Journal of Dermatological Science 59 (2010) 176–183

Epimorphin-derived peptide antagonists remedy epidermal parakeratosistriggered by unsaturated fatty acid

Yoji Okugawa a, J. Jamie Bascom b, Yohei Hirai a,*a Department of Bioscience, School of Science and Engineering, Kwansei Gakuin University, 2-1 Gakuen, Sanda 669-1337, Japanb Life Science Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA

A R T I C L E I N F O

Article history:

Received 31 May 2010

Received in revised form 5 July 2010

Accepted 6 July 2010

Keywords:

Epidermis

Hyperplasia

Epimorphin

Antagonist

Syntaxin

Oleic acid

A B S T R A C T

Background: Unsaturated fatty acid from accumulated sebum disrupts calcium influx in keratinocytes

and triggers epidermal hyperplasia, leading to comedone formation in the skin. Oleic acid, a

representative unsaturated fatty acid, has been shown to be a useful reagent to induce these cellular

alternations, however, the detailed mechanism still remains to be elucidated.

Objectives: This study aimed at the identification of the mediator of unsaturated fatty acid-caused

epidermal hyperplasia so as to generate the effective therapeutic agents.

Methods: The downstream mediator of oleic acid-treatment was identified in the epidermal

keratinocyte and the effect of its antagonistic peptides on the epidermal behaviors was investigated

in culture and in vivo.

Results: In culture, treatment with oleic acid augmented extracellular secretion of epimorphin in HaCaT

keratinocytes and prevented the epidermal terminal differentiation including programmed cell death

and cornified envelope formation. The antagonistic peptide of epimorphin (EPn1: a circular compound

composed of CGSIEQSC), which was newly generated in this study, restored normal keratinocyte

behaviors. In hairless mice, topical application of oleic acid to the dorsal skin caused epidermal

hyperplasia with decreased enucleation in the horny layer, which was dramatically hampered by the

administration of EPn1.

Conclusions: The effects of unsaturated fatty acid are attributed to the overstimulation of epimorphin

signaling and suggest the epimorphin antagonist as a possible therapeutic agent for acne and

hyperkeratotic skin disease.

� 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

Keratinocytes in the basal layer of the skin epidermis continu-ously deliver daughter cells upward that activate differentiationprograms to give rise to structurally distinguishable stratifiedstructures. As the physical barrier, the cornified layer of differenti-ated keratinocytes (corneocytes) prevents a rapid loss of the internalfluid and plays a central role in protection against environmentalhazards such as UV irradiation, microbial and other forms ofparasitic invasion. In this regard, the formation and maintenance ofthe cornified layer that synthesizes barrier components are one ofthe most important aspects of the epidermal keratinocytes.However, the critical factors involved in the control of terminaldifferentiation of the cornified layer are still being elucidated. A well-documented signal that facilitates this process is the massive influxof ionic calcium. Calcium binding to cell surface Ca2+ receptors leads

* Corresponding author. Tel.: +81 79 565 7234; fax: +81 79 565 7234.

E-mail address: y-hirai@kwansei.ac.jp (Y. Hirai).

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

doi:10.1016/j.jdermsci.2010.07.004

to the activation of several cytoplasmic proteases and acceleratesboth differentiation and the apoptotic program (diffpoptosis) [1].Low concentration of endogenous Ca2+ is necessary to preventdifferentiation and to maintain the proliferation of nascentkeratinocytes, whereas higher concentrations yield the oppositeeffect and ultimately stimulate diffpoptosis in the highly differenti-ated cornified cell layers [2].

Unsaturated fatty acids (UFAs) are present in the extracellularspace of the differentiating layer of epidermis and play a role inskin barrier function; however, excess UFAs increase the Ca2+

concentration in the epidermis and lead to precocious keratiniza-tion [3–5]. Additionally, pathological lesions such as acne andatopic dermatitis show a substantial accumulation of UFAs [6–8].Among UFAs, oleic acid has become a main focus since it has beenshown to accumulate in the ceramide of atopic patients. Moreover,its application to rabbit ears induces sizeable comedones display-ing marked hyperplasia and hyperkeratosis in the epithelium ofthe follicular infundibulum [3,7].

To define the downstream signals of UFAs in abnormalepithelial differentiation, we focused on the extracellular form

y Elsevier Ireland Ltd. All rights reserved.

Y. Okugawa et al. / Journal of Dermatological Science 59 (2010) 176–183 177

of epimorphin since this protein dramatically depresses terminaldifferentiation of keratinocytes that is reminiscent of the effect ofUFAs [9]. Epimorphin is abundant in the stromal compartmentwith detectable amount in the epidermis. Calcium influx cues theextracellular secretion of epimorphin and perturbs its signalinggradient in the epidermis [10,11]. Since unsaturated fatty acidleads to accumulation of intracellular calcium [5], we hypothesizedthat oleic acid-induced abnormal differentiation of epidermis isattributed to the perturbation of signaling pathways regulated byextracellular epimorphin.

In this study, we found that translocation followed byextracellular secretion of epimorphin in keratinocytes is regulatedby oleic acids and that inhibition of epimorphin signaling by newlygenerated antagonistic peptides prevents oleic acid-inducingdefects in the epidermal differentiation. The data shown hereprovide novel insights to how excess UFAs stimulate keratinizationdefects in the epidermis and suggests the possible application ofantagonistic peptides of epimorphin as therapeutic agents fortreatment of some skin disorders such as acne and atopic dry skin.

2. Materials and methods

2.1. Cells

A functionally normal human epidermal keratinocyte cell lineHaCaT, a mouse fibroblast cell line PT67 and those that stablyexpressing T7 tagged epimorphin (HaCaT–TE or PT67–TE) orexpressing extracellular epimorphin (HaCaT–EPM1 or HaCaT–EPM2) were maintained in DMEM/HamF12 medium supplemen-ted with 10% FCS [9,10]. In some wells oleic acid dissolved inethanol just before the experiment was added to the culturemedium to the concentration of 0.01, 0.025 or 0.05%, and theethanol concentration in all wells was adjusted to 0.1%. Toinvestigate cell proliferation, the total cellular metabolic activitywas analyzed with WST-1 reagent (Dojin Chemical Co., Kumamoto,Japan) according to the manufacturer’s protocol. In brief, the cellswere incubated with WST-1 reagent solution for 2 h, and theabsorbance at 450 nm of the supernatant correlates with thenumber of metabolically active cells was measured.

2.2. Antibodies

The primary antibodies used for Western blotting were thoseagainst epimorphin [12], involucrin (1:100) (Santa Cruz Biotech-nology Inc., CA), cytokeratin-1 (1:1000) (Covance, Richmond, CA),cytokeratin-5 (1:1000) (Covance, Richmond, CA), DNase1L2(1:200) (gift from Dr. Eckhart, Wien University), caspase-14(1:200) (IMGENEX, San Diego, CA), and b-actin (1:2000) (Sig-ma–Aldrich, St. Louis, MO). For some blots, HRP-labeled monoclo-nal antibodies against T7 or b-actin were directly used fordetection. To retrieve secreted epimorphin, supernatant of HaCaT–TE or PT67–TE cells was incubated with anti-T7 tag monoclonalantibody (Novagen) followed by ProteinG-sepharose (BD Biosci-ence, Tokyo, Japan).

2.3. Generation of synthetic epimorphin inhibitory peptides

Previous studies identified epimorphin functional domain (thepep7 domain: SIEQSCDQDE) for hair follicular morphogenesis andthe successful generation of chemically and structurally modifiedforms as the active agonistic peptides [13,14]. By similar attemptswe newly generated antagonistic circular peptides, that is,NH(CH2)nCO linked to a cysteine residue was connected at theN-terminal of SIEQSC, a part of the epimorphin functional domain,followed by the introduction of an intramolecular disulphidebridge. The step for the intramolecular bridge could be included in

the peptide synthesis procedure. All the peptide synthesis andpurification was carried out at KNC laboratories (Kobe, Japan). Theobtained circular peptides were dissolved in water or 50% ethanol(vehicle) and used for culture and the animal assay, respectively.The antagonistic activity was achieved when the circular peptidehas a linker of n = 1 (glycine) and we named this CGSIEQSC with theintramolecular disulfide bridge as EPn1. As a control peptide, alinear format of SIEQSC with an additional aspartic acid wasgenerated that exhibits no detectable activities (CP) was used.

2.4. Animals and tissues

Hos/hr-1 albino female hairless mice, purchased from Hoshinoanimal (SLC, Japan) at 7 weeks of age, were divided into threegroups (n = 3). We used female animals just because they weregentle and ease of the topical application of agents withoutanesthesia. Dorsal skins of each group of mice were pretreatedwith 100 ml of vehicle (50% ethanol), Epn1 (10 ng/ml in 50%ethanol) or CP (10 ng/ml in 50% ethanol) once a day for 3 days (day1, 2 and 3) before oleic acid-administration. On day 4, 5 and 6,100 ml of oleic acid (10% in ethanol) was topically administrated,but the pretreatment was also applied 5 min prior to and after eacholeic acid-administration. To investigate the epidermal hyperpla-sia the hairless mice were killed on day 8, and punch biopsies wereembedded directly in OCT compound. Five transverse cryosectionswere prepared from each tissue and stained with hematoxylin/eosin (HE) solution. Epidermal thickness was evaluated bymeasuring the depth of epidermis of ten random images. Tocollect and analyze the parakeratotic corneocytes, the skin surfacewas taped up followed by stripping off the adhesive tape (Scotch,Tokyo, Japan) as described [5]. The nuclei in the desquamatedstratum corneum (the outermost scaling horny layers) on the tapewere stained with 10 ng/mL propidium iodide (Sigma, St. Louis,Missouri) for 5 min. The number of remaining nuclei in para-keratotic corneocytes was counted. These studies were conductedin conformity with the policies and procedures of the InstitutionalAnimal Care and Use Committee of Kwansei Gakuin University.

2.5. Three-dimensional cultures

To induce epidermal programmed cell death (anoikis) theaggregates of HaCaT cells were embedded in collagen gels andcultured as described previously [9]. In brief, HaCaT cells weresuspended in 350 ml of DH10 containing 1000 U/mL DNAseI(Sigma-Aldrich, St. Louis, MO) and rotated at 100 rpm in a 24-welldish (ultra low attachment surface, Corning, Tokyo, Japan).Smooth, rounded cell clusters formed within 24 h and wereembedded in collagen gels prepared with 0.5% Type IA collagensolution (KOKEN, Co., Tokyo, Japan) and incubated in DH10 for 4days. The cells in the center of the clusters were physically apartfrom the collagen substrate and showed immediate initiation ofthe differentiation program including anchorage-dependent celldeath and subsequent formation of central space in the clusters. Insome cultures oleic acid was added to medium at 0.01%, or a higherconcentration (0.05%) to effectively affect cells embedded incollagen gels

2.6. The induction of the cornified cell envelope formation

The formation of an insoluble cornified cell envelope (CCE) wasassessed after artificial calcium influx. HaCaT cells (1.0 � 105 cells/ml) cultured with or without 0.02% oleic acid for 3 days weresuspended in serum free DH medium and incubated with thecalcium ionophore A23187 (20 mg/ml) (Sigma–Aldrich, St. Louis,MO) for 5 h at 37 8C. Oleic acid at 0.02% was enough to reproduciblyreduce CCE formation by more than 50%. After washing with PBS,

Fig. 1. Influence of oleic acid on the expression and secretion of epimorphin. (A) HaCaT cells were treated with oleic acid at 0.01 or 0.25% and tested for epimorphin expression

on day 3. As an internal control, b-actin was tested on the same blot. (B) Oleic acid induces epimorphin secretion from HaCaT keratinocytes but not from PT67 fibroblasts. Cells

stably expressing intact epimorphin tagged with T7 peptide at the N-terminus (HaCaT–TE and PT67–TE) were treated with oleic acid and cultured for 3 days. The secreted T7-

tagged epimorphin in the culture medium was retrieved with anti-T7 antibody and protein G-sepharose beads and analyzed with HRP-labeled anti-T7 monoclonal antibody.

Oleic acid slightly up-regulated epimorphin expression in HaCaT cells and caused its secretion as a C-terminally cleaved form. As a positive control, supernatant of HaCaT-TE

or PT67-TE treated with UVB irradiation (10 mJ/cm2) was used [9].

Fig. 2. Generation of epimorphin antagonists. (A) The growth profiles of two

independent HaCaT clones expressing extracellular epimorphin (a high producer

HaCaT–EPM1 and a low producer HaCaT–EPM2) were determined by WST-1 assay.

Expression of extracellular epimorphin caused growth arrest when cells were

grown to achieve the cell–cell contact (after day 3) Addition of an antagonistic

peptide EPn1 (10 mg/ml) but not the control peptide (CP) prevented this effect. Data

means � SD, **P < 0.01. (B) Structures and antagonistic activities of epimorphin

antagonists prepared in this study. Structural variations are derived from the number

of carbon atoms within NH(CH2)nCO2 and the peptide with n = 1 (EPn1) displayed the

most strong antagonistic activity.

Y. Okugawa et al. / Journal of Dermatological Science 59 (2010) 176–183178

the cells were boiled in 2% SDS and 20 mM DTT for 10 min and thenumber of remaining insoluble cells (due to the hard-shelledinsoluble cornified envelope) was counted as described [9].

2.7. Statistical analysis

Results are expressed as mean + SD of three independentexperiments. Data were analyzed using Mann–Whitney U-test,and a P-value of <0.05 was considered statistically significant.

3. Results

3.1. Oleic acid-treatment triggers epimorphin secretion in the

keratinocytes

Oleic acid has been shown to increase the concentration ofextracellular calcium ion leading to perturbation of epidermaldifferentiation programs [3–5]. Our previous studies demonstratedthat epimorphin is secreted from the cell in response to calciuminflux and thereby stimulates keratinocyte differentiation. Toinvestigate a possible link between oleic acid-stimulation andepimorphin signaling pathways in keratinocytes, we tested theexpression and secretion of epimorphin in response to oleic acid-treatment. In response to factors from the dermal fibroblast thehuman keratinocyte HaCaT cell line undergoes appropriateterminal differentiation marked by proliferation, anoikis, andcornification [15,16]. In the presence of oleic acid for three days,HaCaT cell proliferation was markedly decreased as has beenshown in the same cells stimulated by extracellular epimorphin[9]. In addition, the cells showed a slight increase in endogenousepimorphin (Fig. 1A). To investigate whether oleic acid cues theextracellular secretion of epimorphin, we used a transfected cellclone derived from the HaCaT cell line (HaCaT–TE cells) as well as aclone derived from 3T3 fibroblasts (PT67–TE cells) which both

Y. Okugawa et al. / Journal of Dermatological Science 59 (2010) 176–183 179

stably express a T7-tagged form of full-length epimorphin [9,10].Analyses of secreted epimorphin retrieved from their supernatantwith anti-T7 antibody showed that oleic acid leads to epimorphinsecretion selectively from HaCaT cells as a C-terminal cleaved form(Fig. 1B). Provided that this secreted form retains epimorphin’sactivity [9–11], it is conceivable that the secreted form ofepimorphin accounts, at least in part, for oleic acid-dependentperturbation of epidermal differentiation.

3.2. Generation of antagonistic peptides of epimorphin

To further investigate a possible role for epimorphin action inoleic acid-treatment, we engineered epimorphin antagonisticpeptides. As we have previously identified the essential domain

Fig. 3. EPn1 prevents oleic acid-triggered perturbation of the cell death program in H

underwent normal differentiation, which was hampered by oleic acid. (a), experiment

embedded in collagen gels. (b) Morphological appearance of cell aggregates on day 1 and d

Scale bar, 100 mm. (c, left) Induction of several differentiation markers including those fo

(caspase 14, DNase1L2) in the cultured cell aggregates were investigated on day 1 and d

compared at day 4. b-actin, the loading control. Caspase-3 that is related to apoptosis in m

collagen gels underwent terminal differentiation and anoikis, leading to large central spa

the aggregates, this phenotype was rescued by addition of EPn1 but not CP (1 mg/ml). (C)

ml) neutralized this effect. In B and C, phenotypic appearances (upper) and the statistical

100 mm. Data means � SD, *P < 0.05.

for the extracellular action of epimorphin, we prepared a series ofstructurally modified peptides composed of a part of this aminoacid sequence (SIEQSC) and tested for their potential inhibitoryaction on epimorphin signaling. Generally, the rigid structure isnecessary for small peptides to exert biological their functionstherefore we decided to introduce intramolecular bridges in thepeptides. For this purpose, a cysteine residue linked withNH(CH2)nCO was connected with the N-terminus of the peptidefollowed by introduction of an intramolecular disulphide bondwith the C-terminal cysteine residue. To test their antagonisticactivities we used the two independently isolated HaCaTderivatives, which have been introduced with a full-lengthepimorphin containing an N-terminal fusion of an exogenoussignal peptide (HaCaT–EPM1 and HaCaT–EPM2) to stably over-

aCaT keratinocytes. (A) The aggregates of HaCaT cells embedded in collagen gels

al procedure. The cell aggregates were formed by suspension culture for 24 h and

ay 4. Addition of oleic acid (0.05%) resulted in suppression of the luminal formation.

r the early stage (keratin1, 5), intermediate stage (involucrin) and the terminal stage

ay 4. (c, right) Expression of these markers in the cultures with oleic acid (O.A.) was

any cell types but not in keratinocytes was also tested. The cell populations distal to

ces in the aggregates (b, c). (B) Oleic acid-caused perturbation of lumen formation in

Forced expression of epimorphin also suppressed lumen formation but EPn1 (1 mg/

analyses of 100 randomly chosen aggregates (lower) on day 4 were shown. Scale bar,

Fig. 4. EPn1 normalizes oleic acid-caused infant cornified cell envelope (CCE) in

HaCaT cells.

The formation of CCEs in HaCaT cells, treated with oleic acid and EPn1 and then

were subsequently treated with A23187, were measured, and the relative ratios to

that without oleic acid and EPn1 were listed as CCE formation index. (A) The CCE

formations of cells treated with oleic acid, oleic acid plus Epn1 or oleic acid plus CP.

Oleic acid-treatment caused a decreased CCE formation, which is restored by

simultaneous incubation with the EPn1 but not the CP. (B) HaCaT–EPM cells that

express extracellular epimorphin failed to demonstrate CCE formation, however,

EPn1 recovered the CCE formation of this cell type. Data means � SD, *P < 0.05,

**P < 0.01, ***P < 0.001.

Y. Okugawa et al. / Journal of Dermatological Science 59 (2010) 176–183180

express cell surface epimorphin [9]. The transgene product hasbeen shown to arrest the growth of the cells when cell densityreaches to that enough to accomplish the cell–cell signalinginteraction [9]. Using WST-1 assay, we found that the mostcompact form of the circular peptide (n = 1, Mw 840; named EPn1)possesses highest antagonistic activity: it can significantly restoreepimorphin-mediated growth arrest (Fig. 2A). The EPn1 peptideexhibited clear antagonistic action of other epimorphin-dependentcell behaviors as well (see below), while other circular peptidesgave weaker and intact linear peptide (CP) has no effect (Fig. 2B).

3.3. EP can restore oleic acid-induced abnormal epidermal

anoikis in culture

Epidermal basal cells deprived of extracellular matrix associa-tion withdraw from the cell cycle and become committed todifferentiate to irreversibly undergo terminal differentiation.Previously, we have shown that excess expression of extracellularepimorphin severely affects epidermal differentiation, pro-grammed cell death (anoikis), and the cornified envelopeformation [9]. Thus we next analyzed whether oleic acid-treatment also affects keratinocyte anoikis and whether EPn1can restore the phenotypic behavior by using the three-dimen-sional cell cluster assay [9,12,17]. In HaCaT cell clusters embeddedin collagen gels, the central cell populations that were distal to thecollagen matrix launched the differentiation/anoikis program. Thisprogram involved enucleation and cell death, leading to theformation of large central lumina within 3–4 days (Fig. 3Aa and b).These cell aggregates represent a spectrum of cell differentiationgrades based on characteristic epithelial differentiation markers(Fig. 3Ac). In particular, caspase-14 and DNAse1L2, which arespecific to epidermal anoikis, were up-regulated during thisculture period whereas caspases-3 that has a central role in classicalapoptotic pathways in many cell types but not in keratinocytesremained unchanged (Fig. 3Ac). These results indicate that normalkeratinocyte differentiation/anoikis is exhibited in this culturemodel. However, in the presence of oleic acid HaCaT cells displayabnormal expression of several gene markers specific for keratino-cyte differentiation: although low concentration of oleic acid(0.01%) slightly elevated markers for terminal differentiation suchas DNase1L2 and caspase-14 as described previously [18], a higherconcentration of oleic acid (0.05%) severely reduced these markersand increased early differentiation markers keratin 1 and involucrin(Fig. 3Ac). Concomitant with alteration of the mentioned differen-tiation markers, 0.05% oleic acid severely inhibited lumen forma-tion, a feature of HaCaT cells which was partially restored by EPn1(Fig. 3Ab and B). Furthermore, EPn1 clearly neutralized theinhibitory effect of epimorphin on lumen formation, further supportof epimorphin as the downstream mechanism of oleic acid-treatment (Fig. 3C). These results demonstrate that EPn1 restoresoleic acid-induced inhibition of programmed cell death viainhibiting its downstream target epimorphin.

3.4. EPn1 corrects the formation of the cornified cell envelope (CCE) in

keratinocytes

To elucidate the influence of oleic acid and EPn1 on cornifiedcell envelope (CCE) formation, we carried out the CCE formationassay using calcium ionophore A23187 [19–21]. Exposure to oleicacid revealed a marked reduction of CCE formation (to 39–46% ofthe control), which was significantly restored to 73% by pretreat-ment with a very low concentration of EPn1 (10 ng/ml) but not thecontrol peptide (CP) (Fig. 4A). Again, the CCE formation inepimorphin-overexpressing HaCaT cells was dramatically restoredby treatment with EPn1 (Fig. 4B), confirming epimorphin as thedownstream mediator of keratinocyte terminal differentiation.

3.5. Topical application of EPn1 prevents oleic acid-induced skin

disorders in hairless mice

That oleic acid-induced epidermal abnormality including thegrowth arrest, differentiation/anoikis disorders and decreased CCEformation were all rescued by EPn1 in HaCaT keratinocytes, wenext investigated whether EPn1 treatment could abate epidermalhyperplasia or parakeratosis induced by repeated application ofoleic acid in mice (Fig. 5A). We first confirmed that oleic acid-treatment led to epidermal hypertrophy (Fig. 5Ba and b), and thatpretreatment of the skin with CP failed to attenuate this phenotype(Fig. 5Bc). However, when the skin was pretreated with EPn1, amarked reduction of epidermal hyperplasia was evident (Fig. 5Bd).Upon histological analysis of the stratum corneum it was revealedthat oleic acid-treatment severely inhibits enucleation of outer-most epidermis (Fig. 5Ca and b) and that treatment with EPn1significantly abrogated the phenotype (Fig. 5Cc and d). Takentogether, oleic acid disruption of the keratinization program inouter epidermal cells in vivo can be rescued solely by EPn1treatment.

4. Discussion

Unsaturated fatty acids in sebum are known to increase theintracellular concentration of calcium ions through activation in N-methyl-D-aspartate (NMDA) receptor in epidermal keratinocytesleading to abnormal keratinization and disruption of epidermalbarrier function [4,5]. Conversely, disruption of epidermal barrierfunction has been shown to trigger a decrease in calcium

Fig. 5. Effect of EPn1 on epidermal hyperplasia and parakeratosis in hairless mice induced by oleic acid. The dorsal skins of hairless mice were treated with oleic acid, oleic acid

plus Epn1 or oleic acid plus CP (A) and investigated for degree of epidermal thickness (B) and enucleation in the stratum corneum (C). (B, upper) HE staining of transverse

sections from the dorsal skin of mice. Oleic acid-treatment (b) caused the epidermal hyperplasia compared with vehicle-treated control (a), this phenotype was blocked by

topical application of the EPn1 (d), but not by CP (c). Scale bar, 200 mm. (B, lower) Statistical analysis of epidermal thickness. (C upper) Parakeratosis was determined by the

number of remaining nuclei in the stratum corneum collected by the stripped adhesive tape. The nuclei were stained with propidium Iodide. (C lower) Statistical analyses of

the remaining nuclei demonstrate that oleic acid prevented enucleation in the stratum corneum which was dramatically recovered by Epn1 peptide. Scale bar, 200 mm, Data

means + SD, ***P < 0.001.

Y. Okugawa et al. / Journal of Dermatological Science 59 (2010) 176–183 181

concentration in the outer nucleated layers of the epidermis[22,23]. We show in this study that oleic acid induces theexpression and extracellular secretion of epimorphin in epidermalkeratinocytes. Epimorphin has been shown to be presented andsecreted extracellularly by calcium influx and regulates morpho-genesis of wide variety of epithelial tissues including hair follicle,lung, liver and mammary epithelia [10,14,24,25]. In epidermalskin, the forced expression of extracellular epimorphin perturbedthe epidermal differentiation program and inhibited terminaldifferentiation [9], which is reminiscent of unsaturated fatty acid-induced abnormal epidermal keratinocytes. Thus it is conceivablethat epimorphin might function as a downstream signalingmolecule upon treatment of keratinocytes with unsaturated fattyacid. Interestingly, we found that an antagonistic peptide againstepimorphin is capable of rescuing the oleic acid-triggered dysker-atosis and precocious development in keratinocytes. Epimorphinbelongs to a member of t-SNARE protein family involved inintracellular membrane fusion events [17,24]. This begs thequestion, how does a protein with a role in intracellular vesiclefusion also regulate terminal differentiation in keratinocytes? Anumber of t-SNARE proteins including epimorphin are found in theplasma membranes of various cell types that package and store highconcentrations of substances in dense core vesicles for regulated

export. Studies on lung epithelia have shown that secretion of lungalveolar surfactant and maintenance of lipid contents of the lamellarbody, is a complex process involving epimorphin-dependentvesicular fusion with the plasma membrane [26,27]. In the skinepidermis, newly synthesized lipids are delivered by lamellar bodiesto the interstitium of the stratum corneum. It may be thatintracellular epimorphin also participates in association or disasso-ciation of cytoplasmic lamellar bodies to the plasma membrane.Notably, the terminal differentiation of keratinocytes involves largescale accumulation of lamellar bodies at both intra- and extracellu-lar sides of the plasma membrane, the latter of which remarkablyfacilitates the epidermal barrier function [28,29]. Thus, theaccumulation and arrangement of lamellar bodies at both the intra-and extracellular surfaces may be influenced by epimorphin.Concomitantly with the accumulation of lamellar bodies at the cellmembrane, a series of structural proteins, involucrin and loricrin, aresynthesized and subsequently crosslinked by transglutaminase toreinforce the hard-shelled CCE [28,30]. Given that extracellularepimorphin attenuates the expression and processing of transglu-taminase [9], the normal morphology of corneocytes could also beaffected by excessive extracellular epimorphin.

As a consequence, we successfully generated peptide antago-nists against epimorphin signaling by structural modification of

Y. Okugawa et al. / Journal of Dermatological Science 59 (2010) 176–183182

segments within epimorphin’s cell binding site. One antagonistexhibiting the strongest inhibitory effect was the rigid circularmolecule CGSIEQSC that contains an intramolecular disulfidebridge (EPn1). Administration of EPn1 strongly abrogates theeffects of extracellular epimorphin, including growth inhibition,attenuation of anoikis and CCE formation. Presumably, themolecular skew formed by this bridge would be appropriate forcompetitive cell binding with intact epimorphin but does not elicitthe signaling response. A recent report demonstrated that the fouramino acid residues located within epimorphin helices B and C(D79, D101, S145, F141) are essential to elicit epimorphin’sextracellular function for mammary epithelia [31]. The identifiedamino acid sequence of the epimorphin antagonists residesneighboring to the aspartic acid (D101), indicating the regionaround D101 is a central domain of epimorphin for its receptorbinding. It is notable; however, that the inhibitory effects of EPn1on oleic acid-inducing abnormal differentiation were less thanperfect, implying that additional signaling mediators function inparallel or downstream of epimorphin. In epidermal keratinocytes,syntaxin3 and syntaxin4 (which exhibit similar molecularstructures and cellular functions as epimorphin) are expressedat the plasma membrane [32]. Our preliminary experimentsdemonstrate that their extracellular localization is increased inresponse to calcium influx, UV irradiation and apoptosis-inducingfactors (not shown). However, the amino acid sequences corre-sponding to the epimorphin’s cell binding center (SIEQSC) are notwell conserved in these syntaxins (SMEKHI for syntaxin3 andAIEPQK for syntaxin4), implying their cooperative participation inkeratinocyte differentiation would not likely be antagonized byEPn1. Alternatively, oleic acid-treatment may also affects signalingpathways independent of epimorphin and epimorphin relatedfamily proteins. Some reports have shown that unsaturated fattyacids trigger activation of receptors of IL-1a or TNFa, which hasbeen known to influence keratinocyte differentiation [33,34].

Partial parakeratosis is observed in several skin disorders, suchas acne and atopic dermatitis, and is characterized by abnormallipid synthesis, epidermal barrier dysfunction and altered inflam-mator response to irritants [3,7]. While treatments that aim toaccelerate epidermal turnover could be a stopgap remedies forthese types of disorders, a more direct therapeutic approachinvolves modulation of the deregulated signaling pathways. Wepropose that EPn1, a peptide antagonist of epimorphin, is a strongcandidate for the treatment against such epidermal disorders.Previous studies have shown that ubiquitous activation ofepimorphin signaling in the whole body results in the embryoniclethality and that temporal and targeted overexpression in themammary epithelial cells leads to tumor formation [35,36] Incontrast, epimorphin knockout mice are viable and displayrelatively limited phenotypes [37,38], suggesting that the tempo-ral suppression of epimorphin function may have few of adverseeffects. In fact, a very recent study clearly suggested thetherapeutic benefit of the suppression of epimorphin function[39]. Admittedly, peptide formation is known to be stable in vitro

but easily degraded by endogenous proteases in vivo, which hasbeen a serious obstacle for the clinical application. However,peptide instability could be advantageous for the clinical applica-tion as an external medicine for the epidermis considering thatwith frequent application and proximity of the target epidermiscan limit harmful side effects. Our next aim will be to verify thefeasibility of the clinical application of EPn1.

Acknowledgments

We thank Leopold Eckhart for antibodies against DNase1L2, andKenichi Kuriyama, Sadakazu Onishi and Kiichiro Nakajima forhelpful advices.

References

[1] Whitfield JF, Bird RP, Chakravarthy BR, Isaacs RJ, Morley P. Calcium-cell cycleregulator, differentiator, killer, chemopreventor, and maybe, tumor promoter.J Cell Biochem Suppl 1995;22:74–91.

[2] Micallef L, Belaubre F, Pinon A, Jayat-Vignoles C, Delage C, Charveron M, et al.Effects of extracellular calcium on the growth-differentiation switch in im-mortalized keratinocyte HaCaT cells compared with normal human keratino-cytes. Exp Dermatol 2009;18(February (2)):143–51.

[3] Choi EH, Ahn SK, Lee SH. The changes of stratum corneum intersticesand calcium distribution of follicular epithelium of experimentally inducedcomedones (EIC) by oleic acid. Exp Dermatol 1997;6(February (1)):29–35.

[4] Katsuta Y, Iida T, Hasegawa K, Inomata S, Denda M. Function of oleic acid onepidermal barrier and calcium influx into keratinocytes is associated with N-methyl-D-aspartate-type glutamate receptors. Br J Dermatol 2009;160(Janu-ary (1)):69–74.

[5] Katsuta Y, Iida T, Inomata S, Denda M. Unsaturated fatty acids induce calciuminflux into keratinocytes and cause abnormal differentiation of epidermis. JInvest Dermatol 2005;124(May (5)):1008–13.

[6] Ferreri C, Angelini F, Chatgilialoglu C, Dellonte S, Moschese V, Rossi P, et al.Trans fatty acids and atopic eczema/dermatitis syndrome: the relationshipwith a free radical cis–trans isomerization of membrane lipids. Lipids2005;40(July (7)):661–7.

[7] Maeda T. An electron microscopic study of experimentally induced comedoand effects of vitamin A acid on comedo formation. J Dermatol 1991;18(July(7)):397–407.

[8] Oh CW, Myung KB. An ultrastructural study of the retention hyperkeratosis ofexperimentally induced comedones in rabbits: the effects of three comedo-lytics. J Dermatol 1996;23(March (3)):169–80.

[9] Okugawa Y, Hirai Y. Overexpression of extracellular epimorphin leads toimpaired epidermal differentiation in HaCaT keratinocytes. J Invest Dermatol2008;128(August (8)):1884–93.

[10] Hirai Y, Nelson CM, Yamazaki K, Takebe K, Przybylo J, Madden B, et al.Non-classical export of epimorphin and its adhesion to alphav-integrin inregulation of epithelial morphogenesis. J Cell Sci 2007;120(June (Pt. 12)):2032–43.

[11] Radisky DC, Stallings-Mann M, Hirai Y, Bissell MJ. Single proteins might havedual but related functions in intracellular and extracellular microenviron-ments. Nat Rev Mol Cell Biol 2009;10(March (3)):228–34.

[12] Hirai Y, Lochter A, Galosy S, Koshida S, Niwa S, Bissell MJ. Epimorphin functionsas a key morphoregulator for mammary epithelial cells. J Cell Biol1998;140(January (1)):159–69.

[13] Hirai Y, Takebe K, Nakajima K. Structural optimization of pep7, a small peptideextracted from epimorphin, for effective induction of hair follicle anagen. ExpDermatol 2005;14(September (9)):692–9.

[14] Takebe K, Oka Y, Radisky D, Tsuda H, Tochigui K, Koshida S, et al. Epimorphinacts to induce hair follicle anagen in C57BL/6 mice. FASEB J 2003;17(Novem-ber (14)):2037–47.

[15] Breitkreutz D, Stark HJ, Plein P, Baur M, Fusenig NE. Differential modulation ofepidermal keratinization in immortalized (HaCaT) and tumorigenic humanskin keratinocytes (HaCaT-ras) by retinoic acid and extracellular Ca2+. Differ-entiation 1993;54(October (3)):201–17.

[16] Schoop VM, Mirancea N, Fusenig NE. Epidermal organization and differentia-tion of HaCaT keratinocytes in organotypic coculture with human dermalfibroblasts. J Invest Dermatol 1999;112(March (3)):343–53.

[17] Hirai Y. Epimorphin as a morphogen: does a protein for intracellular vesiculartargeting act as an extracellular signaling molecule? Cell Biol Int2001;25(3):193–5.

[18] Hanley K, Jiang Y, He SS, Friedman M, Elias PM, Bikle DD, et al. Keratinocytedifferentiation is stimulated by activators of the nuclear hormone receptorPPARalpha. J Invest Dermatol 1998;110(April (4)):368–75.

[19] Cline PR, Rice RH. Modulation of involucrin and envelope competence inhuman keratinocytes by hydrocortisone, retinyl acetate, and growth arrest.Cancer Res 1983;43(July (7)):3203–7.

[20] Pillai S, Bikle DD. Lanthanum influx into cultured human keratinocytes: effecton calcium flux and terminal differentiation. J Cell Physiol 1992;151(June(3)):623–9.

[21] Takahashi H, Aoki N, Nakamura S, Asano K, Ishida-Yamamoto A,Iizuka H. Cornified cell envelope formation is distinct from apoptosisin epidermal keratinocytes. J Dermatol Sci 2000;23(August (3)):161–9.

[22] Mauro T, Bench G, Sidderas-Haddad E, Feingold K, Elias P, Cullander C. Acutebarrier perturbation abolishes the Ca2+ and K+ gradients in murine epidermis:quantitative measurement using PIXE. J Invest Dermatol 1998;111(December(6)):1198–201.

[23] Menon GK, Elias PM, Lee SH, Feingold KR. Localization of calcium in murineepidermis following disruption and repair of the permeability barrier. CellTissue Res 1992;270(December (3)):503–12.

[24] Radisky DC, Hirai Y, Bissell MJ. Delivering the message: epimorphin andmammary epithelial morphogenesis. Trends Cell Biol 2003;13(August(8)):426–34.

[25] Simian M, Hirai Y, Navre M, Werb Z, Lochter A, Bissell MJ. The interplay ofmatrix metalloproteinases, morphogens and growth factors is necessary forbranching of mammary epithelial cells. Development 2001;128(August(16)):3117–31.

Y. Okugawa et al. / Journal of Dermatological Science 59 (2010) 176–183 183

[26] Chintagari NR, Jin N, Wang P, Narasaraju TA, Chen J, Liu L. Effect of cholesteroldepletion on exocytosis of alveolar type II cells. Am J Respir Cell Mol Biol2006;34(June (6)):677–87.

[27] Abonyo BO, Gou D, Wang P, Narasaraju T, Wang Z, Liu L. Syntaxin 2 and SNAP-23 are required for regulated surfactant secretion. Biochemistry 2004;43(March (12)):3499–506.

[28] Candi E, Schmidt R, Melino G. The cornified envelope: a model of cell death inthe skin. Nat Rev Mol Cell Biol 2005;6(April (4)):328–40.

[29] Proksch E, Brandner JM, Jensen JM. The skin: an indispensable barrier. ExpDermatol 2008;17(December (12)):1063–72.

[30] Nemes Z, Steinert PM. Bricks and mortar of the epidermal barrier. Exp Mol Med1999;31(March (1)):5–19.

[31] Chen CS, Nelson CM, Khauv D, Bennett S, Radisky ES, Hirai Y, et al. Homologywith vesicle fusion mediator syntaxin-1a predicts determinants of epimor-phin/syntaxin-2 function in mammary epithelial morphogenesis. J Biol Chem2009;284(March (11)):6877–84.

[32] Bennett MK, Garcia-Arraras JE, Elferink LA, Peterson K, Fleming AM, HazukaCD, et al. The syntaxin family of vesicular transport receptors. Cell1993;74(September (5)):863–73.

[33] Pupe A, Moison R, De Haes P, van Henegouwen GB, Rhodes L, Degreef H, et al.Eicosapentaenoic acid, a n-3 polyunsaturated fatty acid differentially mod-ulates TNF-alpha, IL-1alpha, IL-6 and PGE2 expression in UVB-irradiatedhuman keratinocytes. J Invest Dermatol 2002;118(April (4)):692–8.

[34] Storey A, McArdle F, Friedmann PS, Jackson MJ, Rhodes LE. Eicosapentaenoicacid and docosahexaenoic acid reduce UVB- and TNF-alpha-induced IL-8secretion in keratinocytes and UVB-induced IL-8 in fibroblasts. J Invest Der-matol 2005;124(January (1)):248–55.

[35] Bascom JL, Fata JE, Hirai Y, Sternlicht MD, Bissell MJ. Epimorphinoverexpression in the mouse mammary gland promotes alveolar hyperplasiaand mammary adenocarcinoma. Cancer Res 2005;65(October (19)):8617–21.

[36] Hirai Y, Radisky D, Boudreau R, Simian M, Stevens ME, Oka Y, et al. Epimorphinmediates mammary luminal morphogenesis through control of C/EBPbeta. JCell Biol 2001;153(May (4)):785–94.

[37] Akiyama K, Akimaru S, Asano Y, Khalaj M, Kiyosu C, Masoudi AA, et al. A newENU-induced mutant mouse with defective spermatogenesis caused by anonsense mutation of the syntaxin 2/epimorphin (Stx2/Epim) gene. J ReprodDev 2008 Apr;54(2):122–8.

[38] Wang Y, Wang L, Iordanov H, Swietlicki EA, Zheng Q, Jiang S, et al.Epimorphin(�/�) mice have increased intestinal growth, decreased suscepti-bility to dextran sodium sulfate colitis, and impaired spermatogenesis. J ClinInvest 2006;116(June (6)):1535–46.

[39] Shaker A, Swietlicki EA, Wang L, Jiang S, Onal B, Bala S, et al. Epimorphindeletion protects mice from inflammation-induced colon carcinogenesis andalters stem cell niche myofibroblast secretion. J Clin Invest 2010;(June(1)):2081–93.