Journal of Dermatological Science 69 (2013) 148–158

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Journal of Dermatological Science

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Impaired tight junctions obstruct stratum corneum formation by altering polarlipid and profilaggrin processing

Takuo Yuki a,*, Aya Komiya a, Ayumi Kusaka a, Tetsuya Kuze b, Yoshinori Sugiyama a, Shintaro Inoue a

a Innovative Beauty Science Laboratory, Kanebo Cosmetics Inc., Kotobuki-cho, Odawara, Kanagawa, Japanb Quality Management Group, Kanebo Cosmetics Inc., Kanagawa, Japan

A R T I C L E I N F O

Article history:

Received 10 September 2012

Received in revised form 21 November 2012

Accepted 30 November 2012

Keywords:

Tight junction

Cutaneous barrier

Stratum corneum

Filaggrin

Intercellular lipid

A B S T R A C T

Background: The stratum corneum (SC) is a well-known structure responsible for the cutaneous barrier.

Tight junctions (TJs) function as a paracellular barrier beneath the SC and are involved in the cutaneous

barrier. It remains unclear how TJs are involved in the cutaneous barrier.

Objective: In order to clarify the role of TJs in the cutaneous barrier, we investigated skin equivalent

models with disrupted TJ barriers focusing on the SC.

Methods: Skin equivalents with disrupted TJ barriers were established using GST-C-CPE, a peptide with

specific inhibitory action against specific claudins. The changes of the SC barrier in the skin equivalents

with disrupted TJ barriers were investigated and compared with control skin equivalents.

Results: An outside-to-inside skin barrier assay revealed a defective SC barrier in skin equivalents with

disrupted TJ barriers. A detailed examination of the SC revealed an increase in the pH of the SC in the skin

equivalent with disrupted TJ barriers. An electron microscopy showed the failure of lamellar structures

to mature and the failure of keratohyalin granules to degrade in the skin equivalents with disrupted TJ

barriers. A thin layer chromatography analysis showed an increase in polar lipids and a decrease in non-

polar lipids. A western blot analysis showed an increase in filaggrin dimer and trimer and a decrease in

filaggrin monomer.

Conclusion: We found that disrupted TJs obstructed the SC formation responsible for the cutaneous

barrier. Our study indicates the possibility that impaired TJ barriers affect polar lipids and profilaggrin

processing by disturbing the pH condition of the SC.

� 2012 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights

reserved.

1. Introduction

As the most external organ of the human body, the mainfunction of the epidermis is to provide a protective barrieragainst water loss and the penetration of infectious agents andallergens. The stratum corneum (SC) is the outermost layer of theepidermis and is largely responsible for this vital barrierfunction. The SC arises from a specific differentiation programof keratinocytes from the basal layer to the spinous and granularlayers of the epidermis [1].

In simple epithelial and endothelial cellular sheets, tightjunctions (TJs) are one mode of cell–cell adhesion [2]. They arecomposed of a network of sealing strands and act as a primarybarrier to the diffusion of solutes through the intercellular space.Each strand is made from a row of transmembrane proteins.

* Corresponding author at: Innovative Beauty Science Laboratory, Kanebo

Cosmetics Inc., 5-3-28 Kotobuki-cho, Odawara, Kanagawa 250-0002, Japan.

Tel.: +81 465 34 6116; fax: +81 465 34 3037.

E-mail address: yuki.takuo@kanebocos.co.jp (T. Yuki).

0923-1811/$36.00 � 2012 Japanese Society for Investigative Dermatology. Published b

http://dx.doi.org/10.1016/j.jdermsci.2012.11.595

Occludin and claudins are major types of transmembrane proteinin TJs, but other types are present [3]. The TJ barrier was found inthe murine epidermis, stratified epithelial cellular sheet, byexamining claudin-1 knockout mice [4]. When the intercellulartracer was injected into the wild-type mice epidermis from thedermal side, it diffused through the paracellular space but wasstopped before reaching the SC. Detailed examination revealed theTJ barrier formation at the sites where the intercellular tracer hadbeen stopped [4]. Compared to the TJs in wild-type mice, TJs in theclaudin-1 knockout mice were permeable to an intercellular tracer.Consequently, the cutaneous barrier assessed by transepidermalwater loss (TEWL) was disrupted in claudin-1 knockout mice.Other than claudin-1 knockout mice, there are several animalswhose claudins were genetically modified [5]. Turksen et al.investigated the cutaneous barrier of claudin-6 transgenic miceand showed their abnormal cutaneous barrier [6–8]. The resultsfrom experiments on claudin-1 knockout mice and claudin-6transgenic mice prompted us to hypothesize that the TJ barriermay be involved in the cutanous permeability barrier. In humans,it has been demonstrated that the TJ barrier is also formed in theepidermis and keratinocytes isolated from the epidermis [9–11].

y Elsevier Ireland Ltd. All rights reserved.

T. Yuki et al. / Journal of Dermatological Science 69 (2013) 148–158 149

Ichthyosis is a heterogeneous group of skin disorders charac-terized by abnormal SC scaling and defects in the cutaneouspermeability barrier [12]. Neonatal ichthyosis-sclerosing cholan-gitis syndrome is a rare autosomal recessive disorder caused byhomozygous mutations in the CLDN1 gene coding for the TJcomponent claudin-1 [13–16], suggesting that TJs are relevant toichthyosis. Atopic dermatitis is the most common inflammatoryskin disease and the SC of atopic dermatitis is dysfunctional due toseveral reasons [17–20]. De Benedetto et al. reported that theexpression of claudin-1 is significantly reduced in the nonlesionalskin of patients with atopic dermatitis compared with nonatopicsubjects, suggesting that reductions in this key TJ barrier proteinmight also affect the cutaneous barrier [21].

As described above, epidermal TJs suggest the associationwith the cutaneous barrier, but detailed mechanisms aremissing. In order to clarify how TJs are involved in the cutaneouspermeability barrier, we studied human skin equivalent modelswith disrupted TJ barriers, focusing on the SC formation. Tomodulate the barrier function of the TJs, we utilized the C-terminal half of Clostridium perfringens enterotoxin (C-CPE, seeSection 2 for details), which is reported to bind to claudins anddisrupt the TJ barrier [22–25]. We then examined the SCformation using an originally established human skin equivalentwith a disrupted TJ barrier.

2. Materials and methods

2.1. Production of GST-C-CPE fusion proteins

Clostridium perfringens enterotoxin (CPE) is a causative agentof symptoms associated with clostridium perfringens foodpoisoning in human beings [26]. Katahira et al. cloned a cDNAencoding the receptor for CPE (CPE-R) of the Vero cells of anenterotoxin-sensitive monkey from the expression library [23].They further showed that previously reported RVP1 (Rat ventralprostate 1 protein) had a marked sequence similarity to CPE-R, andthat RVP1 also functioned as a receptor for CPE [24]. Later, tworelated integral membrane proteins, claudin-1 and -2, wereidentified as novel components of TJ strands [27]. Interestingly,the sequence of claudin-1/-2 was very similar to that of RVP1 andCPE-R, so RVP1 and CPE-R were designated as claudin-3 andclaudin-4, respectively [28]. Further, Sonoda et al. showed that theCOOH-terminal half of CPE (amino acid 184–319; C-CPE) affectedthe expression level of claudin-4 and down-regulated the TJ barrierin MDCK 1 cells [22].

To modulate the barrier function of the TJs, we utilized the C-terminal half of Clostridium perfringens enterotoxin, which isreported to bind to at least claudin-4 and -6 and disrupt the TJbarrier [22–25]. DNA fragments encoding the C-terminal half ofCPE (amino acid 184–319) were amplified from the templateplasmid DNA containing a full length of CPE (kindly provided by Dr.Y. Horiguchi) using primer pairs described below.

50-ccgctcgatAGATGTGTTTTAACAGTTCCATCTAC-30

50-ggaagatctTAAAATTTTTGAAATAATATTGAATAAGGG-30

The amplified product was subcloned into pGEX6P-1 vector(Amersham Pharmacia Biotech, Buckinghamshire, UK). Theobtained fusion protein products were designated GST-C-CPE.These GST fusion proteins were generated in Escherichia coli andpurified using glutathione–Sepharose 4B beads (AmershamPharmacia Biotech, Buckinghamshire, UK). Eluted GST fusionproteins were dialyzed against phosphate-buffered saline (PBS) at4 8C and used for experiments. We also tried to prepare a deletionmutant of GST-C-CPE (amino acid 184–313) for control experi-ments, but it tended to form an aggregate, which made it difficultto control its concentration in solution, therefore, we used only GSTfor control experiments.

2.2. Addition of GST-C-CPE into the medium of the skin equivalent

models

In order to establish skin equivalent models with disrupted TJbarriers, human skin equivalent models (TOYOBO, Osaka, Japan)were cultured according to the manufacturer’s recommendations.Seven days after exposing the cells to the air (air-lift culturing), theskin equivalent models were about to form SCs. At this point,10 mg/ml or 50 mg/ml GST-C-CPE was added into the medium.After 4 days of air-lift culturing with GST-C-CPE, skin equivalentmodels that had formed SCs were harvested for experiments. Weadded 50 mg/ml GST for the control experiments.

2.3. Immunoblotting

For analyses of total protein levels of loricrin, involucrin,transglutaminase-1 and filaggrin in the epidermis, harvested skinmodels were washed with ice cold PBS, and an epidermal sheetwas obtained by slipping the dermis off. The epidermal sheet wassubsequently lysed in the buffer containing 200 mM Tris (pH 7.4),2.0% SDS, protease inhibitor cocktail (Roche, Mannheim, Germany),and phosphatase inhibitor cocktail (Roche, Mannheim, Germany).Equal amounts of total protein (20 mg) were subjected to SDS-PAGE. Samples separated by SDS-PAGE were transferred onto PVDFmembranes and the membranes were soaked in 5% skimmed milkand incubated with the primary antibodies and then HRP-conjugated secondary antibodies. The signal was detected byenhanced chemiluminescence (Thermo Scientific, Rockford, IL) andexposed to X-ray film. The primary antibody used here was anti-involucrin mAb (Thermo Scientific, Cheshire, UK), anti-loricrin pAb(Covance, Emeryville, CA), anti-transglutaminase-1 pAb (NovusBiologicals, Letteton, CO) and anti-filaggrin mAb (clone 15C10)(Leica, Newcastle, UK).

2.4. Histology, immunofluorescence staining, and microscopy

Tissues were fixed in formal saline and embedded in paraffin.For the routine histology, 5-mm sections were cut and picked uponto Superfrost Plus slides (Surgipath, Peterborough, UK). Thesections were subsequently stained by standard methods withHematoxylin and Eosin (H&E) or for Ki67. Ki67 was detected bycitrate buffer (pH 6.0) antigen retrieval, followed by incubationwith DAKO M7240 mouse monoclonal Ki67 (MIB-1) and ABC kit(Vector laboratries). Ki67 positive cells were counted with free-software Image J. For frozen sections, tissues were placed in OCTcompound in a liquid nitrogen-cooled isopentane bath. Frozentissue sections (5 mm) were fixed with 95% ethanol for 30 min andsubsequently fixed with acetone for 2 min, blocked with 1% BSA inPBS and immunolabeled using the following primary antibodies;anti-GST pAb (GE healthcare, Uppsala, Sweden), anti-claudin-4pAb (Invitrogen, Camarillo, CA), anti-occludin mAb and anti-filaggrin mAb (clone 15C10) (Leica, Newcastle, UK). For GSTstaining, harvested skin models were washed three times in PBS torinse unbound GST off, and then, double immunostaining for GSTand claudin-4 was performed using frozen sections.

2.5. GST pull-down assay

Tissues incubated with GST or GST-C-CPE were harvested andthe epidermal sheet lysed in lysis buffer (1% Triton X-100, 50 mMTris/HCl, pH 7.4, 150 mM NaCl, and complete protease inhibitors(Roche, Mannheim, Germany)). The lysates were used for GST pull-down assay using GST SpinTrapTM kit containing glutathionesepharose 4B beads (GE healthcare, Uppsala, Sweden). Afterincubation of the lysates with the glutathione sepharose 4B beads,the beads were washed, and bound and unbound proteins were

T. Yuki et al. / Journal of Dermatological Science 69 (2013) 148–158150

analyzed by SDS-PAGE followed by western blotting. The primaryantibody used here was anti-claudin-1 pAb (Invitrogen, Camarillo,CA), anti-claudin-4 mAb (Invitrogen, Camarillo, CA), anti-E-cadherin mAb (Invitrogen, Camarillo, CA), anti-desmoglein 1 + 2pAb (Progen Biotechnik GmbH, Heidelberg, Germany) and anti-b-actin mAb (Sigma, St. Louis, MO).

2.6. TJ permeability assay

In the human keraitinocytes culture, the TJ-associated barrierwas evaluated by measuring the transepithelial electric resistance(TER) using a Millicell-ERSTM epithelial voltmeter [29].

In the human skin models, the TJ permeability assay wasperformed to assess TJ barrier using EZ-LinkTM Sulfo-NHS-LC-Biotin (M.W. 556.59) (Pierce, Rockford, IL) as a paracellular traceraccording to the previous study [9]. Human skin models wereincubated with 2 mg/ml sulfo-NHS-LC-Biotin in PBS containing1 mM CaCl2 from the dermal side. After a 30-min incubation, theskin models were removed and frozen in liquid nitrogen. Frozensections, 5-mm thick, were fixed in 95% ethanol for 30 min andthen placed in 100% acetone for 2 min. The sections were soaked in1% bovine serum albumin/PBS for 15 min, incubated with anti-occludin mAb for 1 h, washed three times with PBS, and incubatedwith a mixture of FITC conjugated anti-rat pAb (JacksonImmunoResearch, West Grove, PA) and streptavidin-Texas Red(Calbiochem, Farmstadt, Germany) for 1 h.

2.7. Analysis of Lucifer yellow permeability

In order to examine the barrier function of the SC, we performeda Lucifer yellow permeability assay. After 4 days with GST or GST-C-CPE, 200 ml Lucifer yellow (Sigma–Aldrich, Dorset, UK) wasadded to the SC and incubated at 37 8C for 2 h. Then, the skinsamples were fixed in 10% formalin neutral buffer solution (Wako,Oosaka, Japan) and embedded in an OTC compound (Sakurafinetek, Tokyo, Japan). Cryo-sections (7 mm) were inspected underthe fluorescence microscope. For a quantification analysis ofLucifer yellow penetration, the amount of Lucifer yellow leaked tothe medium through the skin equivalent models was measured bya fluorometer (Nihon Molecular Devices, Tokyo, Japan).

2.8. Transmission electron microscopy

Freshly obtained skin models were either transferred to half-strength Karnovsky’s fixative, or immersed first in absolutepyridine for 2 h prior to aldehyde fixation (Behne, Uchida et al.[30]). All samples were postfixed with 1% aqueous osmiumtetroxide (OsO4), containing 0.5% potassium ferrocyanide, andembedded in an Epon–epoxy mixture. For visualization of lamellarmembrane structures, alternate samples were postfixed withruthenium tetroxide (RuO4) [30]. Sections were cut on a ReichertUltracut E microtome and counterstained with uranyl acetate andlead citrate. Sections were viewed in a Zeiss 10 CR electronmicroscope, operated at 60 kV.

2.9. SC pH

SC surface pH was measured with a flat surface electrodeattached to a pH meter (HORIBA, Tokyo, Japan).

2.10. Lipid extraction and high-performance thin-layer

chromatography (HPTLC) analysis

A 12 mm diameter area of the epidermis on the surface wasprepared by punch biopsy and used for the lipid extraction.Unbound lipids were extracted by modifying the Bligh/Dyer

method as follows [31]. Epidermal sheets were obtained from theskin models and were soaked in Bligh/Dyer solution (choloro-form:methanol:H2O = 2:4:1.6) for 16 h at room temperature. Afterremoval of the epidermal sheet by centrifugation, 2 ml chloroformand 2 ml H2O were added, and the lower phase was concentratedby nitrogen gas and served as the unbound lipid sample (unboundlipid). The removed epidermal sheet was subsequently saponifiedin 500 ml 1 N NaOH in 90% methanol at 80 8C for 2 h to release thelipids covalently bound to the SC by ester-like bonds [32].Following removal of the residue by centrifugation and neutrali-zation with HCl, 2.5 ml chloroform and 2.5 ml H2O were added andthe mixture was shaken vigorously. The lower phase wasconcentrated by nitrogen gas and served as the ester-bound lipidsample (Bound lipid). The dried samples were re-dissolved inchloroform/methanol (2/1) solvent and fractionated by HPTLC on20 cm � 10 cm plates (Silica gel 60, Merck, Darmstadt, Germany).For separation of polar lipids (the precursors of mature lipids), thechromatograms were developed with chloroform/methanol/water(70/30/5, v/v/v) [33]. Non-polar lipids (mature lipids) wereresolved twice using chloroform/methanol/glacial acetic acid(190/9/1, v/v/v) as a developing solvent [34]. Lipids werequantitated by photo densitometry (Shimadzu, Kyoto, Japan) atl = 595 nm. The results showed the ratio of the amount of lipid inthe epidermis treated with GST-C-CPE to that of the control.

2.11. Calculations and statistics

All data are expressed as means � SD. Individual groups werecompared using the t-test for all pair-wise comparisons. A level ofP < 0.05 was accepted as statistically significant for all comparisons.

3. Results

3.1. Establishment of human skin equivalent models with disrupted TJ

barriers by GST-C-CPE

Originally, claudin-3 and -4 were identified as the cellularreceptors for Clostridium perfringens enterotoxin (CPE) [23,24].CPE protein is divided into two domains: the NH2-terminal halfwith cytotoxicity and the C-terminal half with cell binding activity.A recombinant C-terminal half of CPE (aa. 184–319 of CPE; C-CPE)binds to these claudins without affecting cell viability [23]. Sonodaet al. previously shown that CPE binds to some claudin familymembers in addition to claudin-3 and -4 and that C-CPE disrupts TJbarrier in MDCK cells through down-regulation of claudin-4,thereby reducing the barrier function of the cellular sheet [22].

The purpose of this study is to establish and examine humanskin equivalents with disrupted TJ barriers using the glutathione S-transferase fused COOH-terminal half of clostridium perfringensenterotoxin (GST-C-CPE) in order to clarify the role of TJs in SCformation. Firstly, we prepared GST fused C-CPE according to themethod used by Moriwaki et al. [25]. In Supplementary Fig. S1, weconfirmed that GST-C-CPE bound to claudin-4 but not claudin-1,and disrupted the TJ barrier in the monolayer culture ofkeratinocytes.

Supplementary data related to this article found, in the onlineversion, at doi:10.1016/j.jdermsci.2012.11.595.

After confirming the effect of GST-C-CPE on the TJ barrier inkeratinocytes, we tried to establish human skin equivalent modelswith disrupted epidermal TJ barriers by using GST-C-CPE. GST-C-CPE was added into the medium of the skin equivalent models thatwere about to form an SC. Then, in the presence of GST-C-CPE,human skin equivalent models were cultured for 4 days. First, toexamine whether or not GST-C-CPE disrupted the epidermal TJbarrier in the skin equivalent model, a TJ permeability assay wasperformed. The skin treated with GST showed that occludin was

T. Yuki et al. / Journal of Dermatological Science 69 (2013) 148–158 151

expressed in the stratum granulosum (SG) and the diffused sulfo-NHS-LC-biotin (M.W. 556.59) was halted at the SG (Fig. 1A). Themerged image showed that diffusion of sulfo-NHS-LC-biotin washalted at the occludin expression sites (Fig. 1A), indicating thatTJs function as a paracellular barrier in skin treated with GST. Inskin treated with GST-C-CPE, occludin was expressed in the SG,as in the case of the control skin (Fig. 1A). However, the diffusionof sulfo-NHS-LC-biotin was not halted at the occludin expressionsites and reached the SC (Fig. 1A), indicating that the barrier[(Fig._1)TD$FIG]

Fig. 1. Generation of human skin equivalent models with disrupted TJ barriers. (A) TJ p

stained with occludin (green). Bars: 40 mm. (B) The proteins associated with GST or GST-

equivalent models treated with GST (50 mg/ml), none of the proteins appeared in the bou

only claudin-4 appeared in the bound fraction. (C) Immunohistochemistry using an ant

using an antibody for occludin (green), the TJ-specific marker, and claudin-4 (red). Bar

function of TJs is disrupted in skin treated with GST-C-CPE. Then,to examine whether or not GST-C-CPE binds to claudins, a GSTpull-down assay was performed using the epidermal sheets ofthe skin equivalent models. In skin incubated with GST, claudin-4was detected in the unbound fraction (Fig. 1B). In skin incubatedwith GST-C-CPE, claudin-4 was detected in the bound fraction,not in the unbound fraction (Fig. 1B). In skin treated with GST-C-CPE, claudin-1, E-cadherin, desmoglein 1/2 and b-actin were notdetected in the bound fraction (Fig. 1B). Immunohistochemical

ermeability assay using sulfo-NHS-LC-Biotin (red) was performed, concomitantly

C-CPE were examined by GST pull-down assay and western blot analysis. In the skin

nd fractions. In the skin equivalent models treated with GST-C-CPE (10 or 50 mg/ml),

ibody for claudin-4 (green) and GST (red). Bar: 40 mm. (D) Immunohistochemistry

s: 40 mm. (E) A MTT assay. Error bars indicate mean � SD.

T. Yuki et al. / Journal of Dermatological Science 69 (2013) 148–158152

analyses showed that, in the GST-treated skin equivalent models,claudin-4 was expressed in the granular layer but the signal forGST was not detected (Fig. 1C). In the GST-C-CPE-treated skinequivalent model, claudin-4 appeared to be less expressed andGST was detected clearly compared to the control skin (Fig. 1C).Further, the GST signal mostly colocalized with claudin-4 in theplasma membrane in the skin equivalent models treated withGST-C-CPE (Fig. 1C). These findings indicate that GST-C-CPEbinds to claudin-4 in the epidermis, but not to claudin-1, E-cadherin or desmoglein. In order to confirm whether claudin-4was eliminated from the TJs by GST-C-CPE treatment, doubleimmunostaining of claudin-4 with occludin was performed,because occludin is highly concentrated at the TJs and is anexcellent marker for TJs identified to date [4,35,36]. In Fig. 1D, inthe skin equivalent model treated with GST, occludin waspredominantly expressed at the cell–cell border in the granularlayer as dots. Claudin-4 was also expressed in the granular layerand colocalized with occludin at the cell–cell border in thegranular layer (Fig. 1D). The skin equivalent model treated withGST-C-CPE showed that the localization of occludin remained asdots in the granular layer. However, colocalization of claudin-4with occludin had decreased, possibly due to a decrease inclaudin-4 expression (Fig. 1D). These results suggest that GST-C-CPE eliminates claudin-4 from TJs in the skin equivalent.

Cell viability was assessed using skin equivalent models treatedwith GST or GST-C-CPE by an MTT assay. No difference wasdetected in the MTT values between GST- and GST-C-CPE-treatedskin equivalent models (Fig. 1E).

[(Fig._2)TD$FIG]

Fig. 2. Analysis of epidermal proliferation and differentiation markers in skin treated with

There were no differences in the localization and the number of Ki67 positive cells. The nu

at least 3 sections per group. Error bars indicate mean � SD. Bars: 20 mm. (B) Western

performed. In immunohistochemistry, involucrin and loricrin were described in red and blu

Altogether, we were able to establish skin equivalent modelswith disrupted TJ barriers without affecting cell viability.

3.2. Disrupted TJ barrier did not affect cell proliferation and

differentiation markers

In order to examine the effect of GST-C-CPE on cell proliferationin the epidermis, Ki67 staining was performed. Several basal cellswere positive for Ki67 in the skin equivalent models with disruptedTJ barriers and in the control skin equivalent model, but there wasno difference between them (Fig. 2A). The number of Ki67-positivecells we counted did not differ between them either (Fig. 2A).Further, the expression and localization of loricrin and involucrinwere examined in order to ascertain whether or not GST-C-CPEaffects the differentiation markers. As shown in Fig. 2B, theexpression levels of differentiation markers were not changed byGST-C-CPE. Double immunolabeling for involucrin and loricrinshowed that their localization was also unchanged. These findingsindicated that GST-C-CPE does not affect the expression of cellproliferation and differentiation markers.

3.3. SC barrier was weakened in the skin equivalent model with

disrupted TJ barrier

In order to examine the barrier function of the SC, weperformed a Lucifer yellow penetration assay of the SC. In50 mg/ml GST-treated skin, Lucifer yellow did not permeate intothe SC (Fig. 3A). On the contrary, Lucifer yellow reached the

GST-C-CPE. (A) The epidermal proliferation marker was evaluated by Ki67 staining.

mber of Ki67-positive cells/250 mm in the basement membrane was counted using

blot and immunohistochemistry using an antibody for involucrin and loricrin were

e, respectively. The broken line represents the epidermis/dermis border. Bars: 40 mm.

[(Fig._3)TD$FIG]

Fig. 3. The SC barrier is disturbed in the skin equivalent models treated with GST-C-CPE. (A) Fluorescent dye Lucifer yellow was applied to the SC and the penetration of the dye

was investigated in thin sections under a fluorescence microscope. (a) 50 mg/ml GST-treated skin. (b) 50 mg/ml GST-C-CPE-treated skin. The upper and lower broken lines

represent the SC/SG and epidermis/dermis borders, respectively. The experiment was repeated three times with essentially identical results. Bars: 100 mm. (B) The amount of

fluorescent dye leaked to the media that attached to the dermis was measured. The mean values are displayed in relation to 50 mg/ml GST-treated skin. Result shown as

mean � SD. Data pooled from three independent experiments is shown. *P < 0.05.

T. Yuki et al. / Journal of Dermatological Science 69 (2013) 148–158 153

granular layer by permeating through the SC in the 50 mg/ml GST-C-CPE-treated skin equivalent models (Fig. 3A). Measurement ofLucifer yellow in the medium that passed through the skinequivalent model revealed that the penetration of Lucifer yellowhad increased in the 10 and 50 mg/ml GST-C-CPE-compared to the50 mg/ml GST-treated skin equivalent model (Fig. 3B).

3.4. Morphological and biochemical changes of SC in skin with

disrupted TJ barriers

After confirming no effects of GST-C-CPE on cell viability, theexpression of proliferation and differentiation markers and themorphological and biochemical changes of the SC were examined.Hematoxylin and eosin (H&E) staining showed that the SC in the skinwith a disrupted TJ barrier was thicker than the SC in the control skin(Fig. 4A). No difference in the number of layers of SC were observedin electron microscopy (data not shown), but the thickness of theindividual corneocytes appeared thicker in the SC of the skin with adisrupted TJ barrier than in the SC of the normal skin (Fig. 4A).

In human skin, the surface of the SC is kept acidic due to anabundance of proton donors. Further, there is much evidence thatthe acidification of the SC is important for both permeabilitybarrier formation and cutaneous antimicrobial defense [37–40].Therefore, we examined whether SC acidification was impaired inthe skin with a disrupted TJ barrier. The skin surface pH, measuredby a flat surface electrode, significantly increased and neutralizedas the TJ barrier disrupted (Fig. 4B), demonstrating that SCacidification is impaired in the epidermis with a disrupted TJbarrier.

3.5. Lipid lamellae formation was impaired in skin with disrupted TJ

barrier

It is well known that lipid processing from polar lipid(precursor lipid) to non-polar lipid (matured lipid) requires two

acidic-dependent lipid hydrolases: b-glucocerebrosidase andacid sphingomyelinase, which are activated by extracellularacidity [41]. So we examined whether polar-to-non polar lipidprocessing was impaired. Polar lipid analysis of epidermalextracts showed that glucosylceramide, cholesterol sulfate andsphingomyelin increased as the TJ barrier disrupted (Fig. 5A). Innon-polar lipid analyses, we found no obvious changes in theunbound lipids (fatty acid, cholesterol and ceramides), butcovalently bound lipids (v-hydroxy-fatty acid, v-hydroxy-ceramide) decreased as the TJ barrier was disrupted (Fig. 5A).Ultrastructural examination after ruthenium tetroxide (RuO4)post-fixation revealed a stacked and patterned lamellar structurein the SC of the control epidermis. On the other hand, in the SC ofthe skin with a disrupted TJ barrier, lamellar structure did notstack and pattern, but appeared disaggregated and disorganized(Fig. 5B). Our results show that a disrupted TJ barrier is sufficientto cause a lipid lamellae defect.

3.6. Aberrant profilaggrin processing in skin with disrupted TJ barrier

Urocanic acid, a catabolite of filaggrin, is essential for SCacidification [42]. Matsui et al. reported that the optimum pH ofthe SASPase, one of the processing enzymes for filaggrin, is acidiccondition [43]. There have been several reports on the relationshipbetween SC pH and filaggrin processing, which led us to examinethe filaggrin processing in the skin equivalent models withdisrupted TJ barriers. Immunoblotting of epidermal extractsshowed that the levels of profilaggrin were lower in the epidermiswith a disrupted TJ barrier, but dimeric and trimeric filaggrin werehigher (Fig. 6A). It was more noteworthy that the level ofmonomeric filaggrin was significantly lower in the epidermiswith a disrupted TJ barrier (Fig. 6A). The immunofluorescencesignal intensity for filaggrin in the living cell layers of theepidermis with a disrupted TJ barrier was similar to that ofthe control epidermis. However, focusing on the SC, we discovered

[(Fig._4)TD$FIG]

Fig. 4. Morphological and biochemical changes in the SC in skin treated with GST-C-CPE. (A) Light and electron microscopy of the SC. The experiment was repeated three times

with essentially identical results. Bars of light microscopy: 40 mm. Bars of electron microscopy: 5 mm. (B) Skin surface pH was measured using a flat surface electrode. SC pH

increased significantly and neutralized in skin treated with GST-C-CPE. Result shown as mean � SD. One representative experiment of three is shown. **P < 0.01, ***P < 0.001.

T. Yuki et al. / Journal of Dermatological Science 69 (2013) 148–158154

[(Fig._5)TD$FIG]

Fig. 5. Abnormal epidermal lipid quantification and organization in skin treated with GST-C-CPE. (A) HPTLC was performed on polar lipids (precursor lipids) and non-polar

lipids (mature lipids). Non-polar lipids were divided into unbound lipids and covalently bound lipids. (a) Polar lipids. (b) Unbound lipids of non-polar lipids. (c) Bound lipids of

non-polar lipids. GS: glucosylceramide; CS: cholesterol sulfate; SM: shingomyelin; FA: fatty acid; Chol: cholesterol; CER: ceramide; v-(OH)-FA: v-(hydroxyl)-fatty acid; v-

(OH)-CER: v-hydroxy-ceramide. The mean values were displayed in relation to 50 mg/ml GST treated skin. Result shown as mean � SD. Data pooled from three independent

experiments is shown. *P < 0.05, **P < 0.01. (B) Typical electron micrographs of the SC. (a) Extracellular lemellae (arrowhead) were observed in the GST treated skin. (b) Focal

nonlamellar domains with disrupted extracellular lamellae (arrow) were observed in the GST-C-CPE treated skin. Bars: 100 nm.

T. Yuki et al. / Journal of Dermatological Science 69 (2013) 148–158 155

that the epitopes recognized by the filaggrin pAb remained in theepidermis with a disrupted TJ barrier, but not in the controlepidermis (Fig. 6B). Using electron microscopy, we observedkeratohyalin granules in the SGs of both the epidermis with anormal and that with a disrupted TJ barrier. In the upper layers of thecontrol SC, keratohyalin granules disappeared. However, in theupper layers of the SC with a disrupted TJ barrier, relatively highlevels of keratohyalin granules were present, providing furtherevidence of defective profilaggrin processing (Fig. 6C). These resultsindicate that a disrupted TJ barrier results in the accumulation ofpremature processed dimeric and trimeric filaggrin.

4. Discussion

Functional TJs that prevent the diffusion of paracellular tracers(M.W. �600 Da) were formed just beneath the SC in both humanand mouse epidermises [4,9,11]. Correspondingly, in human skinequivalents derived from human keratinocytes, functional TJs are

formed just beneath the SC [9]. Utilizing a human skin equivalent,we previously clarified that extraneous stimuli such as UVBirradiation or microbe derivative invasion induced changes in theTJ barrier [9,29], suggesting that human skin equivalents are usefulfor investigating the role of TJs in the epidermis. In this study, wefirst established a skin equivalent model with a disrupted TJ barrierusing GST-C-CPE. GST-C-CPE was reported to bind to at leastclaudin-4 and -6 [25]. In fact, we confirmed that GST-C-CPE bindsto claudin-4, accompanied by a disrupting of the TJ barrier (Fig. 1;Supplementary Fig. S1). The sensitivity assessment for CPE of Ltransfectants expressing claudin-5, -6, -7, -8, -10 or -14 revealedthat claudin-6, -7, -8 and -14 had a binding affinity to CPE [44].Claudin-1, -4, 7, 8, 11, 12 and 17 mRNA were expressed in theepidermis [45], suggesting that GST-C-CPE disrupted the TJ barrierby binding to claudin-4 and several other claudins (e.g. claudin-7and -8). Detailed examination of the established skin equivalentmodels revealed a failure of polar lipid (precursor lipid) processingand profilaggrin (precursor filaggrin) processing, accompanied by a

[(Fig._6)TD$FIG]

Fig. 6. Epidermis treated with GST-C-CPE lacks proteolytically processed filaggrin monomer. (A) A western blot analysis of filaggrin was performed. GST-treated skin

equivalent models showed filaggrin polymer (proFlg), trimer (3 � Flg), dimer (2 � Flg) and monomer (1 � Flg). Filaggrin polymer and monomer decreased, but filaggrin

trimer and dimer increased by GST-C-CPE treatment. One representative of three independent experiments is shown. (B) Immunofluorescent staining of filaggrin (red) was

performed. The experiment was repeated three times with essentially identical results. Bars: 100 mm. (C) Electron micrographs of the SC in skin equivalent models treated

with GST or GST-C-CPE. One representative of three independent experiments is shown.

T. Yuki et al. / Journal of Dermatological Science 69 (2013) 148–158156

deterioration of the SC barrier and pH, in the epidermis with thedisrupted TJ barrier (Figs. 3, 5 and 6). Metabolites of polar lipid andprofilaggrin are indispensable for the SC, which is responsible forthe cutaneous barrier [46,47]. Therefore, our study indicates thatTJs can control SC formation through polar lipid and profilaggrinprocessing.

It is widely accepted that the SC is dysfunctional as a result ofone or more of the following defects in patients with atopicdermatitis as compared with normal controls: (1) significantlyelevated SC pH [48,49]; (2) reduced levels of SC lipids [50,51]; and(3) acquired or genetic defects in filaggrin [17,19]. Recently it hasbeen reported that the TJ barrier may be weakened by changes inthe localization and expression levels of TJ components [21]. Going

by our data, we suggest that an impairment of the TJs contributesto abnormal SC formation in atopic dermatitis.

The SC pH is reported to increase in ichthyosis valugaris andatopic dermatitis [18,52]. Several proton donors have beenindentified as potential contributors to SC acidity [37,39,42]. Inaddition to these earlier studies, our study shows that TJs alsocontribute to SC acidity, because a disrupted TJ barrier results inan increased and neutralized SC pH (Fig. 4B). Why then are TJsassociated with SC acidity? Krien PM et al. suggest that urocanicacid, a product of filaggrin proteolysis, may play a key role inmaintenance of the acidic pH of the SC [42]. So the possibleexplanation is that impaired processing of filaggrin decreasedurocanic acid of the SC, which neutrized the SC pH. Another

T. Yuki et al. / Journal of Dermatological Science 69 (2013) 148–158 157

possible explanation is that TJs might stop the flow ofintercellular fluid possessing a buffering capacity from thegranular layer to the SC, which can maintain SC acidity. Furtherstudy is needed to clarify the detailed mechanism of SC acidityalteration by impaired TJs.

In the skin with a disrupted TJ barrier, lipid abnormalitieswere observed (Fig. 5A and B). The precursors of lipids that formthe extracellular lamellar membranes are secreted from theouter epidermal keratinocytes [53]. Following secretion, theselamellar-body-derived lipid precursors are further metabolizedin the SC extracellular spaces by enzymes including lipidhydrolases such as b-glucocerebrosidase, acidic sphingomyeli-nase, secretory phospholipase A2 and acidic/neutral lipase. Inthis metabolic process, the acidification of the SC is important[54]. We observed that the amount lipid precursors was higherin skin with disrupted TJ barrier (Fig. 5A), suggesting that anincrease and neutralization of SC pH could lead to themisprocessing of lipid precursors.

Our data demonstrated the relation of the TJ barrier toprocessing profilaggrin. In the skin with a disrupted TJ barrier,a decrease in filaggrin monomer was observed (Fig. 6A). Moreover,filaggrin trimer and dimer accumulated while profilaggrindecreased, which suggests that the processing of profilaggrin totrimer and dimer is promoted, while the processing of trimer anddimer to monomer is inhibited. The same anomalies of profilag-grin processing have been described in several murine models(SASPase �/�, CAP1/Prss8 �/�, ELA2 Tg) [43,55,56]. Sugawara etal. recently demonstrated that misprocessing of profilaggrin wasalso observed in claudin-1 knockout mice, an animal model of TJdysfunction, corresponding with our data (personal communica-tion). Why then are TJs associated with profilaggrin processing?Considering the evidence against the altered pH condition of theSC (Fig. 4B), we speculated that the altered ion environment of theSC (calcium, magnesium, sodium, potassium and proton etc.)affects enzymatic activity for profilaggrin processing.

Our study proposes that impaired TJs obstruct the developmentof the cutaneous barrier at least through polar lipid andprofilaggrin misprocessing, though detailed mechanisms remainunclear. In the near future, the clarification of the relationshipbetween the TJ barrier and SC ion environment could deepen ourunderstanding of how the TJ barrier controls SC formation.

Funding

No funding from public and private sources.

Acknowledgements

The authors wish to thank Dr. Y. Horiguchi (Osaka University,Osaka, Japan) for his generous gift of C-CPE cDNA. We also thankDr. M. Furuse (Kobe University, Kobe, Japan) for his helpfulinsights.

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