Journal of Dermatological Science 59 (2010) 107–114

Perturbation of lamellar granule secretion by sodium caprate implicatesepidermal tight junctions in lamellar granule function

Shohei Kuroda a, Masumi Kurasawa a, Koji Mizukoshi a, Tetsuo Maeda a, Takuya Yamamoto a,Ai Oba a, Mari Kishibe b, Akemi Ishida-Yamamoto b,*a Pola Chemical Industries, Inc., 560 Kashio-cho, Totsuka-ku, Yokohama 244-0812, Japanb Department of Dermatology, Asahikawa Medical College, 2-1-1-1 Midorigaoka Higashi, Asahikawa 078-8510, Japan

A R T I C L E I N F O

Article history:

Received 13 November 2009

Received in revised form 5 June 2010

Accepted 9 June 2010

Keywords:

Lamellar granule

Tight junction

LEKTI

Keratinocyte

BODIPY-FL

A B S T R A C T

Background: Polarized secretion of lamellar granules (LGs) delivers various lipids, proteases, and

protease inhibitors into the stratum corneum (SC) of the epithelium. Disruption of LGs is associated with

severe cutaneous diseases, but the mechanism of their polarized secretion is not known. On the other

hand, recent study shows epidermal tight junctions (TJs) localize in stratum granulosum (SG), and TJs are

involved in polarized molecule secretion. Thus, we hypothesized epidermal TJs relate to polarized LGs

secretion.

Objective: To assess the possibility that epidermal TJs are involved in polarized LGs secretion.

Methods: In order to examine LGs secretion, we used fluorescent ceramide (BODIPY1 FL C5-ceramide)

and a natural LG cargo, lympho-epithelial Kazal-type-related inhibitor (LEKTI), in cultured normal

human epidermal keratinocytes and a reconstructed human epidermis. We investigated their alteration

using the medium-chain fatty acid sodium caprate (C10), TJs inhibitor. In addition, LG distribution was

observed by electron microscopy.

Results: C10 significantly inhibited secretion of both fluorescent ceramide and LEKTI in cultured normal

human epidermal keratinocytes and a reconstructed human epidermis. C10 also disturbed the polarized

localization of fluorescent ceramide and LEKTI in the reconstructed epidermis. Electron microscopy

revealed that a large number of LGs remained in corneocytes in the C10-treated epidermis, rather than

being secreted.

Conclusion: Our data indicate that C10 perturbs the polarized secretion of LGs. Our study therefore

suggests that epidermal TJs are possibly involved in polarized LG secretion and provides new insights

into potential of treatments for skin diseases caused by abnormal LG secretion.

� 2010 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights

reserved.

Contents lists available at ScienceDirect

Journal of Dermatological Science

journa l homepage: www.e lsev ier .com/ jds

1. Introduction

Epidermal LGs, also known as keratinosomes, lamellar bodies,membrane-coating granules, or Odland bodies, are specializedorganelles of the keratinizing stratified squamous epithelia thattransport various molecules, including lipids, proteases, proteaseinhibitors, and structural proteins [1–4] and are thought to beessential for barrier formation and desquamation [5,6]. Recentstudies show that several severe cutaneous disorders, includingHarlequin ichthyosis [7,8], lamellar ichthyosis type 2 [9], andNetherton syndrome [10] are caused by LG-related abnormalities.Therefore, it is important to understand the mechanisms of LG

* Corresponding author. Tel.: +81 166 68 2523; fax: +81 166 68 2529.

E-mail address: akemi@asahikawa-med.ac.jp (A. Ishida-Yamamoto).

0923-1811/$36.00 � 2010 Japanese Society for Investigative Dermatology. Published b

doi:10.1016/j.jdermsci.2010.06.001

physiology. Recent studies showed that there are specificphenotypes caused by abnormalities in a specific cargo; forexample, KLK7-overexpression induces severe inflammation [11]and cathepsin D-deficiency causes abnormal SC [12]. On the otherhand, abnormal LG secretion found in diseases such as cerebraldysgenesis, neuropathy, ichthyosis, and keratoderma (CEDNIK)syndrome results in different phenotypes. Thus, specific patholog-ical mechanisms may exist in diseases caused by abnormalities inLG-transport/secretion.

Only a small number of investigations have focused on themechanisms by which LGs transport and secrete cargo. Elias et al.[1] characterized the most superficial granular keratinocytes assecretory granular cells. They suggested that LGs are transportedvia the trans-Golgi network in the stratum spinosum (SS) andstratum granulosum (SG) and are only secreted toward the stratumcorneum (SC) from the topmost SG cells. Rassner et al. [13] also

y Elsevier Ireland Ltd. All rights reserved.

S. Kuroda et al. / Journal of Dermatological Science 59 (2010) 107–114108

demonstrated that nascent organelles of LGs originate in a trans-Golgi-like reticulum. We have demonstrated that various LGcontents, including glycosylceramides and LEKTI, are distributed asseparate aggregates and are delivered to the apical regions of SGkeratinocytes [3,14]. Sprecher et al. suggested that soluble N-ethylmaleimide-sensitive factor attachment protein receptor(SNARE) proteins are involved in the maturation and secretionof LGs [15]. They demonstrated the presence of countless clearvesicles in the spinous and granular cells of the skin of patientswith cerebral dysgenesis, neuropathy, ichthyosis, and keratoderma(CEDNIK) syndrome; CEDNIK syndrome is caused by a mutation inthe SNAP-29 gene, which encodes a SNARE protein involved invesicular transport. Sprecher et al. [15] also found that numerousvesicles containing glycosylceramides were retained abnormallywithin corneocytes, indicating abnormal LG secretion, andsuggested that the ichthyosis in CEDNIK syndrome is caused byabnormal LG maturation and secretion. However, the simplequestion of why LGs are transported toward the apical membraneand are secreted from there remains unanswered.

We hypothesized that epidermal tight junctions (TJs) areinvolved in the polarized secretion of LGs. TJs have been implicatedin two major cell functions: the barrier function and cellpolarization, including polarized transport and secretion ofmolecules. Epidermal TJs form in the SG of human and mouseepidermis and inhibit the permeation of biotinylated markers intothe mouse epidermis [16,17]. The crucial role of TJs in forming anepidermal barrier has been demonstrated in mice deficient inclaudin-1 (a TJ-related protein), which die of transepidermaldehydration [17] and a rare human disease associating withichthyosis and neonatal sclerosing cholangitis (NISH syndrome)[18]. However, the effects of claudin-1 disruption on cell polaritywere not addressed in these studies. Here, we investigated therelationship between epidermal TJs and polarized LG secretion, acell polarity-related event. We found that the medium-chain fattyacid sodium caprate (C10), which disrupts TJs in culturedkeratinocytes [19], disrupted the intracellular distribution of LGmolecules and inhibited their secretion.

2. Materials and methods

2.1. Cells and cell culture

Normal human epidermal keratinocyte cells (HEKs) wereobtained from Kurabo (Osaka, Japan) and maintained in HuMe-dia-KG2 culture medium (Kurabo) supplemented with 5 mg/mlinsulin, 0.5 mM hydrocortisone, 200 pg/ml human recombinantepidermal growth factor, and 0.2% bovine pituitary extract. Toinduce differentiation, HEKs were first cultured in completeHuMedia-KG2 culture medium containing 0.15 mM CaCl2 forseveral days and then switched into HuMedia-KG2 culturemedium supplemented with 1.45 mM CaCl2, as described previ-ously [20]. A cultured reconstructed human epidermis derivedentirely from human foreskin keratinocytes, EpiDermTM (EPI-200,MatTek, MA, USA) was pre-incubated in assay medium EPI-100(MatTek) for 5 h before use. This reconstructed human epidermisexpresses abundant LGs with lamellar structures [21].

2.2. Disruption of TJs

C10 (molecular weight 194.25) was obtained from Sigma–Aldrich Corp., MO, USA; prepared as a 1 M aqueous stock solution;and stored at 4 8C. Before each experiment, the stock solution wasdiluted 1:1000 in Humedia-KG2 containing 1.45 mM CaCl2 toprepare 1 mM C10 in medium for application to the cells. Purifiedwater was used as a vehicle control in each experiment. We havepreviously reported that C10 inhibits the barrier function and

conformation of TJs, and these functions recover after the C10 iswashed out [19]. C10 was applied to the cells for the time periodsindicated in each experiment.

2.3. Uptake of ceramide

BSA-conjugated N-[5-(5,7-dimethyl BODIPY)-1-pentanoyl]-D-erythro-sphingosine (DMB-Cer; BODIPY1 FL C5-ceramide, Molec-ular Probes, Eugene, OR) is similar to the NBD-labeled ceramideused in previous investigations on epidermal keratinocytes [1,22],but is more photostable. DMB-Cer was dissolved in purified waterto create a 0.5 mM stock solution. Cultured HEKs were labeledwith 7.5 mM DMB-Cer in Humedia-KG2 containing 1.45 mM Ca2+

at 4 8C for 2 h in the dark. After the labeling period, the mediumcontaining DMB-Cer was removed. The cells were rinsed 3 timeswith fresh medium and then incubated for 30 min to acclimatizethem. The EpiDermTM tissues were incubated in assay mediumwith 5 mM DMB-Cer at 37 8C for 4 h, and then labeled at 4 8C for1 h. After further incubation for 20 h in order to let DMB-Cerpermeate throughout the epidermis, the EpiDermTM was relabeledat 4 8C for 30 min. In both HEKs and EpiDermTM labeled with DMB-Cer, the medium was exchanged with fresh medium containing 5%defatted BSA (essentially fatty acid-free BSA, Sigma) to removeexcess DMB-Cer attached to the surface of the plasma membrane[23], and the tissue was incubated for a further 10 min. Themedium containing defatted BSA was exchanged 3 times.

2.4. Quantitative assessment of DMB-Cer secretion

To measure secretion from HEKs, the cells were incubated forseveral hours, and then the medium was collected for quantifica-tion of secreted DMB-Cer. To measure secretion from EpiDermTM,cultured epithelia labeled with DMB-Cer were carefully peeled offfrom the supporting membrane and incubated in the medium forseveral hours. The medium was collected after the indicatedperiods of time. For quantitative analysis of secreted DMB-Cer ineach experiment, cultures were split into two groups (n = 5 in eachgroup). The first group of the cultures was labeled with DMB-Cer,whereas the second group was left unlabeled as the reference forthe fluorescence measurement. Fluorescence in each conditionedmedium was measured with an ARVO SX multilabel counter(PerkinElmer, Waltham, MA, USA), and the amount of secretedDMB-Cer was quantified by subtracting the mean value of theunlabeled group from that of DMB-Cer labeled group.

2.5. Histochemical analysis of DMB-Cer distribution

HEKs were cultured on glass-bottom dishes (Iwaki, Japan) andlabeled with DMB-Cer as described above. The most importantspectroscopic property of the DMB fluorophore is an aggregation-dependent shift from green to red fluorescence emission due tointermolecular excimer formation. Consequently, structures thataccumulate large amounts of DMB-labeled sphingolipids, such asthe Golgi complex [24] or the lysosomes of fibroblasts frompatients with sphingolipid storage disease [25,26] exhibit redfluorescent staining that is clearly distinct from low-abundancegreen fluorescent staining. In the present study, living cells labeledwith DMB-Cer were excited at 488 nm with an argon laser, and thefluorescence was observed at either 505–530 or >561.5 nm usingan LSM510-META confocal microscope (Carl Zeiss, Overkochen,Germany). The images were analyzed using ZEN ver1.0 software(Carl Zeiss).

EpiDermTM tissues labeled with DMB-Cer were frozen inoptimal cutting temperature compound (Sakura Finetek, Tokyo,Japan). Frozen sections, 20 mm thick, were collected on glass slides,immediately mounted with Fluoromount-G (SouthernBiotech,

Fig. 1. C10 treatment decreases DMB-Cer secretion in HEKs and EpiDermTM tissue.

(a) Differentiated HEKs labeled with DMB-Cer were treated with 1 mM C10 for 8 h

(‘treatment’ group), or treated with 1 mM C10 for 4 h and then incubated for

another 4 h without C10 (‘removal’ group). (b) EpiDermTM tissues were treated with

1 mM C10 for 18 h (‘treatment’ group) or for 12 h and then incubated for 6 h

without C10 (‘removal’ group). Data represent mean � SD of five independent

experiments. Dunnett’s multiple comparison test was performed at the endpoint of the

experiments. *P < 0.05.

S. Kuroda et al. / Journal of Dermatological Science 59 (2010) 107–114 109

Birmingham, AL, USA), and then examined immediately. Lengthyobservation and mounting water soluble materials on the sampleswere carefully avoided in order to prevent leakage of DMB-Cerfrom the tissue.

2.6. Western blotting

A previously described protocol [27] was used with somemodification. HEKs were incubated with 1.45 mM CaCl2 for variousperiods of time as indicated in each experiment. At the end of eachtreatment period, cells were lysed in an appropriate volume of ice-cold lysis buffer (50 mM Tris–HCl, pH 8.0, 150 mM NaCl, 5 mMEDTA, 1% NP-40, 1 mM PMSF, and 1 mg/ml each of antipain,chymostatin, leupeptin, and pepstatin). Cell extracts were soni-cated on ice and clarified from the insoluble material bycentrifugation at 15,000 � g, at 4 8C for 5 min. Conditionedmedium was collected at each time period and concentrated byovernight precipitation at�20 8C in the presence of 4 ml of acetoneper milliliter of medium. Proteins were recovered by centrifuga-tion at 4 8C for several minutes, resuspended in a suitable volumeof ice-cold lysis buffer, and sonicated. Protein samples werequantified using a Protein Assay DC Kit (Bio-Rad, Hercules, CA,USA), and equal amounts of protein extracts (20 mg/lane) for eachsample were resolved with 12.5% SDS-polyacrylamide gel electro-phoresis. Following transblotting onto Immobilon-P membranes(Millipore, Bedford, MA, USA) and blocking in Block Ace (DSPharma Biomedical, Osaka, Japan), the membranes were incubatedwith 10 mg/ml mouse anti-human LEKTI monoclonal antibody(clone 1C11G6, Zymed, San Francisco, USA [28]) or 2.5 mg/mlrabbit anti-human actin polyclonal antibody (Abcam, Cambridge,UK) as an internal control. The membranes were then incubatedwith horseradish peroxidase-conjugated goat anti-mouse IgG orgoat anti-rabbit IgG (GE Healthcare Bio-Sciences Corp., Piscataway,NJ, USA). Immunoreactive bands were detected using ImmobilonWestern substrate (Millipore, Bedford, MA, USA) and exposure toX-ray film (Fuji-Film, Tokyo, Japan).

2.7. Immunofluorescence microscopy

The immunofluorescence microscopy method has been de-scribed elsewhere [20]. Briefly, HEKs grown on glass coverslips or 6-mm thick vertical sections of EpiDermTM were rinsed in PBS, fixedwith 2% paraformaldehyde at 4 8C for 1 h, and then rinsed in PBScontaining 100 mM glycine (PBS-G) at 4 8C for 1 h. After anadditional rinse in PBS-G containing 0.05% Triton X-100 at roomtemperature for 5 min, the samples were blocked with PBS-Gcontaining 10% Block Ace as blocking buffer. The samples were thenincubated with mouse anti-human LEKTI antibody diluted to 5 mg/ml in the blocking buffer at 4 8C overnight. The samples were thenwashed several times in PBS-G and incubated with Cy3-conjugatedgoat anti-mouse IgG antibody (GE Healthcare, Tokyo, Japan) dilutedto 2 mg/ml in the blocking buffer, at 4 8C for 1 h. The samples wereobserved with an 63� oil-immersion objective and photographedwith an LSM510-META confocal microscope (Carl Zeiss). The imageswere analyzed using ZEN ver1.0 software (Carl Zeiss).

2.8. Ultrastructural observation

A conventional transmission electron microscopy method wasused to observe LGs. Briefly, skin samples were fixed with half-strength Karnovsky’s fixative, postfixed with either 1% osmiumtetroxide or 0.2% buffered ruthenium tetroxide (Polysciences, Inc.,Warrington, PA), dehydrated in a graded series of ethanol, andembedded in an Epon epoxy resin mixture [29]. Ultrathin sectionsstained with uranyl acetate and lead citrate were examined with aJEM-1210 electron microscope (JEOL Ltd., Tokyo, Japan).

2.9. Statistics

In Fig. 1, significance was tested using JMP ver 6.0 (SAS, Cary,NC, USA) by Dunnett’s multiple comparison test at the endpoint ofthe experiments. Values of P � 0.05 were considered statisticallysignificant.

3. Results

3.1. C10 treatment perturbs DMB-Cer secretion and distribution

We used C10 to study the relationship between TJs andpolarized LG secretion because it is a well-known absorptionenhancer that induces dilatation of TJs in monolayers of theintestine-derived cell line Caco-2 [30]. In addition, we have foundthat C10 inhibits the barrier function of epidermal TJs by disturbingtheir structure in cultured keratinocytes [19]. We first quantifiedsecreted DMB-Cer by using a fluorometer and then observed DMB-Cer distribution by confocal microscopy. In both HEKs (Fig. 1a) andEpiDermTM (Fig. 1b), DMB-Cer secretion was dramatically de-creased by C10 treatment, but recovered after C10 removal. Incontrol HEKs, DMB-Cer was distributed throughout the cytoplasmand was at particularly high-density around the nucleus (Fig. 2a),suggesting that it associates with the Golgi apparatus. Asincubation progressed, the DMB-Cer signal gradually disappeared(Fig. 2b and c). C10 treatment prevented this process. The high-density DMB-Cer signal was present at the same intensity after 4 htreatment with C10 as before the treatment (Fig. 2d). Removal ofC10 resulted in the disappearance of the high-density DMB-Cer.Surface graphics of fluorescence, calculated and drawn with ZENsoftware (Fig. 2, lower panels) also supported this finding. In the[(Fig._1)TD$FIG]

[(Fig._2)TD$FIG]

Fig. 2. Highly concentrated DMB-Cer distribution in HEKs still remains after C10 treatment. Differentiated HEKs were cultured in a glass-bottom dish and labeled with DMB-

Cer, then treated with 1 mM C10 and visualized with confocal microscopy. The color of the signal changes from green to red depending on the density of DMB-Cer. Green color

indicates lower density and red color indicates higher density. Upper photographs: Merged images of high (red, excitation >561.5 nm) and low (green, excitation 530–

505 nm) densities of DMB-Cer and Hoechst 33342 (blue, nuclear counterstaining). Bottom figures: Whole Z-stuck images of DMB-Cer from bottom to top cell layers

constructed by ZEN software. (a–c) Cells without C10 treatment at 0 (a), 4 (b), and 8 h (c) in culture medium. (d) Cells treated with C10 for 4 h. (e) Cells incubated in culture

medium without C10 for 4 h after treatment with C10 for 4 h. Scale bar = 20 mm.

S. Kuroda et al. / Journal of Dermatological Science 59 (2010) 107–114110

control EpiDermTM tissues (Fig. 3), the DMB-Cer signal was highlyconcentrated on the apical side of the SG, whereas it was morebroadly distributed in the C10-treated EpiDermTM. The DMB-Cerconcentration reverted to the apical side of the SG after C10removal (arrows in Fig. 3c).

3.2. C10 treatment decreases LEKTI secretion without altering

intracellular pro-LEKTI expression

HEKs and EpiDermTM were incubated with C10 for severalhours and then incubated without C10 (see legend in Fig. 4), andpro-LEKTI and LEKTI fragments were detected with Westernblotting (Fig. 4). Total cell extracts of both HEKs and EpiDermTM

produced two bands in the higher-molecular-weight rangebetween 100 and 150 kDa, reported previously to be pro-LEKTI[31]. By contrast, 3 bands reported to be LEKTI fragments [32] weredetected in the lower-molecular-weight range between 10 and

25 kDa in the blot of conditioned medium. b-Actin, an internalcontrol, was expressed at the same level in each experiment. C10treatment did not alter the protein expression of intracellular pro-LEKTI in total cell extracts at any time point in either HEKs orEpiDermTM. By contrast, the secreted LEKTI fragments in condi-tioned medium, especially the 25-kDa band and to a lesser extentthe 10- and 15-kDa bands, were considerably decreased in HEKs.All low-molecular-weight bands were decreased in EpiDermTM.C10 removal resulted in recovery of the secreted LEKTI fragmentbands (Fig. 4a, C10R in the 4h-8 h lanes and Fig. 4b, C10R in the 12–18 h lanes).

Next, we assessed the expression of LEKTI, which includes bothpro-LEKTI and fragmented LEKTI, in EpiDermTM using histochem-istry. LEKTI was distributed from the lower SS to the topmost SG inall groups, regardless of C10 treatment. In control tissue, LEKTI waspolarized at the apical side in the SS and even more so in the SG.However, the polarized localization disappeared after C10

[(Fig._3)TD$FIG]

Fig. 3. C10 treatment disturbs DMB-Cer distribution in EpiDermTM. EpiDermTM labeled with DMB-Cer were incubated with 1 mM C10 for 12 h and then further incubated

without C10 for 6 h. A cross-section of each sample was observed with confocal microscopy. Upper row shows merged images of high (red) and low (green) density DMB-Cer

localization. Bottom row shows magnified images of the boxed areas in the upper row. (a) Control and (b) C10-treated EpiDermTM after C10 treatment for 12 h. (c) EpiDermTM

incubated for 6 h without C10 after 12 h of C10 treatment. Arrows: DMB-Cer localization in the apical side of the cells. N, nucleus. Scale bar = 20 mm.

[(Fig._4)TD$FIG]

Fig. 4. C10 treatment inhibits LEKTI fragment secretion without change of pro-LEKTI expression in HEKs and EpiDermTM. Differentiated HEKs (a) were incubated with 1 mM

C10 for 4 h, and then without C10 for 4 h. EpiDermTM (b) was incubated with 1 mM C10 for 12 h, and then without C10 for 6 h. Conditioned medium and total cell extracts

were collected at each time period, and Western blotting was performed with a mouse anti-human LEKTI antibody and donkey anti-mouse IgG antibody. A rabbit anti-b-actin

antibody was used as an internal control. Upper figures show immunoblotting of total cell extracts and lower figures show that of conditioned medium. Control, cells treated

with vehicle; C10, C10-treated cells; C10R, cells incubated in culture medium without C10 after C10 treatment for the time period indicated.

S. Kuroda et al. / Journal of Dermatological Science 59 (2010) 107–114 111

treatment, and LEKTI was distributed throughout the intracellularspace (arrow in bottom picture in Fig. 5). Cytoplasmic LEKTIstaining was even detected in some corneocytes (arrow in middlepicture in Fig. 5). The apical bias of LEKTI staining was recoveredafter C10 removal (arrowheads in Fig. 5c).

3.3. Corneocytes contain numerous LGs after C10 treatment

LGs are 0.1–0.3 mm ovoid granules containing a lamellarstructure [33]. In Karnovsky’s fixative- and osmium-fixed speci-mens, we observed only a few vesicles of this size (LG-likegranules) in control corneocytes (Fig. 6a), but numerous LG-likegranules appeared in C10-treated corneocytes (Fig. 6b). Rutheniumfixation [21] of the same samples revealed that these granules hada lamellar structure (Fig. 6c), confirming that they are indeed LGs.In SG cells, the difference in LG distribution between C10-treatedcells and controls was not apparent in electron microscopicobservations.

4. Discussion

In the present study, we demonstrated that TJs contribute to thepolarized secretion of LGs. The mechanisms and physiological

regulators of polarized LG secretion had been a largely unexploredtopic. Similarly, while the epidermal barrier function of TJs is wellrecognized, their roles in LG secretion had not been addressedbefore. To investigate that relationship, we used two LG markers:the fluorescent ceramide DMB-Cer and LEKTI, a natural cargo ofLGs [14]. We confirmed that DMB-Cer colocalized with LEKTIbeforehand (data not shown). Ceramide synthesized de novo ismetabolized to glycosylceramide and loaded into LGs [3,34,35].We assumed that DMB-Cer, which can be glucosylated inkeratinocytes [36], should behave in the same way as naturalceramide and associate with LGs. Here we showed that C10, whichdisrupts the barrier function and structure of TJs, significantlyperturbed polarized secretion and distribution of DMB-Cer andLEKTI in cultured HEKs and also in EpiDermTM. Furthermore,numerous LGs were observed in SC cells of EpidermTM by C10treatment. Although the distributions of LG in SG cells in electronmicroscopy observation did not clearly change by C10 treatment,there was clear difference in its distributions in fluorescentmicroscopy observation. Moreover, the amount of (putatively notsecreted) LGs was obviously increased. Therefore, it suggests thatC10 perturbed LG distributions in not only SC cells but also SG cells.Thus, these data suggests that perturbation of TJs by C10 inducedabnormal LGs secretion.

[(Fig._5)TD$FIG]

Fig. 5. C10 treatment disturbs LEKTI distribution in EpiDermTM. EpiDermTM was treated with 1 mM C10 for 12 h and then incubated without C10 for 6 h. Immunofluorescence

microscopy was performed with a mouse anti-human LEKTI antibody and Cy3-conjugated goat anti-mouse IgG antibody. (a) Control tissue, (b) tissue treated with C10 for

12 h and (c) tissue incubated for 6 h without C10 after C10 treatment for 12 h. Nuclei were stained with DAPI. Red: LEKTI; blue: nucleus. Scale bar = 20 mm. Dotted line

represents the interface between the SC and SG. Arrowheads show LEKTI localization at the apical side of SG cells. Arrows show LEKTI remaining in a corneocyte (middle

picture) and in the intracellular space in the granular cells (bottom picture). SC, stratum corneum; SG, stratum granulosum; SS, stratum spinosum; SB, stratum basale.[(Fig._6)TD$FIG]

Fig. 6. C10 treatment disturbs on LG distribution in SC and SG layer in EpiDermTM. EpiDermTM was incubated with 1 mM C10 for 12 h, and then observed by an electron

microscope. (a) Control, (b) and (c) C10-treated tissue. Panels (a) and (b) were taken from specimens fixed with osmium and show the interface between SC and SG. Panel (c)

shows lamellar structure in LG-like granules revealed by ruthenium fixation in both SC and SG cells. Each scale bar indicates 200 nm.

S. Kuroda et al. / Journal of Dermatological Science 59 (2010) 107–114112

S. Kuroda et al. / Journal of Dermatological Science 59 (2010) 107–114 113

Whether TJs actually regulate cell polarity is somewhatcontroversial. The relationship between TJs and polarity was oncethought to be very simple: cellular proteins delivered to a givenplasma membrane domain retains their localization because TJsfunction as a fence preventing lateral diffusion and randomizationof membrane molecules [37,38]. However, the story has turned outto be not so simple, because some membrane molecules canachieve polarization and redistribution despite a lack of TJs [39].Those reports imply that the TJ-action on LG secretion would be acomplicated one. There are at least two possible mechanisms. Oneis involvement of TJ-related signal transduction for moleculartransport; the other is formation of plasma membrane polarity forexocytosis including LG secretion. Concerning the latter one,docking proteins anchored in the plasma membrane such as SNAREproteins are important for the fusion between vesicular andplasma membrane during exocytosis. TJs divide the plasmamembrane into apical and basolateral domains by enclosing thecell periphery in a belt-like fashion, which is considered to be ableto keep polarized localization of docking proteins. It would beinteresting to see C10-effects on these docking proteins in thefuture studies. Although further investigations are needed, thepresent study contributes to understanding the relation betweenTJs and polarity-related cellular events, including LG secretion. Ourresults may also help to understand the mechanisms of LGsecretion in lung alveolar type II cells, which are well-polarizedcells with TJs [40,41] that secrete LGs containing surfactant only tothe luminal surface.

Understanding the mechanisms of LG transportation andsecretion is critical for the development of effective means tocure LG-related skin abnormalities, e.g. unveiling mechanismsgreatly contributes to develop more improved cure for diseasesincluding skin barrier disruption. Improving or promoting LGssecretion expects to construct more cross-linked structurebetween ceramide and cornified envelopes and attributes efficientskin barrier function, which we believe very useful idea for skinwellness. For the treatment of skin diseases characterized bydefective LG secretion, downregulation of caveolin-1 which arrestsLG secretion might be an option [42].

In this study, we used C10 to disrupt TJs. The mechanism ofthese effects was stated to be via phospholipase C activation andupregulation of intracellular Ca2+, which would lead to calmodulindependent contraction of actin–myosin filaments attached to theintracellular domain of TJs [43]. Thus, there is a possibility that notTJs disruption but other factors in this mechanism affect polarizedmolecule transport in HEKs. Although more specific investigationssuch as knockdown of TJ components are needed to verify thefunctions of TJs on polarized LG secretion, our study strongly helpsimproving consideration for the mechanism of polarized LGsecretion.

Acknowledgements

We thank all the members of our laboratory for theirinformative help and advice.

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