Journal of Dermatological Science 54 (2009) 17–24

Dichotomous effect of ultraviolet B on the expression of corneodesmosomalenzymes in human epidermal keratinocytes

Megumi Nin a, Norito Katoh a,*, Satoshi Kokura b, Osamu Handa b,Toshikazu Yoshikawa b, Saburo Kishimoto a

a Department of Dermatology, Kyoto Prefectural University of Medicine, Graduate School of Medical Science, Kawaramachi-Hirokoji, Kamigyo-ku, Kyoto 602-8566, Japanb Department of Medicine, Kyoto Prefectural University of Medicine, Graduate School of Medical Science, Kyoto, Japan

A R T I C L E I N F O

Article history:

Received 9 July 2008

Received in revised form 5 November 2008

Accepted 13 November 2008

Keywords:

Stratum corneum

Corneodesmosome

LEKTI

KLK5

KLK7

UVB

A B S T R A C T

Background: The stratum corneum chymotryptic enzyme (SCCE) and the stratum corneum tryptic

enzyme (SCTE) are serine proteases of the kallikrein family that are encoded by the KLK7 and KLK5 genes,

respectively. They might play a role in desquamation by cleaving corneodesmosomal proteins. Their

activities are regulated by lympho-ephithelial Kazal-type related inhibitor (LEKTI), which is encoded by

the SPINK5 gene.

Objective: To elucidate the role of these molecules in ultraviolet B (UVB)-induced desquamation of the

stratum corneum, we examined the effect of UVB irradiation on the expression of these enzymes in

human epidermal keratinocytes.

Methods: The effects of UVB on SCCE (KLK7), SCTE (KLK5) and LEKTI (SPINK5) expression in keratinocytes

were estimated using HaCaT cells, normal human epidermal keratinocytes and a reconstructed human

epidermis model.

Results: UVB irradiation significantly reduced the expression of SPINK5 and increased the expression of

KLK5 and KLK7, as assessed by real-time PCR, and reduced the expression of LEKTI and increased the

expression of SCTE and SCCE by Western blotting and/or immunofluorescence analysis. Protease activity,

as assessed by in situ zymography, was enhanced in the epidermis following exposure to UVB irradiation

compared with sham-irradiated epidermis.

Conclusion: These results suggest that reduced expression of LEKTI and increased expression of SCCE and

SCTE in human epidermal keratinocytes after UVB irradiation may contribute to desquamation of the

stratum corneum.

� 2008 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. int l .e lsev ierhea l th .com/ journa ls / jods

1. Introduction

Ultraviolet radiation causes various harmful effects on the skin,such as sunburn, immunosuppression, carcinogenesis, photoaging[1], and disruption of the epidermal barrier [2]. In addition,exposure of the skin to ultraviolet B (UVB) irradiation inducesdesquamation of the stratum corneum [3]. However the mechan-ism underlying desquamation after UVB exposure remains unclear.

The structural integrity of the stratum corneum is maintained bythe presence of modified desmosomes, called corneodesmosomesthat lock the corneocytes together and provide tensile strength forthe stratum corneum to resist shear forces [4]. The corneodesmo-somes are considered to play a role as a barrier against the

* Corresponding author. Fax: +81 75 251 5586.

E-mail address: nkatoh@koto.kpu-m.ac.jp (N. Katoh).

0923-1811/$30.00 � 2008 Japanese Society for Investigative Dermatology. Published b

doi:10.1016/j.jdermsci.2008.11.004

penetration of irritants and allergens [4]. Three proteins have beendescribed as components of the extracellular face of corneodesmo-somes: the two desmosomal cadherins, desmoglein 1 and desmo-collin 1 [5], and corneodesmosin [6]. A number of different proteasesof the serine, cysteine, or aspartic protease families have beenidentified in the stratum corneum, and are considered to play a rolein desquamation by cleaving corneodesmosomal protein [7–9].Representative proteases include the stratum corneum chymotryp-tic enzyme (SCCE) and the stratum corneum tryptic enzyme (SCTE)[10–12], which are serine proteases of the kallikrein family encodedby KLK7 and KLK5 genes, respectively [13]. They are both highlyexpressed in granular keratinocytes and are present in theintercellular spaces of the stratum corneum [14,15]. SCCE has beenshown to hydrolyze corneodesmosin and desmocollin 1, and SCTE isalso capable of cleaving desmoglein 1. While both SCCE and SCTE areproduced as inactive precursors, SCTE is capable of activating SCCE[16], in addition to self-activation [17–19].

y Elsevier Ireland Ltd. All rights reserved.

Table 1Primers used for real-time quantitave polymerase chain reaction.

Human GAPDH

Forward 5-ACCACAGTCCATGCCATCACT-3

Reverse 5-CCATCACGCCACAGTTTCC-3

Human KLK5

Forward 5-TGAATTCTATAGTGCCTGGGTCTCA-3

Reverse 5-CCAAGTAGAGAGGAACCACAAGGA-3

Human KLK7

Forward 5-CATGCTCGTGAAGCTCAATAGC-3

Reverse 5-GGAGGGCAGCCTGACTTTCT-3

Human SPINK5

Forward 5-CAAACAAATACACACATCCGCAGTA-3

Reverse 5-TGGCTGCGGTGGTTCCT-3

M. Nin et al. / Journal of Dermatological Science 54 (2009) 17–2418

The activities of these proteases in desquamation are regulatedby several protease inhibitors that are coexpressed to balance therate of corneodesmosome cleavage. Lympho-ephithelial Kazal-type related inhibitor (LEKTI) is a representative serine proteaseinhibitor, which is encoded by the SPINK5 (serine proteaseinhibitor Kazal-type 5) gene [20]. LEKTI plays an important rolein regulating desquamation of the stratum corneum duringhomeostasis by inhibiting the activity of SCTE and SCCE in theepidermis.

In this study, we examined the effect of UVB irradiation on theexpression of LEKTI/SPINK5, SCTE/KLK5 and SCCE/KLK7 in normalhuman epidermal keratinocytes in order to elucidate the role ofthese molecules in UVB-induced desquamation of the stratumcorneum. We demonstrated a dichotomous effect of UVB irradia-tion on the expression of these corneodesmosomal enzymes.

2. Materials and methods

2.1. Cell culture

Spontaneously immortalized human keratinocytes, HaCaT cells[21], were grown in 250-cm2 cell culture flasks with Dulbecco’smodified essential medium (Gibco-BRL, Gaithersburg, MD, USA)supplemented with 10% fetal bovine serum and 100 units/mLpenicillin and 100 mg/mL streptomycin (Gibco-BRL) at 37 8C in ahumidified atmosphere containing 5% CO2. Normal humanepidermal keratinocytes (NHEK) from neonatal foreskin werepurchased from Cascade Biologics (Portland, OR, USA) andmaintained according to the manufacturer’s instructions. Briefly,NHEK grown to 80% confluence and subcultured at 2500 cells/cm2

in 250-cm2 cell culture flasks were maintained in EpiLife Mediumsupplemented with Coating Matrix Kit, EpiLife Defined GrowthSupplement (Cascade Biologics), 100 units/mL penicillin and100 mg/mL streptomycin (Gibco-BRL) at 37 8C in a humidifiedatmosphere containing 5% CO2. A reconstructed human epidermismodel from individual normal human keratinocytes was pur-chased from LabCyte EPI-MODEL (J-TEC, Aichi, Japan) and culturedaccording to the manufacturer’s instructions.

2.2. UVB irradiation

The UVB source was a bank of six fluorescent sunlamps (FL20S-E-30; Toshiba Medical Supply, Tokyo, Japan) with an emissionspectrum of 275–375 nm, mainly in the UVB range, peaking at305 nm and including a small amount of UVA and UVC (energy:UVA, 30%; UVB, 54%, UVC, 0.2%). The UVB irradiation was measuredusing a UV-radiometer (UVR-3036/S2; TOPCON Corporation,Tokyo, Japan). Prior to UVB irradiation, the cells were washedtwice with phosphate-buffered saline (PBS). After UVB irradiation,cells were incubated in cell culture medium at 37 8C for varioustime periods.

2.3. Cell viability

Cell viability of HaCaT cells, NHEK and the reconstructed humanepidermis after UVB irradiation was measured using a TACSTM MTTAssay (R&D System, Inc., MN, USA) according to the manufacturer’sinstructions. Cell viability was expressed relative to that ofuntreated control cultures.

2.4. RNA extraction, cDNA synthesis, and real-time quantitative

polymerase chain reaction

Total RNA was extracted from UVB (0–50 mJ/cm2) and sham-irradiated HaCaT cells and NHEK after 0–24 h using TRI REAGENT(Sigma, Saint Louis, MO, USA). An aliquot (1 mg) of the extracted

RNA was reverse-transcribed using a first strand cDNA synthesiskit (Amersham Biosciences, Buckinghamshire, UK) according to themanufacturer’s instruction. Real-time PCR was carried out using7300 Real-time PCR system (Applied Biosystems, Foster City, CA,USA) using SYBR1 Green. The reaction mixture (RT-PCR kit, TakaraBiochemicals) contained 12.5 ml Premix Ex Taq 2.5 ml SYBR1

Green, custom-synthesized primers, ROX reference dye, and cDNA(equivalent to 20 ng total RNA) to a final reaction volume of 25 ml.Table 1 summarizes the sequences of primers that were designedusing Primer Express software (Applied Biosystems). The PCRsettings were as follows: the initial denaturation for 15 s at 95 8Cwas followed by 40 cycles of amplification for 3 s at 95 8C and 31 sat 60 8C, with subsequent melting curve analysis increasing thetemperature from 60 to 95 8C. Quantification of gene expressionwith real-time PCR data was determined relative to GAPDH.

2.5. Western blotting

Protein levels of human LEKTI, SCTE and SCCE were measuredby Western blotting. Briefly, cells were lysed in a suitable volumeof ice-cold lysis buffer (CelLytic-MT Lysis/Extraction Reagent;Sigma). The homogenate was centrifuged at 10,000 rpm for 5 minat 4 8C and the supernatant was collected. Protein samples (20 mg)were separated on a sodium dodecyl sulfate polyacrylamide gel byelectrophoresis and then transferred to nitrocellulose membranes.The blots were incubated with primary antibodies against LEKTI(1/200; Santa Cruz Biotechnology, Santa Cruz, CA, USA), SCCE (1/100; Santa Cruz Biotechnology), SCTE (1/1000; Protein Tech Group,Chicago, IL, USA) or b-actin (1/10,000; Sigma), followed byincubation with horseradish-peroxidase-conjugated secondaryantibodies (1/2000; Amersham) and visualization using an ECLkit (Amersham). The band densities were calculated with Image Jsoftware and normalized to the protein content of b-actin.

2.6. Immunofluorescent cell staining

For immunofluorescence analysis, the reconstructed humanepidermis was irradiated with UVB (50 mJ/cm2), cultured at 37 8Cfor 72 h, and then snap frozen in liquid nitrogen for storage at�80 8C. The frozen sections (14 mm) were fixed in 4% paraformal-dehyde, blocked with 10% horse serum, and incubated withpolyclonal rabbit antibodies for LEKTI (1/50), SCCE (1/50) and SCTE(1/100). For the negative control, rabbit IgG (1/100) was applied(Jackson ImmunoResearch, Inc. Laboratories, PA, USA). Biotiny-lated goat anti-rabbit IgG (Vector Laboratories, Inc., Burlingame,CA, USA) was used as secondary antibody. Green fluorescence wasgenerated by labeling with streptavidin-FITC (BD Biosciences, SanJose, CA, USA). Images were obtained with a Biozero BZ-8100microscope (Keyence Company, Osaka, Japan).

Fig. 1. Cell viability of HaCaT cells, NHEK and reconstructed human epidermis after

UVB irradiation at various doses. HaCaT cells, NHEK and reconstructed human

epidermis were exposed to different doses of UVB and incubated for 48 and/or 72 h.

Cell viability was analyzed by the MTT assay in HaCaT cells (a), NHEK (b), and

reconstructed human epidermis (c). Data represent one of three independent

experiments performed in triplicate with similar findings, and are expressed as the

mean � S.D., *p < 0.05, **p < 0.01 versus 0 mJ/cm�2.

M. Nin et al. / Journal of Dermatological Science 54 (2009) 17–24 19

2.7. In situ zymography

Protease activity was estimated by in situ zymography aspreviously described [22]. Briefly, the reconstructed humanepidermis was incubated in cell culture medium at 37 8C for 72 hafter UVB irradiation (50 mJ/cm2). The frozen sections (14 mm) wererinsed with 1% Tween-20 in PBS and incubated with 100 ml ofBODIPY-FL-casein substrate (10 mg/mL in 10 mM Tris–HCl, pH 7.8;Molecular Probes) at 37 8C for 3 h. After removal of excess ofsubstrate solution, nuclei were stained with 40,6-diamidino-2-phenylindole (DAPI). Images were obtained with a Biozero BZ-8100microscope (Keyence Company). In some experiments, proteaseinhibitors were added, including 200 mM 4-(2-aminoethyl)-benze-nesulfonylfluoride (AEBSF) and 20 mg/mL aprotinin (Calbiochem,San Diego, CA, USA) to inhibit serine protease, 200 mM pepstatin toinhibit asparate protease (Sigma–Aldrich), and EDTA to inhibitmetalloprotease (DS pharma Biochemical Co., Ltd., Osaka, Japan).

2.8. Statistical analysis

The results are expressed as the mean � S.D. Cell viability andmRNA levels were analyzed by the Bonferroni test. Protein levelswere analyzed using the paired t-test. All statistical analyses wereperformed using Statcel 2 software (OMS publisher, Saitama, Japan)and differences between the groups were considered significant if thep-value was less than 0.05.

3. Results

3.1. Effect of UVB irradiation on cell viability

We first analyzed the effect of UVB irradiation on cell viability inHaCaT cells, NHEK and in the reconstructed human epidermismodel. UVB irradiation induced a loss of cell viability in a dose- andtime-dependent manner, as shown in Fig. 1a–c. UVB irradiation ata dose of 50 mJ/cm2 had no effect on cellular viability of HaCaT cellsup to 48 h, whereas a higher dose and/or longer duration after UVBirradiation caused significant reductions in cellular viability(Fig. 1a). UVB irradiation at a dose of 30 mJ/cm2 had no effecton cellular viability of NHEK up to 48 and 72 h, whereas a higherdose of UVB irradiation caused a significant reduction in cellularviability (Fig. 1b). In the reconstructed human epidermis model,UVB irradiation at a dose of 100 mJ/cm2 induced loss of cellviability after 72 h, as assessed by MTT assay (Fig. 1c).

3.2. In vitro modulation of mRNA expression of corneodesmosomal

enzymes by UVB irradiation in human keratinocytes

To determine whether UVB irradiation would affect theexpression of corneodesmosomal enzymes in human epidermalkeratinocytes, expression of the mRNA of each of these enzymeswas analyzed using real-time PCR. RNA was extracted from HaCaTcells and NHEK, 0–24 h after UVB irradiation (0–50 mJ/cm2). Theexpression of SPINK5 mRNA was significantly reduced after 12 or24 h of UVB irradiation at a dose of 30 mJ/cm2 (Fig. 2a–d). Incontrast, expression of KLK5 and KLK7 mRNA was increased afterUVB irradiation (30 mJ/cm2) (Fig. 2e–l). These results indicate thatUVB irradiation dichotomously regulates the mRNA expression ofthese corneodesmosomal enzymes in human epidermal keratino-cytes.

3.3. In vitro modulation of LEKTI, SCTE and SCCE by UVB irradiation in

human keratinocytes

To determine whether UVB would affect the expression of thecorneodesmosomal enzymes at the protein level, we next analyzed

the expression of LEKTI, SCTE and SCCE in HaCaT cells and NHEK byWestern blotting. LEKTI, SCTE, and SCCE generated bands at140 kDa, around 35 kDa and about 31 kDa, respectively. Theexpression of LEKTI was significantly reduced after 72 h of UVBirradiation (30 mJ/cm2) in HaCaT cells and NHEK (Fig. 3a and b). Bycontrast, the level of SCTE was increased significantly after 24 h ofUVB irradiation (30 mJ/cm2) in HaCaT cells and NHEK (Fig. 3c andd). The level of SCCE protein did not change significantly (data notshown). LEKTI and SCTE were not detected in the medium ofDMEM and EpiLife.

3.4. Expression of LEKTI, SCTE and SCCE in the reconstructed human

epidermis model

To understand the effect of UVB irradiation on the spatialexpression of corneodesmosomal enzymes in the human epider-mis, we examined whether the expression patterns of LEKTI, SCTEand SCCE were altered in UVB-irradiated reconstructed humanepidermis by immunofluorescence. LEKTI expression was observed

Fig. 2. UVB irradiation dichotomously affected the expression of mRNA for corneodesmosomal enzymes in human keratinocytes. SPINK5, KLK5 and KLK7 mRNA expression in

the HaCaT cells (a, e, and i) and NHEK (c, g, and k) were analyzed by real-time PCR 0, 1, 2, 6, 12, and 24 h following UVB irradiation at a dose of 30 mJ/cm2. In addition,

expression of the mRNA of each of these genes was determined in HaCaT cells (b, f, and j) and NHEK (d, h, and l) after 12 and 24 h, respectively, following UVB irradiation at

doses of 0, 10, 30 and 50 mJ/cm2. Reactions were performed in triplicate. Data represent one of the five independent experiments with similar results and are expressed as the

mean � S.D., *p < 0.05, **p < 0.01 versus 0 mJ/cm2 or 0 h.

M. Nin et al. / Journal of Dermatological Science 54 (2009) 17–2420

Fig. 3. In vitro modulation of LEKTI, SCTE after UVB irradiation in HaCaT cells and NHEK. HaCaT cells and NHEK were exposed to the indicated dose of UVB, incubated for

different times, and their lysates were prepared for Western blotting. The protein levels of LEKTI (a and b) and SCTE (c and d) were measured at the indicated time points after

UVB exposure at a dose of 30 mJ/cm2 in HaCaT cells (a and c) and NHEK (b and d). The histograms show the percent change in protein level relative to the control sample

without UVB exposure. The band densities were calculated using Image J software and normalized to the protein content of b-actin. Data represent one of the five

independent experiments performed in triplicate with similar results and are expressed as the mean � S.D., *p < 0.05, **p < 0.01 versus 0 mJ/cm2 or 0 h.

M. Nin et al. / Journal of Dermatological Science 54 (2009) 17–24 21

much less frequently in the granular and cornified layers of UVB-irradiated epidermis compared with sham-irradiated epidermalmodel. By contrast, SCTE and SCCE were highly expressed in theacanthotic and granular layers of UVB-irradiated epidermis,whereas these enzymes were only weakly expressed in thegranular layers of sham-irradiated epidermis (Fig. 4).

3.5. Protease activity in the reconstructed human epidermis model

examined by in situ zymography

Reduced expression of LEKTI, in addition to increased expres-sion of SCTE and SCCE in the UVB-irradiated epidermis may resultin up-regulation of protease activity in the epidermis. To confirmthis hypothesis, we carried out in situ zymography with BODIPY FL-casein. As expected, protease activity in the reconstructed humanepidermis was increased after 72 h of UVB irradiation (50 mJ/cm2)when compared with sham-irradiated epidermis. The serineprotease inhibitors, aprotinin and AEBSF, completely suppressedthe observed proteolytic activity, suggesting that the UVB-

enhanced protease activity was mediated most probably by serineproteases, including SCTE and SCCE. Pepstatin (asparate proteaseinhibitor) and EDTA (metalloprotease inhibitor) were ineffective(Fig. 5).

4. Discussion

It is generally accepted that intercellular lipids, naturalmoisturizing factors and sebum play crucial roles in maintainingthe barrier of the skin [23]. In addition, the lower part of thestratum corneum forms another barrier to the penetration ofirritants and allergens [4]. Desquamation of corneocytes reducesskin barrier function by allowing penetration of irritants andallergens. Desquamation of corneocytes can only occur once thecorneodesmosome has been broken down by skin-specificproteases, such as SCCE and SCTE, which are kept under controlby specific protease inhibitors including LEKTI [4]. Although it hasbeen reported that UVB irradiation induces desquamation ofcorneocytes [3], the effect of UVB irradiation on the expression of

Fig. 4. LEKTI, SCTE and SCCE expression after UVB irradiation in reconstructed human epidermis. Expression of LEKTI, SCTE and SCCE protein after UVB exposure at a dose of

50 mJ/cm2 in reconstructed human epidermis was visualized by immunofluorescence. (a, c, and e) Normal skin. (b, d, f, and g) UVB-irradiated skin. Green indicates LEKTI (a

and b), SCTE (c and d) and SCCE (e and f). Control IgG (g). Scale bars, 300 mm. The above are representative findings from six independent experiments.

M. Nin et al. / Journal of Dermatological Science 54 (2009) 17–2422

these corneodesmosomal enzymes has not been shown. Toelucidate the role of these molecules in UVB-induced desquama-tion of the stratum corneum, we examined the effect of UVBirradiation on the expression of these enzymes in humanepidermal keratinocytes.

In this study, we demonstrate for the first time that UVBirradiation at a dose that had no effect on cellular viability reducedthe expression of LEKTI/SPINK5 and increased SCTE/KLK5 and SCCE/KLK7 in human epidermal keratinocytes. In addition, proteaseactivity in the UVB-irradiated epidermis was enhanced and mostprobably mediated by serine proteases when assessed using in situ

zymography. It is suggested that imbalance of the corneodesmo-somal enzymes and enhanced protease activity in the epidermalkeratinocytes after UVB irradiation would contribute to thereduction of corneocyte adhesion and increased desquamationof the stratum corneum.

In normal skin, LEKTI, SCTE and SCCE were all found in thelamellar granule system, but were localized distinctly [26]. LEKTIwas detected intracellularly and extracellularly in deeper epider-mal layers than SCCE, which was found in more superficialdifferentiated keratinocytes [26]. In addition, LEKTI is expressedand released earlier than SCTE and SCCE. These findings areconsistent with a role of LEKTI in preventing premature proteolysisof extracellular matrix proteins or cell surface adhesion molecules,

and controlling the timing of desquamation [24]. Nethertonsyndrome is caused by mutations in the SPINK5 gene. In patientswith Netherton syndrome, excessive desquamation of corneocytesresults in dysfunction of the skin permeability barrier, congenitalichthyosiform erythroderma and atopic features [25]. In theepidermis of these patients, SCTE-like and SCCE-like activities areincreased [26,27]. In addition, SCTE and SCCE expression wereextended to the cell layers in which desmoglein 1 and desmocollin1 expression were reduced, suggesting that these proteasesparticipate in the premature degradation of corneodesmosomalcadherins [27]. In our study, SCTE and SCCE expression were alsoextended to the cell layers by immunofluorescence. It is thereforesuggested that one of the mechanisms by which UVB irradiationincreased the expression and activities of SCCE and SCTE aremediated by a reduction of LEKTI expression.

Transgenic mice overexpressing human SCCE in suprabasalepidermal keratinocytes exhibit increased TEWL and, moreinterestingly, dermal inflammation [28]. SCCE may be capable ofconverting the precursor protein of interleukin-1b, a proinflam-matory cytokine, into its active form [29]. SCTE, on the other handactivates SCCE proenzyme and possibly also phospholipase A2,which leads to the synthesis of inflammation-mediating prosta-glandins [30]. SCTE may also activate proteinase-activatedreceptor 2 (PAR2), which mediates inflammatory responses [31].

Fig. 5. Protease activity after UVB irradiation in reconstructed human epidermis. Reconstructed human epidermis was exposed to 50 mJ/cm2 UVB and incubated for 72 h.

Protease activity was examined by in situ zymography using FITC-conjugated casein substrate. Nuclei were also stained with DAPI (c and d). A normal skin (a, c, and e), UVB-

irradiated skin (b, d, and f). Aprotinin, AEBSF, pepstatin, and EDTA were also added (supplementary Fig. 5). Scale bars, 300 mm. The above findings are representative of six

independent experiments.

M. Nin et al. / Journal of Dermatological Science 54 (2009) 17–24 23

It is hypothesized that activation of PAR2 by SCTE activatesphospholipase A2 and then prostaglandins, all of which wouldcontribute to evoke inflammation [34]. Taken together, bothproteases are likely of great importance in skin desquamation aswell as inflammation [32]. It is thus suggested that enhancedexpression of these corneodesmosomal enzymes might contributeto skin inflammation by UVB.

The mechanism by which UVB irradiation dichotomouslyaffects the expression of corneodesmosomal enzymes is to dateunknown. Recently, it has been demonstrated that UVB irradia-tion induces changes of the epidermal calcium gradient, whichresults in increased cytosolic calcium in the lower epidermis [33].The expression of SCCE was increased in epidermal keratinocytescultured under high-calcium conditions [34]. These resultssuggest that the UVB-induced alteration of the epidermal calciumgradient may be one of the mechanisms by which UVB enhancesthe expression of SCCE and possibly SCTE. On the other hand, toour knowledge, the system regulating LEKTI expression inkeratinocytes has not been reported. It may be interesting toexamine the relationship between the expression of thesecorneodesmosomal enzymes and UVB-induced events, such asalteration of the calcium gradient, and up-regulation of NF-kB andAP-1 [35].

In conclusion, the results presented herein suggest that UVBexposure in our daily life could disrupt the homeostatic regulationof corneodesmosomal enzymes resulting in desquamation. Altera-tions in these enzymes may also be related to UVB-induced skininflammation. Our study provides further evidence regarding thenecessity to avoid excessive exposure to sunlight.

Acknowledgements

This work was supported in part by a Grant-in-Aid for ScientificResearch from the Japan Society for the Promotion of Science (NK),and Health and Labour Science Research Grants for Research on

Allergic Disease and Immunology from the Ministry of Health,Labour, and Welfare of Japan (NK).

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.jdermsci.2008.11.004.

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