide application (Fig. 6). The gradual increase in Texas Red-labeled dextrans in lateral and basal regions surrounding the epithelial cells by 30 min after occludin peptide treatment suggests an increase in paracellular flow of dextrans across the epithelium. No uptake in columnar cells was detected, and infrequent uptake into basal cells was observed. A similar trend was observed with 300 µM occludin peptide application, although the extent of paracellular diffusion of fluorescently labeled dextrans was reduced. These data suggested that apical application of 1000 µM occludin peptide does not permeabilize the apical cell membrane and that the observed increase in LDH release was not associated with increased cellular permeability to large molecular tracers. No difference in Texas Red dextran permeability was observed with the apical application of 1000 µM scrambled peptide compared with vehicle control cultures. Because it has been previously demonstrated that the sodium salt of the medium-chain fatty acid C10 alters tight junction barrier function in airway epithelial cells (Coyne et al., 2003), a control experiment using C10 was performed. Cellular uptake of the 70-kDa dextran into the epithelium was observed within 1 min of apical application of C10, which was not detected in occludin peptide-treated cells.
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Specificity of Occludin Peptide on Tight Junction Protein Expression and Localization. To determine whether tight junction modulation by apical exposure of WD HAE cells to occludin peptide was a result of the specific effects on occludin, alterations in tight junction-associated protein expression and distribution were assessed by Western blot analyses and immunofluorescent localization of occludin, claudin-1, claudin-4, and ZO-1 in control and occludin peptide-treated cultures. No consistent changes in the total amounts of occludin, claudin-1, or claudin-4 were detected by Western blotting in WD HAE cells immediately after or 24 h after occludin peptide treatment (Fig. 7, A and B, respectively). However, in WD HAE cells apically exposed to 1000 µM occludin peptide, there were subtle changes in the redistribution of occludin and ZO-1 immediately after occludin peptide application, with some loss of the chicken wire occludin staining pattern in the XY plane of occludin peptidetreated but not control cultures (Fig. 7C). Alterations in the localization of claudins-1 and -4 at a similar time point were less prominent, if any. Immunofluorescent localization of occludin, ZO-1, claudin-1, and claudin-4 showed no changes in their distribution at 24 h after apical exposure to occludin peptide. No changes in the expression or localization of these tight junction proteins were observed in cultures apically treated with 1000 µM scrambled peptide. These data suggest that although treatment with the occludin peptide significantly reduces RT, it does not cause a significant cellular redistribution in the tight junction proteins of polarized WD HAE cells. Because ZO-1 colocalizes with occludin and is known to bind occludin, changes in ZO-1 localization immediately after occludin peptide application probably resulted from the subtle redistribution of occludin.
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Effect of Occludin Peptide on Tight Junction Fence Function. Although the redistribution of occludin immediately after occludin peptide application did not seem to be dramatic, we further assessed the specificity of occludin disruption by measuring the fence function in occludin peptidetreated and control cultures. The apical membrane lipids of WD HAE cells were labeled with BODIPY-sphingomyelin, and changes in apical membrane lipids before and after addition of vehicle, scrambled peptide, or occludin peptide were measured. Although very subtle disruptions of the apical membrane at 30 min after occludin peptide application may have occurred, the fluorescent-labeled lipid generally remained confined to the apical domain of the plasma membrane and did not diffuse to the lateral membrane (Fig. 8), suggesting that apical treatment of primary airway epithelia with a high concentration of occludin peptide does not significantly alter the fence function of tight junctions, even though occludin expression remained unaltered. A relative comparison with an altered fence function using a mediumchain fatty acid C10 demonstrated that no significant alterations in the fence function of tight junctions occurred after occludin peptide application relative to the C10 positive control.
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Discussion |
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). Several lines of evidence show that occludin plays an important role in tight junction functions. Overexpression of chicken occludin in MDCK cells was shown to increase tight junction strand number with a corresponding elevation in transepithelial resistance (McCarthy et al., 1996). A truncated form of occludin was shown to increase tight junction permeability to low molecular weight molecular tracers in MDCK cells (Balda et al., 1996). Suppression of occludin by stable expression of short interfering RNA has been associated with changes in the gate functions of tight junctions in MDCK cells (Yu et al., 2005).
Synthetic occludin peptides provide further evidence for their role in tight junction functions. The transepithelial resistance of X. laevis epithelial cells in vitro was shown to decrease by the administration of a synthetic occludin peptide into the culture medium (Wong and Gumbiner, 1997). Peptides corresponding to the first extracellular loop of occludin also increased the tight junction permeability to mannitol in Caco-2 cells (Tavelin et al., 2003), suggesting that occludin is a good target for tight junction modulation. Nothing is known about the effect of synthetic occludin peptides on tight junction permeability in airway epithelial cells. In this study, we investigated the effects of apical application of occludin peptide on paracellular permeability in WD HAE cells.
Our study showed that occludin peptide rapidly decreased RT in WD HAE cells, with a significant reduction within minutes of apical exposure to 300 or 1000 µM occludin peptide. The occludin peptide-induced decrease in RT was reversible and returned to normal levels within 24 h, confirming the reversibility of this strategy. The significant decrease in RT resulted in an alteration in tight junction permeability, as evidenced by a corresponding increase in paracellular permeability to molecular tracers. Significant increases in the diffusion of both low and high molecular weight dextrans were observed in primary airway epithelia previously treated with 1000 µM occludin peptide. To further test the effects of the occludin peptide on tight junction permeability to macromolecules, we measured the transduction efficiencies of adenoviral and adeno-associated viral vectors in WD HAE cells pre-exposed to apical occludin peptide. Significant increases in gene transfer mediated by both adenoviral and AAV vectors were detected in occludin peptide-treated WD HAE cells. This finding suggested that occludin peptide alters paracellular permeability and increases translocation of vectors to the basolateral membrane, resulting in enhanced binding of viral vectors to viral receptors.
Although it has been previously demonstrated that EGTA and the medium chain fatty acids C10 and C12 can enhance dextran diffusion and gene transfer efficiency (Gregory et al., 2003; Johnson et al., 2003), these agents have been shown to have varying toxicity profiles. C12 is toxic to airway epithelial cells, as evidenced by the presence of high levels of LDH in the culture media even at 72 h after C12 treatment. Our data with apical delivery of occludin peptide showed minimal, transient toxicity with the highest concentration of 1000 µM at 6 h after treatment, which was resolved by 24 h. The transient release of LDH raised the question whether the increase in permeability to dextrans and viral vectors after occludin peptide application was due to transcellular permeability rather than an increase in tight junction permeability. To address this question, we analyzed Texas Red dextran permeability in live WD HAE cells after occludin peptide treatment (Fig. 6). Penetration of a fluorescently labeled 70-kDa dextran in live WD HAE cultures was visualized by scanning images in the XZ plane by confocal microscopy. The apical application of 1000 µM occludin peptide did not result in cellular uptake throughout the epithelium, evidenced by the absence of fluorescence within the epithelial cells even at 30 min after occludin peptide treatment. XZ images captured at various time points after occludin peptide treatment showed the passage of Texas Red-labeled dextrans through the paracellular rather than the trans-cellular pathway. Similar results were observed with 300 µM occludin peptide treatment.
When the apical membranes of the WD HAE cells were labeled with BODIPY-sphingomyelin followed by occludin peptide treatment, a slight dissipation of label intensity at the apical membrane was detected in occludin peptidetreated but not control cultures. This loss of intensity may have accounted for the increased LDH release because the lipophilic amino acid may transiently interact with the apical membrane to release the peptide from the prodrug complex (lipoamino acid plus peptide). This interaction might increase the release of small molecules such as LDH but not increase permeability to large dextrans. Although we could not exclude the possibility that some BODIPY-sphingomyelin was detected in the lateral membranes of occludin peptidetreated cells because of exchange of some apical membrane BODIPY-sphingomyelin into the P-buffer, followed by reinsertion into the lateral membrane, we did not readily detect lateral diffusion of BODIPY-sphingomyelin in occludin peptide-treated cells. As a control for diffusion of BODIPY-sphingomyelin from the apical to the lateral surface, a relative comparison to altered fence function induced by sodium caprate (C10) was performed indicating lateral diffusion of labeled membrane lipid in C10-treated cultures, whereas no significant diffusion of label to the lateral membrane space was detected on apical application of occludin peptide.
Thus, exposure of WD HAE cells to 1000 µM occludin peptide resulted in a mild, transient increase in LDH release that was not associated with significant alterations in the structural components of the epithelia (Figs. 6 and 8). The high levels of LDH in the culture media of vehicle control cells at 24 h possibly resulted from the cumulative loss of cells in these terminally differentiated cultures that typically senesce at 8 weeks after plating. Ciliated cells are also highly metabolic and deplete energy stores while secreting lactate, which may in turn affect cell viability. However, the key point is that the LDH levels were similar in both treatment and control (scrambled peptide and vehicle) groups at 24 h, suggesting that the toxicity induced by high concentrations of occludin peptide is transient and quickly resolves.
To further determine whether occludin peptide application affected the expression and distribution of tight junction proteins, Western blot analysis and immunolocalization of tight junction-associated proteins was performed on control and occludin peptide-treated cultures. Immunolocalization analysis immediately after occludin peptide application showed a subtle redistribution of occludin and ZO-1, with no significant changes in claudin-1 and claudin-4 localization. Changes in the distribution of occludin and ZO-1 may be due to specific targeting of occludin by the occludin peptide, resulting in disruption of the occludin-ZO-1 complex found at tight junctions. Although interferon-
increases paracellular permeability in intestinal epithelial cells by inducing endocytosis of occludin, junctional adhesion molecule A, and claudin-1 (Utech et al., 2005), assessment of whether this mechanism occurred during enhanced paracellular permeability after occludin peptide application was beyond the scope of this study.
At 24 h after apical application of occludin peptide, there was no evidence of redistribution of any of these tight junction proteins. Furthermore, no significant alterations in the expression of occludin, claudin-1, and claudin-4 were observed in Western blot analyses of WD HAE cells immediately after or 24 h after apical exposure to occludin peptide. The lack of significant changes in these tight junction protein components suggests that the occludin peptide-induced alterations in tight junction permeability were due primarily to specific disruption of occludin.
C14-OP90–103 used in this study is a racemic mixture of two diastereomers, D-C14-OP90–103 and L-C14-OP90–103, which vary in their stability (Tavelin et al., 2003). The L-isomer containing L-2-amino dodecanoic acid (L-C14-OP) not only released OP90–103 at a 15-fold faster rate than the D-isomer that contained D-2-amino dodecanoic acid (D-C14-OP) but also decreased RT by 40-fold (Tavelin et al., 2003). Because only intact OP90–103 was released by either isomer, it indicated that the lipoamino acid moiety prevented the released OP90–103 from degradation. In the present study, a high concentration of occludin peptide was required to increase paracellular permeability to dextrans and gene transfer vectors. However, this high concentration resulted in modest cellular toxicity in occludin peptide-treated WD HAE cells. Use of the more active L-isomer of C14-OP90–103 rather than a mixture of the both the D-C14-OP90–103 and L-C14-OP90–103 isomers may be a safer alternative to effectively modulate tight junction permeability with minimal toxicity.
Based on our results, apical occludin peptides may represent a better class of tight junction modulators in airway epithelial cells that specifically target the extracellular domains of tight junction proteins, resulting in enhanced tight junction permeability with minimal toxicity. Although further studies to elucidate the safety profile of occludin peptide in lung epithelia are needed, specific modulation of tight junctions by occludin peptide could prove to be a valuable alternative strategy for the efficient delivery of pharmacological agents and viral vectors for the treatment of lung diseases.
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Acknowledgements |
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Footnotes |
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Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
ABBREVIATIONS: ZO, zona occludens; ECL, extracellular loop; OP, occludin peptide; WD HAE, well differentiated human airway epithelia; FITC, fluorescein isothiocyanate; GFP, green fluorescent protein; Ad, adenovirus; AAV, adeno-associated virus; X-Gal, 5-bromo-4-chloro-3-indolyl-b -D-galactopyranoside; LDH, lactate dehydrogenase; PBS, phosphate-buffered saline; BSA, bovine serum albumin; RT, transepithelial resistance.
Address correspondence to: Dr. Larry G. Johnson, Professor of Medicine and Director, Pulmonary and Critical Care Medicine, The University of Arkansas for Medical Sciences, 4301 W. Markham St., Mail Slot 555, Little Rock, AR 72205. E-mail lgjohnson{at}uams.edu
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