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Research Article

Claudin-binder C-CPE mutants enhance permeability of insulin across human nasal epithelial cells

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Pages 2703-2710 | Received 18 Mar 2015, Accepted 08 May 2015, Published online: 02 Jun 2015

Abstract

Objective: Intranasal insulin administration has therapeutic potential for Alzheimer's disease and in intranasal administration across the nasal mucosa, the paracellular pathway regulated by tight junctions is important. The C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE) binds the tight junction protein claudin and disrupts the tight junctional barrier without a cytotoxic effect. The C-CPE mutant called C-CPE 194 binds only to claudin-4, whereas the C-CPE 194 mutant called C-CPE m19 binds not only to claudin-4 but also to claudin-1.

Methods: In the present study, to investigate the effects of C-CPE mutants on the tight junctional functions of human nasal epithelial cells (HNECs) and on the permeability of human recombinant insulin across the cells, HNECs were treated with C-CPE 194 and C-CPE m19.

Results: C-CPE 194 and C-CPE m19 disrupted the barrier and fence functions without changes in expression of claudin-1, -4, -7, and occludin or cytotoxicity, whereas they transiently increased the activity of ERK1/2 phosphorylation. The disruption of the barrier function caused by C-CPE 194 and C-CPE m19 was prevented by pretreatment with the MAPKK inhibitor U0126. Furthermore, C-CPE 194 and C-CPE m19 significantly enhanced the permeability of human recombinant insulin across HNECs and the permeability was also inhibited by U0126.

Conclusion: These findings suggest that C-CPE mutants 194 and m19 can regulate the permeability of insulin across HNECs via the MAPK pathway and may play a crucial role in therapy for the diseases such as Alzheimer's disease via the direct intranasal insulin administration.

Introduction

Intranasal insulin administration has therapeutic potential for Alzheimer's disease because of its direct movement into the brain without movement into the systemic blood circulation (Brünner et al., Citation2013). The drug delivery system across the nasal mucosa is being reconsidered. In intranasal administration across the nasal mucosa, the paracellular pathway regulated by tight junctions is important.

The nasal epithelium is a highly regulated, impermeable barrier formed by tight junctions (Takano et al., Citation2005; Holgate, Citation2008; Kojima et al., Citation2013a). Tight junctions are the most apical components of intercellular junctional complexes and they have fence and barrier functions in normal epithelial and endothelial cells (Schneeberger & Lynch, Citation1992; Gumbiner, Citation1993; Cereijido et al., Citation1998). The claudin family, which consists of at least 27 members, is solely responsible for forming tight junction strands and has four transmembrane domains and two extracellular loops (Tsukita et al., Citation2001). The second extracellular loop is the receptor of Clostridium perfringens enterotoxin (CPE) (Fujita et al., Citation2000). CPE bound to its receptor causes changes in the membrane permeability via complex formation on the plasma membrane followed by the induction of apoptosis (McClane et al., Citation2004). Claudin-3, -4, -6, -7, -8, and -14, but not claudin-1, -2, -5, and -10, are sensitive to CPE (Fujita et al., Citation2000). In human nasal epithelial cells (HNECs) in vivo and in vitro, tight junction proteins occludin, JAM-A, tricellulin, claudin-1, -4, -7, -8, -12, -13, -14, and ZO-1 and -2 form well-developed tight junction strands (Takano et al., Citation2005; Koizumi et al., Citation2008; Kojima et al., Citation2013b).

On the contrary, the C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE; amino acids 184–319) binds to claudin-4 and disrupts the tight junctional barrier without a cytotoxic effect (Sonoda et al., Citation1999). Recently, it was found that a C-CPE mutant with 10 amino acids at the N-terminal of C-CPE (C-CPE 194) had highly solubility in phosphate-buffered saline (PBS) and binding ability with claudin-4 (Uchida et al., Citation2010; Takahashi et al., Citation2011). Furthermore, a C-CPE mutant called C-CPE m19, which has binding ability not only with claudin-4 but also with claudin-1, was found after screening claudin binders from a C-CPE mutant-displaying library by using claudin-displaying budded baculovirus (Takahashi et al., Citation2012). However, the detailed effects of C-CPE 194 and C-CPE m19 on normal nasal epithelial cells remained yet unknown.

In the present study, we investigated the effects of C-CPE mutants on the tight junctional functions of normal HNECs and the permeability of human recombinant insulin across the cells. C-CPE 194 and C-CPE m19 disrupted the barrier and fence functions without cytotoxicity and changes in expression of claudins. Furthermore, C-CPE 194 and C-CPE m19 significantly enhanced the permeability of human recombinant insulin across HNECs. The effects of these C-CPE mutants were prevented by the MAPKK inhibitor U0126.

Materials and methods

Reagents and inhibitors

Rabbit polyclonal anti-claudin-1, anti-claudin-4, anti-claudin-7, and anti-occludin antibodies and mouse monoclonal anti-claudin-4 and occludin antibodies were obtained from Zymed Laboratories (San Francisco, CA). An insulin-FITC-labeled human recombinant and rabbit polyclonal anti-actin antibody were purchased from Sigma-Aldrich, Inc. (St. Louis, MO). A rabbit polyclonal anti-phospho-pERK1/2 antibody was obtained from Cell Signaling (Beverly, MA). A rabbit polyclonal anti-ERK1/2 antibody was purchased from Promega Corporation (Madison, WI). A mouse monoclonal anti-his-tag antibody was purchased from Medical & Biological Laboratories Co., Ltd. (Nagoya, Japan). Alexa Fluor 488 (green)-conjugated anti-rabbit IgG and Alexa Fluor 594 (red)-conjugated anti-mouse IgG antibodies were purchased from Molecular Probes, Inc. (Eugene, OR). Inhibitors of panPKC (GF109203X), mitogen-activated protein kinase kinase (MAPKK) (U0126), p38 MAPK (SB203580), phosphatidylinositol 3-kinase (PI3K) (LY294002), and c-Jun N-terminal kinase (JNK) (SP600125) were purchased from Calbiochem-Novabiochem Corporation (San Diego, CA).

Preparation of C-CPE mutants

The plasmids encoding C-CPE 194 and C-CPE m19 were kind gifts from the Laboratory of Bio-Functional Molecular Chemistry, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan, as part of our joint research (Uchida et al., Citation2010; Takahashi et al Citation2012). They were transfected into Escherichia coli BL-21 (DE 3) and protein expression was stimulated by the addition of isopropyl-d-thiogalactopyranoside. The cells were then harvested and lysed in buffer A (10 mM Tris–HCl, pH 8.0, 400 mM NaCl, 5 mM MgCl2, 0.1 mM phenylmethanesulfonyl fluoride, 1 mM 2-mercaptoethanol, and 10% glycerol). The fusion protein was isolated from the cell lysates by affinity chromatography with HisTrap™ HP (GE Healthcare, Little Chalfont, UK). The buffer was exchanged with PBS by gel filtration and then the purified protein was stored at −80 °C until use. C-CPE protein was quantified with a BCA protein assay kit in which BSA served as the standard (Pierce Chemical, Rockford, IL).

Cell culture and treatments

The cultured HNECs were derived from the mucosal tissues of patients who underwent inferior turbinectomy at the Sapporo Hospital of the Hokkaido Railway Company, or the KKR Sapporo Medical Center Tonan Hospital. Informed consent was obtained from all patients and this study was approved by the ethics committees of Sapporo Medical University, the Sapporo Hospital of Hokkaido Railway Company, and the KKR Sapporo Medical Center Tonan Hospital.

The procedures for primary culture of human nasal epithelial cells were as reported previously (Koizumi et al., Citation2008). Some primary cultured HNECs were transfected with the catalytic component of telomerase, the human catalytic subunit of the telomerase reverse transcriptase (hTERT) gene as described previously (Kurose et al., Citation2007; Kojima et al., Citation2013a). The cells were plated on 35-mm or 60-mm culture dishes (Corning Glass Works, Corning, NY), coated with rat tail collagen (500 µg of dried tendon/ml 0.1% acetic acid). They were cultured in serum-free bronchial epithelial cell basal medium (BEBM, Lonza Walkersville, Inc., Walkersville, MD) supplemented with bovine pituitary extract (1% v/v), 5 μg/ml insulin, 0.5 μg/ml hydrocortisone, 50 μg/ml gentamycin, 50 μg/ml amphotericin B, 0.1 ng/ml retinoic acid, 10 μg/ml transferrin, 6.5 μg/ml triiodothyronine, 0.5 μg/ml epinephrine, 0.5 ng/ml epidermal growth factor (Lonza Walkersville, Inc., Walkersville, MD), 100 U/ml penicillin and 100 μg/ml streptomycin (Sigma-Aldrich, St. Louis, MO) and incubated in a humidified, 5% CO2:95% air incubator at 37 °C. In this experiment, second and third passaged cells were used.

The HNECs were treated with C-CPE 194 and C-CPE m19 at 2 or 4 μg/ml until 24 h and were pretreated with the inhibitors of signal transduction pathways each at 10 μM 30 min before treatment with 2 μg/ml C-CPE 194 and C-CPE m19 for 24 h.

Western blot analysis

For Western blotting of total cell lysates, the dishes were washed with PBS and 300 µl of sample buffer (1 mM NaHCO3 and 2 mM phenylmethylsulfonyl fluoride) was added to 60 - or 35-mm culture dishes. The cells were scraped and collected in microcentrifuge tubes and then sonicated for 10 s. The protein concentrations of the samples were determined using a BCA Protein Assay Reagent Kit (Pierce Chemical Co., Rockford, IL). The samples were prepared by using 2 × SDS sample buffer (Cosmo Bio Co., Tokyo, Japan).

Aliquots of 15 µg of protein/lane for each sample were separated by electrophoresis in 4/20% SDS polyacrylamide gels (Cosmo Bio Co., Tokyo, Japan). After electrophoretic transfer to nitrocellulose membranes (Immobilon; Millipore, Billerica, MA), the membranes were saturated with blocking buffer (Tris-buffered saline [TBS] with 0.1% Tween 20 and 4% skim milk) for 30 min at room temperature and incubated with polyclonal anti-claudin-1, anti-claudin-4, anti-claudin-7, anti-phospho-ERK1/2, anti-ERK1/2, anti-actin, and monoclonal anti-his-tag antibodies (1:1000) for 1 h at room temperature. The membranes were incubated with horseradish peroxidase-conjugated anti-rabbit IgG (Dako A/S, Copenhagen, Denmark) at room temperature for 1 h. The immunoreactive bands were detected using an ECL Western blotting analysis system (GE Healthcare, Little Chalfont, UK).

Immunoprecipitation

The dishes were washed with PBS twice and 300 μl of NP-40 lysis buffer (50 mM Tris–HCl, 2% NP-40, 0.25 mM Na-deoxycholate, 150 mM NaCl, 2 mM EGTA, 0.1 mM Na3VO4, 10 mM NaF, and 2 mM PMSF) was added to the 60 mm dishes. The cells were scraped off, collected in microcentrifuge tubes and then sonicated for 10 s. Cell lysates were incubated with protein A-Sepharose CL-4B (Pharmacia LKB Biotechnology, Inc., Uppsala, Sweden) for 1 h at 4 °C and then clarified by centrifugation at 15 000g for 10 min. The supernatants were incubated with the anti-his-tag antibody bound to protein A-Sepharose CL-4B overnight at 4 °C. After incubation, the immunoprecipitates were washed extensively with the same lysis buffer and subjected to Western blot analysis with anti-claudin-1, anti-claudin-4, and anti-claudin-7 antibodies.

Immunocytochemistry

The cells were grown on 35-mm glass-base dishes (Iwaki, Chiba, Japan) coated with rat tail collagen. They were fixed with cold acetone and ethanol (1:1) at −20 °C for 10 min. After rinsing in PBS, the sections and cells were incubated with polyclonal anti-claudin-1 (1:100), monoclonal anti-claudin-4 (1:100), polyclonal anti-claudin-7 (1:100), and monoclonal anti-occludin (1:100) antibodies at room temperature for 1 h and then with Alexa Fluor 488 (green)-conjugated anti-rabbit IgG (1:200) and Alexa Fluor 594 (red)-conjugated anti-mouse IgG (1:200) at room temperature for 1 h. DAPI (Sigma-Aldrich, St. Louis, MO) was used for counterstaining of nuclei in the cells. The specimens were examined using a confocal laser scanning microscope (LSM5; Carl Zeiss, Jena, Germany).

Cell viability assay

The cytotoxic effects of C-CPE (194 and m19) were assessed with cell counting kit-8 (DOJINDO, Japan) in accordance with the instructions of the manufacturer. Cells were seeded at 3–5 × 103 cells/well (depending on the growth rate and size) in 96-well plates (Corning Life Science, Corning, NY) and precultured for 48 h in a CO2 incubator at 37 °C. Then 10 μl of cell counting kit-8 solution was added to each well and the cells were incubated for 2 h at 37 °C. The optical density of each well was measured at 450 nm with an iMark microplate reader (Bio-Rad, Hercules, CA). The ratio of absorbance was calculated and presented as mean ± SD of more than triplicate experiments.

Measurement of transepithelial electrical resistance (TEER)

The cells were cultured to confluence on the inner chambers of 12-mm Transwell 0.4 -µm pore-size filters (Corning Life Science, Corning, NY). The C-CPE mutants and the inhibitors were treated into inner chambers. TEER was measured using an EVOM voltmeter with an ENDOHM-12 (World Precision Instruments, Sarasota, FL) on a heating plate (Fine, Tokyo, Japan). The values are expressed in standard units of ohms per square centimeter and presented as the mean ± SD of triplicate experiments. For calculation, the resistance of blank filters was subtracted from that of filters covered with cells.

Measurement of permeability

To determine the paracellular flux, the cells were cultured on 12 mm Transwell, 0.4 µm pore-size filters (Corning Inc., Corning, NY), and then the medium containing 500 μg/ml FITC-labeled dextran (MW:4 kDa, Sigma-Aldrich, St. Louis, MO) or 1 mg/ml FITC-labeled human insulin (MW: 6.5 kDa, Sigma-Aldrich, St. Louis, MO) was added to the inner chamber. Samples were collected from the outer chamber at 15 min, 30 min, 1 h, 2 h, 3 h, and 4 h and were measured with a Wallac 1420 multilabeled counter (PerkinElmer, Turku, Finland) and an Infinite M1000 Pro (TECAN, Männedorf, Switzerland).

Diffusion of BODIPHY-sphingomyelin

For the measurement of the tight junctional fence function, we used diffusion of BODIPY-sphingomyelin (Balda et al., Citation1996) with some modification. Sphingomyelin/BSA complexes (5 nM) were prepared in P buffer (10 nM HEPES, pH 7.4, 1 mM sodium pyruvate, 10 mM glucose, 3 mM CaCl2, and 145 mM NaCl) using BODIPY-FL-sphingomyelin (Molecular Probes, Eugene, OR) and defatted BSA. Cells plated on glass-bottom microwell plates (Mat Tek Corp., Ashland, MA) were loaded with BODIPY-sphingomyelin/BSA complex for 1 min on ice, after which they were rinsed with cold DMEM and mounted in DMEM on a glass slide. The samples were analyzed by confocal laser scanning microscopy (LSM5; Carl Zeiss, Jena, Germany). All pictures shown were generated within the first 5 min of analysis.

Data analysis

Signals were quantified using Scion Image Beta 4.02 Win (Scion Co., Frederick, MA). Each set of results shown is representative of at least three separate experiments. Results are given as means ± SEM. Differences between groups were tested by analysis of variance followed by a post hoc test and an unpaired two-tailed Student's t test.

Results

His-tag C-CPE 194 and C-CPE m19

To confirm peptide of C-CPE 194 and C-CPE m19, we performed Western blotting by using an anti-his-tag antibody. The peptides of C-CPE 194 and C-CPE m19 were confirmed as bands of approximately 15 kDa ().

Figure 1. (A) Western blotting for anti-his-tag antibody in the peptide of C-CPE 194 and C-CPE m19. (B) Cell viability assay in HNECs after treatment with C-CPE 194 or C-CPE m19 at 1–4 μg/ml for 24 h. Barrier function examined by TEER values (C and D), paracellular flux using FITC-dextran 4 kDa (E) and fence function examined by diffusion of labeled BODIPY-sphingomyelin (F) in HNECs after treatment with 2 μg/ml C-CPE 194 or C-CPE m19 for 24 h. *p < 0.05, **p < 0.01 versus control. 194: C-CPE 194, m19: C-CPE m19.

Figure 1. (A) Western blotting for anti-his-tag antibody in the peptide of C-CPE 194 and C-CPE m19. (B) Cell viability assay in HNECs after treatment with C-CPE 194 or C-CPE m19 at 1–4 μg/ml for 24 h. Barrier function examined by TEER values (C and D), paracellular flux using FITC-dextran 4 kDa (E) and fence function examined by diffusion of labeled BODIPY-sphingomyelin (F) in HNECs after treatment with 2 μg/ml C-CPE 194 or C-CPE m19 for 24 h. *p < 0.05, **p < 0.01 versus control. 194: C-CPE 194, m19: C-CPE m19.

Cytotoxic effects of C-CPE mutants in HNECs

To investigate the cytotoxic effects of the C-CPE mutants in HNECs, the cells were treated with C-CPE 194 or C-CPE m19 at 2 or 4 μg/ml for 24 h, and the cell viability assay was performed. Cytotoxic effects were not observed at any concentration of C-CPE 194 or C-CPE m19 ().

Effects of C-CPE mutants on barrier function and fence function in HNECs

To investigate the effects of the C-CPE mutants on the barrier function in HNECs, the cells were treated with 2 μg/ml C-CPE 194 or C-CPE m19 for 24 h and the barrier function was measured by TEER values and fluxes of FITC-dextran. The TEER values were decreased after treatment with C-CPE 194 - and C-CPE m19 in a time-dependent manner () and the significant decrease of TEER values at 24 h after treatment was observed compared with the control (). The permeability of FITC-dextran was significantly increased at 24 h after treatment with 2 μg/ml C-CPE 194 or C-CPE m19 (). To investigate the effects of the C-CPE mutants on the fence function in HNECs, BODIPY-sphingomyelin diffusion in the membrane was measured in HNECs 24 h after treatment with 2 μg/ml C-CPE 194 or C-CPE m19. In the control, the BODIPY-sphingomyelin was effectively retained in the apical domain. In HNECs after treatment with C-CPE 194 or C-CPE m19, the probe diffused through the tight junctions, strongly labeled the basolateral surfaces and appeared to penetrate the cells ().

Effects of C-CPE mutants on the expression and the localization of tight junction proteins in HNECs

To investigate whether C-CPE affected the expression of tight junction proteins claudin-1, -4, -7, and occludin in HNECs, the cells were treated with 2 μg/ml C-CPE 194 or C-CPE m19 for 4, 8, and 24 h. In Western blots, no change in expression of the tight junction proteins was observed in the cells after treatment with C-CPE 194 or C-CPE m19 compared with the control ().

Figure 2. Western blottting (A) and immunocytochemistry (B) for claudin-1, -4, -7, and occludin in HNECs after treatment with 2 μg/ml C-CPE 194 or C-CPE m19 for 24 h. CLDN: claudin, OCLN: occludin, 194: C-CPE 194, m19: C-CPE m19. Bar = 20 μm.

Figure 2. Western blottting (A) and immunocytochemistry (B) for claudin-1, -4, -7, and occludin in HNECs after treatment with 2 μg/ml C-CPE 194 or C-CPE m19 for 24 h. CLDN: claudin, OCLN: occludin, 194: C-CPE 194, m19: C-CPE m19. Bar = 20 μm.

To investigate the effect of C-CPE on localization of claudin-1, -4, -7, and occludin in HNECs, the cells were treated with 2 μg/ml C-CPE 194 or C-CPE m19 for 24 h and immunocytochemistry was performed. In the treated cells, no change in localization of the tight junction proteins was observed ().

Behavior of C-CPE mutants after treatment in HNECs

To investigate the behavior of the C-CPE mutants in HNECs after treatment, Western blotting was performed after coimmunoprecipitation by using an anti-his-tag antibody. Claudin-4-binding C-CPE 194 was decreased from 4 h to 24 h after treatment, whereas claudin-7-binding C-CPE 194 was detected at 4 and 8 h after treatment (). Claudin-4- and -7-binding C-CPE m19 were detected at 4, 8, and 24 h after treatment (). In the present study, claudin-1 was not detected in any sample after treatment with C-CPE mutants ().

Figure 3. Western blotting for claudin-1, -4, and -7 after coimmunoprecipitation using an anti-his-tag antibody in HNECs after treatment with 2 μg/ml C-CPE 194 or C-CPE m19 for 4, 8, and 24 h. The corresponding expression levels are shown as bar graphs. **p < 0.01 versus control. CLDN, claudin.

Figure 3. Western blotting for claudin-1, -4, and -7 after coimmunoprecipitation using an anti-his-tag antibody in HNECs after treatment with 2 μg/ml C-CPE 194 or C-CPE m19 for 4, 8, and 24 h. The corresponding expression levels are shown as bar graphs. **p < 0.01 versus control. CLDN, claudin.

C-CPE mutants transiently increase the activity of ERK1/2 phosphorylation in HNECs

To investigate whether C-CPE mutants affected MAPK signaling in HNECs, the cells were treated with 2 μg/ml C-CPE 194 or C-CPE m19 for 4, 8, and 24 h and Western blotting was performed using a ERK1/2 phosphorylation antibody. In both C-CPE 194 and C-CPE m19, an increase of ERK1/2 phosphorylation was observed at 4 h after treatment (). When the cells were pretreated with MAPKK inhibitor U0126 after treatment with 2 μg/ml C-CPE 194 or C-CPE m19 for 4 h, the increase of ERK1/2 phosphorylation induced by C-CPE mutants was completely prevented by U0126 ().

Figure 4. (A) Western blotting for phospho-ERK 1/2 and ERK 1/2 in HNECs after treatment with 2 μg/ml C-CPE 194 or C-CPE m19 for 4, 8, and 24 h. (B) Western blotting for phospho-ERK 1/2 and ERK 1/2 in HNECs at 4 h after treatment with 2 μg/ml C-CPE 194 or C-CPE m19 with or without 10 μM U0126. The corresponding expression levels of A and B are shown as bar graphs. **p < 0.01 versus control, ##p < 0.01 versus 194 or m19. 194: C-CPE 194, m19: C-CPE m19.

Figure 4. (A) Western blotting for phospho-ERK 1/2 and ERK 1/2 in HNECs after treatment with 2 μg/ml C-CPE 194 or C-CPE m19 for 4, 8, and 24 h. (B) Western blotting for phospho-ERK 1/2 and ERK 1/2 in HNECs at 4 h after treatment with 2 μg/ml C-CPE 194 or C-CPE m19 with or without 10 μM U0126. The corresponding expression levels of A and B are shown as bar graphs. **p < 0.01 versus control, ##p < 0.01 versus 194 or m19. 194: C-CPE 194, m19: C-CPE m19.

A MAPKK inhibitor prevents downregulation of barrier function induced by C-CPE mutants in HNECs

To investigate which signaling pathways were associated with the downregulation of the barrier function induced by C-CPE mutants in HNECs, the cells were pretreated with MAPKK inhibitor U0126, panPKC inhibitor GF109203X, c-Jun N-terminal kinase (JNK) inhibitor SP600125, phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 and p38 MAPK inhibitor SB203580, each at 10 μM, before treatment with 2 μg/ml C-CPE 194 or C-CPE m19. The TEER value was enhanced by only U0126 and the decrease of the TEER value induced by C-CPE 194 or C-CPE m19 was prevented by U0126 ().

Figure 5. TEER values (A) and paracellular flux using FITC-insulin (B) in HNECs pretreated with or without 10 μM U0126 at before treatment with or without 2 μg/ml C-CPE 194 or C-CPE m19 for 24 h. *p < 0.05, **p < 0.01 versus control, #p < 0.05, ##p < 0.01 versus 194 or m19.

Figure 5. TEER values (A) and paracellular flux using FITC-insulin (B) in HNECs pretreated with or without 10 μM U0126 at before treatment with or without 2 μg/ml C-CPE 194 or C-CPE m19 for 24 h. *p < 0.05, **p < 0.01 versus control, #p < 0.05, ##p < 0.01 versus 194 or m19.

C-CPE mutants enhance the permeability of FITC-insulin across HNECs

To investigate whether C-CPE mutants could enhance the permeability of FITC-insulin across HNECs, the cells of the inner chamber were treated with FITC-labeled human insulin (MW: 6.5 kDa) after treatment with C-CPE 194 or C-CPE m19 for 24 h. FITC-insulin of the outer chamber in C-CPE 194 and C-CPE m19 was significantly increased in a dose-dependent manner compared with the control (). The increase of FITC-insulin by both C-CPE mutants was significantly prevented the MAPKK inhibitor U0126 ().

Discussion

In the present study, we demonstrated for the first time that claudin-binder C-CPE mutants enhanced the permeability of insulin across HNECs via the MAPK signaling pathway. It is reported that C-CPE does not have a cytotoxic effect in vitro (Sonoda et al., Citation1999). C-CPE disrupts the tight junctional barrier function with changes of claudins in various types of cells (Sonoda et al., Citation1999; Masuyama et al., Citation2005; Gao et al., Citation2011; Matsuhisa et al., Citation2012; Takahashi et al., Citation2012).

In the present study, the C-CPE mutants C-CPE 194 and C-CPE m19 disrupted the barrier and fence functions of tight junctions without cytotoxicity or changes in protein expression and localization of claudin-1, -4, -7, and occludin. However, the detailed mechanisms of disruption of the tight junctional barrier by C-CPE mutants remain unclear.

It has been thought that the tight junctional functions are regulated via distinct signal transduction pathways (Stuart & Nigam, Citation1995; González-Mariscal et al., Citation2008; Kojima et al., Citation2009). Furthermore, the signal transduction from tight junctions also regulates epithelial cell proliferation, gene expression, differentiation, and morphogenesis (Matter & Balda, Citation2003; Tsukita et al., Citation2008). When HNECs were pretreated with inhibitors of various signal transduction pathways (MAPKK inhibitor U0126, panPKC inhibitor GF109203X, JNK inhibitor SP600125, PI3K inhibitor LY294002, and p38 MAPK inhibitor SB203580) before treatment with C-CPE 194 and C-CPE m19, MAPPK inhibitor U0126 prevented the disruption of the barrier. Furthermore, C-CPE 194 and C-CPE m19 enhanced the activity of ERK1/2 phosphorylation. These results suggest that C-CPE mutants binding claudins enhance the activity of MAPK in HNECs and the disruption of the barrier function of tight junctions by C-CPE mutants in part via the MAPK signaling pathway. Because in the present study, the barrier function of normal HNECs was enhanced by MAPKK inhibitor U0126.

Western blotting after coimmunoprecipitation showed that C-CPE 194, but not C-CPE m19, binding claudin-4 and -7 was markedly decreased in HNECs from 24 h after treatment. Although the mechanisms are not unclear, it is noteworthy that C-CPE 194 rapidly disappears from the cells, indicating their potential use in the drug deliver system. It is thought that C-CPE 194 may be safe to use than C-CPE m19.

In conclusion, the claudin-binder C-CPE mutants disrupted the barrier and fence functions of tight junctions without cytotoxicity in HNECs and enhanced the permeability of insulin across the epithelial cells. It is possible that C-CPE mutants could play a crucial role in direct insulin therapy such as intranasal insulin administration for Alzheimer's disease.

Declaration of interest

This work was supported by the Ministry of Education, Culture, Sports Science, and Technology, and the Ministry of Health, Labour and Welfare of Japan.

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