c 2016 British Association of Dermatologists · 2020. 6. 18. · SKALP is expressed by hyper-proliferative keratinocytes, such as in psoriasis, wound healing and cell culture conditions,

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McGovern, Jacqui, Meinert, Christoph, de Veer, Simon, Hollier, Brett,Parker, Tony, & Upton, Zee(2017)Attenuated kallikrein-related peptidase activity disrupts desquamation andleads to stratum corneum thickening in human skin equivalent models.British Journal of Dermatology, 176(1), pp. 145-158.

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This is the peer reviewed version of the following article: McGovern, J., Meinert, C., deVeer, S., Hollier, B., Parker, T. and Upton, Z. (2017), Attenuated kallikrein?related pepti-dase activity disrupts desquamation and leads to stratum corneum thickening in humanskin equivalent models. Br J Dermatol, 176: 145-158. doi:10.1111/bjd.14879, which hasbeen published in final form at https://doi.org/10.1111/bjd.14879. This article may be usedfor non-commercial purposes in accordance with Wiley Terms and Conditions for Use ofSelf-Archived Versions.

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This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/bjd.14879 This article is protected by copyright. All rights reserved.

Received Date : 16-Feb-2016

Revised Date : 08-Jun-2016

Accepted Date : 20-Jun-2016

Article type : Original Article

Attenuated kallikrein-related peptidase activity disrupts desquamation and leads to

stratum corneum thickening in human skin equivalent models

J.A. McGovern1,2, C. Meinert3, S.J. de Veer4, B.G. Hollier1, T.J. Parker1,2, and Z. Upton1,2, 5

1Tissue Repair and Regeneration Program, Institute of Health and Biomedical Innovation,

Queensland University of Technology, Kelvin Grove, Queensland, Australia

2School of Biomedical Sciences, Faculty of Health, Queensland University of Technology,

Kelvin Grove, Queensland, Australia

3Cartilage Regeneration Laboratory, Institute of Health and Biomedical Innovation,

Queensland University of Technology, Kelvin Grove, Queensland, Australia

4Molecular Simulation Group, Institute of Health and Biomedical Innovation, Queensland

University of Technology, Kelvin Grove, Queensland, Australia

5Institute of Medical Biology, Agency for Science, Technology and Research (A*STAR),

Biomedical Grove, Singapore

Running title: Attenuated KLK activity disrupts desquamation in HSEs

Manuscript word count (5069); Figures (5); Tables (1).

Key words: Desquamation, Kallikrein-related peptidase 5, Kallikrein-related peptidase 7,

Human Skin Equivalent; Stratum Corneum Thickening

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This article is protected by copyright. All rights reserved.

Corresponding author: Dr Jacqui McGovern, Institute of Health and Biomedical Innovation,

Queensland University of Technology, 60 Musk Avenue, Kelvin Grove, Queensland, 4059,

Australia. Phone: 617 31380028; Fax 617 31386030; E-mail: [email protected]

Funding source: This project was supported by an Australian Postgraduate Research Award

(JAM) and the Tissue Repair and Regeneration Program, Institute of Health and Biomedical

Innovation, Queensland University of Technology.

Conflicts of interest: None declared.

What’s already known about this topic?

• Corneocytes are sloughed from the skin surface in a process known as desquamation,

and this process is impaired in human skin equivalent (HSE) models

• The key enzymes involved in desquamation are kallikrein-related peptidase (KLK) 5

and KLK7, which cleave the extracellular cell-cell junction proteins corneodesmosin

(CDSN), desmocollin 1 (DSC1) and desmoglein 1 (DSG1)

• KLK5 and KLK7 are inhibited by lympho-epithelial Kazal-type-related inhibitor

(LEKTI) and secreted antileukoprotease (SKALP)

What does this study add?

• Altered desquamation is accompanied by aberrant localisation of cell-cell junction

proteins and protease inhibitors in human skin equivalent (HSE) models

• KLK7 protein is over-expressed, yet both KLK5 and KLK7 are not activated, and

overall epidermal proteolytic activity is lower in HSE models than native skin.

• SKALP and LEKTI proteins are present in the stratum corneum in HSEs, potentially

inhibiting KLK7 where it should be active. CDSN protein persists in the stratum

corneum, which suggests that it is not degraded

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Abbreviations

3D Three dimensional

BCA Bicinchoninic acid

CDSN Corneodesmosin

DAB 3,3-Diaminobenzidine

DED De-epidermised dermis

DMEM Dulbecco’s modified eagle medium

DSC1 Desmocollin 1

DSG1 Desmoglein 1

FCS Foetal calf serum

FGM Full Greens media

FITC Fluorescein isothiocyanate

HSE Human skin equivalent

IHC Immunohistochemistry

KLK Kallikrein-related peptidase

LEKTI Lympho-epithelial Kazal-type-related inhibitor

PCR Polymerase chain reaction

qRT-PCR Real time reverse transcriptase polymerase chain reaction

RPL32 60S ribosomal protein L32

SDS-PAGE Sodium dodecyl sulphate polyacrylamide gel electrophoresis

SKALP Skin-derived antileukoproteinase

SLPI Secreted leukocyte protease inhibitor

SPINK5 Serine protease inhibitor Kazal-type 5

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Summary

Background:

Epidermal homeostasis is maintained through the balance between keratinocyte

proliferation, differentiation and desquamation, however human skin equivalent (HSE)

models are known to excessively differentiate. In native tissue, proteases such as

kallikrein-related peptidase (KLK) 5, and KLK7 cleave the extracellular components of

corneodesmomes; proteins corneodesmosin (CDSN), desmocollin 1 (DSC1) and

desmoglein 1 (DSG1), loosening the cellular connections and enabling desquamation.

The actions of KLK7 are tightly controlled by protease inhibitors; skin-derived

antileukoproteinase (SKALP), and lympho-epithelial Kazal-type-related inhibitor

(LEKTI) which also inhibits KLK5, localising protease activity to the stratum corneum.

Objectives:

To investigate the mechanisms which inhibit the desquamation cascade in HSE models.

Methods:

Human skin tissue and HSE models were investigated using gene microarray, real-time

PCR, immunohistochemistry, and Western blot analysis to examine key components of

the desquamation pathway. To elucidate proteolytic activity in both HSEs and native skin,

in situ and gel zymography was performed.

Results:

Histological analysis indicated that HSE models form a well-organised epidermis, yet

develop an excessively thick and compact stratum corneum. Gene microarray analysis

revealed that the desquamation cascade was dysregulated in HSE models and this was

confirmed using real-time PCR and immunohistochemistry. Immunohistochemistry and

Western blot indicated overexpression of LEKTI and SKALP in HSEs. Although KLK7

was also highly expressed in HSEs, zymography indicated that protease activation and

activity was lower than in native skin.

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Conclusions:

These findings demonstrate that stratum corneum thickening is due to inhibited KLK5

and KLK7 activation and a subsequent lack of corneodesmosomes degradation in the

HSE model epidermis.

Introduction

Epidermal stratification commences with the migration of proliferative keratinocytes from the

basal layer, followed by differentiation to form the stratum spinosum and stratum

granulosum. This process culminates in the formation of the stratum corneum, a terminally

differentiated layer of cornified keratinocytes (corneocytes) which are gradually sloughed

during desquamation. A tightly controlled balance between keratinocyte proliferation and

corneocyte desquamation maintains epidermal homeostasis and a constant epidermal

thickness.1

Desquamation involves the degradation of corneodesmosomes; specialised desmosomes

located in the stratum corneum, the extracellular domain of which is comprised of

corneodesmosin (CDSN), desmocollin 1 (DSC1) and desmoglein 1 (DSG1).2,3 Progressive

corneodesmosome degradation loosens cell-to-cell adhesion, and allows the release of

corneocytes from the apical surface of the stratum corneum (Fig. 1). Proteolytic activity

conferred by trypsin-like kallikrein-related peptidase 5 (KLK5) and chymotrypsin-like KLK7

are essential for the epidermal desquamation process to occur.4 These enzymes act on the

three protein components (CDSN, DSC1 and DSG1) that provide the extracellular link in

corneodesmosomes.2,5,6

KLK5 and KLK7 are expressed by keratinocytes in the stratum granulosum and are secreted

to the intercellular space within lamellar granules.7,8 The activity of KLK5 and KLK7 is

confined to the stratum corneum through the concurrent production and secretion of

inhibitors: lymphoepithelial kazal-type related inhibitor (LEKTI), secreted leukocyte protease

inhibitor (SLPI; also called antileukoprotease), and skin-derived antileukoproteinase

(SKALP, also known as elafin).

LEKTI is produced as a multi-domain protein, which is cleaved into inhibitory fragments

before they are transported by lamellar granules into the extracellular space in the stratum

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granulosum.8,9 The complex between LEKTI fragments and KLK5 or KLK7 dissociates as

the pH changes from neutral (pH 6·8-7·5) in the stratum granulosum to acidic (pH 4·5-5·3) in

the stratum corneum.10 Once KLK5 and KLK7 disassociate from the inhibitory LEKTI

fragments, these proteases are able to degrade the corneodesmosomes and instigate the

desquamation process (Fig. 1).

SKALP is expressed by hyper-proliferative keratinocytes, such as in psoriasis, wound healing

and cell culture conditions, but is absent from normal, quiescent epidermis.11 It plays an

important role in vivo in protection from excessive cutaneous inflammation and extracellular

matrix proteolysis.12 The SKALP protein is secreted from lamellar granules by keratinocytes,

and is then cross-linked to cornified envelope proteins by transglutaminase.13,14

Human skin equivalent (HSE) models are a useful tool to study skin in the laboratory.

However, excessive thickening of the stratum corneum is a common occurrence in HSE

models, since they presumably lack an effective desquamation process.15,16 It has long been

known that HSEs produce SKALP,17 which is usually an indicator for the hyper-proliferative

state of the HSE epidermis. This study investigated the aberrant production of protease

inhibitors in HSEs and the potential disruption to the desquamation cascade in this model

system. We report herein that although KLK7 was over-expressed at the protein level

compared to native skin, activation of KLK5 and KLK7 was reduced in HSEs. In addition,

KLK5 and KLK7 proteolytic activity was further inhibited by the abundant and diffuse

epidermal presence of the protease inhibitors LEKTI and SKALP, leading to abrogation of

CDSN degradation and thickening of the stratum corneum in HSE models.

Materials and Methods

Antibodies – Polyclonal antibodies against CDSN (HPA044730) and DSC1 (HPA012891)

were purchased from Prestige Antibodies (Sigma-Aldrich, St. Louis, MO, USA). Polyclonal

anti-KLK7 (NBP1-31428), anti-LEKTI (NBP1-90509) and anti-SKALP (NBP1-85690) and

monoclonal anti-Cytokeratin 1 (NB100-2756) were purchased from Novus Biologicals

(Littleton, CO, USA). Polyclonal anti-Loricrin (ab24722) and monoclonal anti-Ki-67

(M7240) were purchased from Abcam (Cambridge, UK) and Dako (Glostrup, Denmark),

respectively.

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Skin Samples – Surgical discard skin was obtained from adults undergoing elective

abdominoplasty or mammoplasty surgery. The tissue was obtained with informed patient

consent, institutional (QUT HREC 1300000063) and hospital (UnitingCare Health; 2003/46)

ethical approval and handled in accordance with the Helsinki Declaration. After collection,

the skin was stored at room temperature in phosphate buffered saline supplemented with 400

IU mL-1 penicillin, 400 µg mL-1 streptomycin, and 1 µg mL-1 Amphotericin B (all Invitrogen,

Mulgrave, VIC, Australia) for 2-4 hours, until further processing in the laboratory. Separate

skin samples were utilised for the native skin immunohistochemistry (IHC), generation of

human skin equivalent (HSE) models and zymography experiments, with a minimum of two

skin donors per experiment.

Culture of primary keratinocytes – Primary human keratinocytes were isolated from native

skin and cultured according to Rheinwald and Green (1975).18 The culture media termed full

Green’s media (FGM) contained DMEM and Ham’s-F12 (both Invitrogen) at a 3:1 ratio (v/v)

supplemented with 10% foetal calf serum (FCS; Hyclone), 2 mmol L-1 L-Glutamine, 50 IU

mL-1 penicillin, 50 µg mL-1 streptomycin, 0·01% (v/v) non-essential amino acids, 10 ng mL-1

epidermal growth factor (EGF; all from Invitrogen), 0·2 µmol L-1 triiodothyronine, 180 µmol

L-1 adenine, 0·1 µg mL-1 cholera toxin, 0·4 µg mL-1 hydrocortisone, 5 µg mL-1 transferrin and

1 µg mL-1 insulin (all from Sigma-Aldrich).

Generation of the HSE models – The HSE models were generated as described previously.19-

21 Briefly, sterile stainless-steel rings (Aix Scientifics, Aachen, Germany) with an internal

diameter of 6·7 mm were placed on the papillary side of 1·4 cm × 1·4 cm de-epidermised

dermis (DED) pieces. Keratinocytes (2 × 104; passage 1) were seeded inside the rings and

incubated for 48 hours submerged in FGM at 5% CO2 and 37 °C. The rings were removed

and the HSEs were lifted to the air-liquid interface to allow for epidermal stratification. The

air-exposed HSEs were cultured in FGM for 0, 3, 5 and 9 days, respectively. Samples were

then fixed in 10% neutral buffered formalin and paraffin embedded for subsequent

histological and immunohistochemical analysis.

Whole human genome microarray analysis – Total epidermal RNA was isolated from HSE

models after 0, 3, 5 and 9 days culture, respectively, at the air-liquid interface using standard

RNA extraction protocols (TRIzol®, Invitrogen) and contaminating genomic DNA was

removed using DNase I (Invitrogen) following the manufacturer’s instructions. Due to the

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low yield, equivalent amounts of RNA were pooled from each of three biological replicate

HSEs to obtain a total of 1 µg of RNA necessary for microarray analysis. RNA sample

labelling, conversion to cRNA, biotin labelling and hybridisation to the Illumina whole

genome (HT-12 v3·0) BeadChip array (Illumina Inc., San Diego, CA, USA) was performed

at the Institute for Molecular Biosciences Microarray Facility (University of Queensland,

Brisbane, Australia). The BeadChip array was scanned using the Illumina Beadstation 500.

The resulting data was processed using Illumina Genome Analyser system software. Signal

intensity data was uploaded into GeneSpring GX 10·0 software (Agilent Technologies,

Mulgrave, VIC, Australia) for differential gene expression analysis, as we have previously

described.22

Gene expression analysis (qRT-PCR) – The epidermis was removed from native skin by

incubation with Dispase II (Invitrogen) for 30 minutes at 37 °C before collection into

TRIzol® (Invitrogen). Total RNA was collected from the central portion of the HSE models

using a 6 mm punch biopsy (Kai Medical, Tokyo, Japan) and TRIzol®. The RNA was

transcribed to cDNA using the SuperScript III® First-Strand Synthesis SuperMix (Invitrogen)

following DNase I (Invitrogen) treatment, all following the manufacturer’s instructions.

Primers for KLK7 and 60S ribosomal protein L32 (RPL32) were kindly donated by Dr

Daniela Loessner23 and Ms Lipsa Mohanty, respectively. All other oligonucleotide primers

for PCR were purchased from Invitrogen or Integrated DNA Technologies (Coralville, IA,

USA) and are outlined in Supplementary Table 1. The DNA was amplified with SYBR Green

PCR MasterMix (Invitrogen) on an ABI Prism 7500 PCR machine. The cycle threshold (Ct)

value of each gene was normalised to the housekeeping gene RPL32 using the delta Ct

method (2-ΔCt).

Immunohistochemistry – Formalin-fixed, paraffin-embedded sections (5 µm) of both human

skin and HSE models were processed for immunohistochemistry (IHC). Heat-induced

epitope retrieval was performed in a Decloaking Chamber™ (Biocare Medical) and IHC was

performed using the MACH 4™ Universal Detection System (Biocare Medical) as described

previously.24 Primary antibodies against keratin 1 (1:500), loricrin (1:1000), Ki-67 (1:100),

KLK7 (1:400), DSC1 (1:1000), CDSN (1:1000), LEKTI (1:500) and SKALP (1:500) were

diluted in Da Vinci Green Diluent™ (Biocare Medical) and applied to the sections for 60

minutes at 37 °C or overnight at 4 °C. Positive immunoreactivity was visualised using 3,3-

diaminobenzidine (DAB, Biocare Medical), then counterstained with Hematoxylin-G1 (HD

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Scientific) before images were captured on an Olympus BX41 microscope with a mounted

digital camera (MicroPublisher 3·3 RTV, Olympus, Q-Imaging).

In Situ Zymography – Native human skin samples and HSEs were submerged in Tissue-Tek®

O.C.T.™ compound (Sakura Finetek, AJ, The Netherlands) and snap frozen in liquid

nitrogen. Fresh, frozen sections (5 µm) were captured on Poly-L-Lysine slides (Menzel-

Gläser, Braunschweig, Germany) and washed with distilled water containing 2% (v/v)

Tween-20 prior to adding 0·5 µg mL-1 quenched FITC-casein (Sigma-Aldrich) and 0·5%

agarose in reaction buffer (50 mmol L-1 Tris-HCl, pH 8·0).25 The sections were cover-slipped

(Menzel-Gläser), incubated for 72 hours at 37 °C and visualised using a Leica SP5 Laser

Scanning Microscope (Leica Microsystems, North Ryde, NSW, Australia).

Casein zymography – To examine proteolytic activity, the epidermis of native skin and HSEs

were isolated and finely minced in 1 mol L-1 acetic acid as described by Furio et al. (2014).26

Following extraction at 4 °C for 24 – 48 hours, the samples were clarified by centrifugation at

13,000 × g for 20 minutes before the supernatant was frozen at -80 °C, freeze-dried and

resuspended in distilled water. Protein content was determined using the bicinchoninic acid

(BCA) protein assay reagent (Pierce, Thermo Fisher Scientific, Rockford, IL, USA). A total

of 10 µg protein was loaded onto casein/acrylamide co-polymerised gels (12% acrylamide,

0·1% α-casein; Sigma-Aldrich). Following SDS-PAGE, the gels were washed twice for 30

minutes with 2·5% Triton X-100 and incubated for 72 hours in reaction buffer (50 mmol L-1

Tris-HCl, pH 8·0) at 37 °C. The gels were stained with 1% Coomassie Brilliant blue G250

and photographed.

KLK7 Western Immunoblotting – Immunodetection of KLK7, LEKTI, and SKALP was

performed using the same epidermal extracts as described above. Briefly, 15%

polyacrylamide gels were equally loaded with 10.µg of protein and subjected to SDS-PAGE.

The separated proteins were transferred to nitrocellulose membranes, blocked with 5% skim

milk powder in Tris buffered saline containing 0.1% Tween 20 (TBS-T), and incubated for 1

hour at room temperature with anti-KLK7, or overnight at 4 °C with anti-LEKTI and anti-

SKALP (all 1:1000 dilution). Enhanced chemiluminescent detection of KLK7, LEKTI, and

SKALP was performed using a HRP-conjugated anti-rabbit secondary antibody (1:200; Cell

Signaling Technology, Danvers, MA, USA). Gels stained with Coomassie blue were used as

total protein loading control.

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Statistics – The qRT-PCR data are presented as box plots that show percentiles 0, 25, 50

(median), 75 and 100. All other data are presented as mean ± standard deviation. Statistical

analyses were performed using SPSS software (version 21, IBM Corporation, USA).

Differences between groups were determined using analysis of variance (ANOVA) and

Tukey’s or Dunnet’s T3 post-hoc tests as appropriate, with significance accepted where

P

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in the HSE model were highly proliferative until day 3, after which keratinocyte proliferation

steadily declined, yet maintained a higher proliferative index than was observed in native

skin.

Keratinocyte proliferation and subsequent differentiation are important processes which

ultimately lead to the formation of the stratum corneum. Keratin 1 (K1) is expressed in

keratinocytes as differentiation commences and is one of the first markers to appear during

epidermogenesis in HSE models.21,28,29 In both native skin and HSE sections K1 was present

in all suprabasal epidermal layers, excluding the stratum corneum (Fig. 2c). The percentage

of the HSE epidermis which expressed K1 increased significantly (P < 0·0001) between days

3 and 5; however, this was significantly lower (P < 0·01) than observed in native skin (Fig.

2g). Together, these data revealed that a smaller proportion of the epidermis in the HSEs had

entered into post-mitotic differentiation than in native skin.

Loricrin is a terminal differentiation marker expressed by keratinocytes committed to forming

the stratum corneum30,31 and was identified predominantly in the stratum granulosum in

native skin (Fig. 2d). In sections from the HSE model, loricrin appeared diffusely distributed

throughout the upper suprabasal epidermal layers (Fig. 2d), but was absent at day 0 (Fig. 2d).

The percentage of epidermal loricrin immunoreactivity significantly increased between days

0 and 3 (P < 0·0001), and days 3 and 5 (P < 0·0001), with immunoreactivity at days 5 and 9

being significantly higher (both P < 0·0001) than observed in native skin (Fig. 2h). Together,

these results suggested that the HSE supported enhanced proliferation and terminal

differentiation, which led to the formation of a stratum corneum to a greater extent than found

in native skin.

Microarray screening suggests that desquamation was altered in developing HSE

models

Gene microarray analysis was performed to screen temporal changes in the gene expression

profile as the HSE epidermis matured. Following acquisition, pre-processing and

normalisation of the microarray data in GeneSpring, a list of over 2,600 genes that were

differentially regulated compared to day 0 was generated. Expression of genes such as FLG,

EVPL and PPL, which encode the epidermal structural proteins filaggrin, envoplakin and

periplakin, respectively, increased as the epidermis developed (Table 1). Furthermore, genes

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encoding transglutaminases; TGM1, TGM3 and TGM5, associated with cornified envelope

cross-linking, were up-regulated during epidermal differentiation. Interestingly,

corneodesmosome genes CDSN, DSC1 and DSG1 were up-regulated as the epidermis

matured. In particular, DSC1 exhibited a greater than 29-fold up-regulation after 9 days

compared to day 0. The proteases KLK5 and KLK7 demonstrated a slight down-regulation

trend over time, whereas expression of desquamation inhibitor SPINK5 remained relatively

stable throughout the course of the experiment (Table 1). Together these data suggested that

the desquamation cascade was altered in the HSE epidermis compared to native skin and led

us to further investigate these genes using quantitative real time PCR (qRT-PCR).

HSE models exhibited increased corneodesmosomal gene expression

Based on the results of the microarray analysis, a selection of desquamation-associated genes

was investigated using qRT-PCR and compared to expression levels in native skin (Fig. 3).

KLK5 and KLK7 were expressed at significantly higher levels in the HSE model compared to

native skin (all time points compared to native skin, P < 0·05, except for KLK7, day 0

compared to native skin was not significant; Fig. 3a, b), however, no significant temporal

differences in either KLK5 or KLK7 expression were observed over the 9 days of culture in

the HSE model. Expression of SPINK5, which encodes the protein LEKTI, was significantly

higher after 3 days culture at the air-liquid interface when compared to native skin or the day

0 HSE model (P < 0·05; Fig. 3c). The expression of PI3, the gene encoding SKALP, was

seemingly lower in native skin compared to HSE models, but the difference was not

statistically significant (Fig. 3d). Expression of CDSN was significantly higher in the HSE

model when compared to native skin at all time points (P < 0·05) except for day 0 (Fig. 3e).

Furthermore, CDSN gene expression levels in HSEs at days 5 and 9 were significantly higher

compared to day 0 (P < 0·05, Fig. 3e). While DSC1 expression was similar between the HSE

and native skin at day 0 and day 3, transcript levels were approximately 50-fold higher than

native skin at days 5 and 9 (P < 0·01; Fig. 3f), respectively. Compared to native skin, DSG1

gene expression was significantly higher in HSEs at day 3 and day 5 (P < 0·05), but not day 9

(Fig. 3g). Taken together, the microarray and qRT-PCR data suggested the general trend that,

at the mRNA level, corneodesmosomal components and KLK5 and KLK7 were expressed at

a higher level in HSE models compared to native skin, whereas expression of protease

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inhibitors SPINK5 and PI3 was not significantly different between HSE models and native

skin at most investigated time points.

Desquamation-related proteins exhibit aberrant expression and localisation in HSE

models

The expression and localisation of KLK7, LEKTI, SKALP, CDSN and DSC1 protein was

examined using IHC (Fig. 4). In native skin, KLK7 immunoreactivity was localised to the

apical stratum granulosum and the stratum corneum (Fig. 4a). While KLK7 was undetectable

in the day 0 HSE, it was abundant throughout the stratum spinosum and stratum granulosum

and was localised to the peri-cellular space from days 3 to 9 (Fig. 4a). Similarly, LEKTI was

present in the upper stratum spinosum and stratum granulosum, but was undetectable in the

stratum corneum of native human skin and HSE models (Fig. 4b). SKALP immunoreactivity

was observed in all supra-basal epidermal layers, and displayed discontinuous staining

throughout the stratum corneum in HSE models, but was undetectable in native skin (Fig.

4c). In native skin, CDSN was present through the upper stratum granulosum and at the

interconnections between the basket weave structures of the stratum corneum (Fig. 4d). In

HSE models, CDSN expression was located in the stratum granulosum and stratum corneum

from days 3 to 9 (Fig. 4d). DSC1 was present in the upper stratum granulosum, but absent in

the stratum spinosum and stratum basale in native skin (Fig. 4e). In contrast DSC1 was

undetectable using IHC at days 0 and 3, but was present at the cellular periphery in all

suprabasal layers in the HSE model at day 5 and day 9 (Fig. 4e). Furthermore, DSC1

immunoreactivity was noticeably reduced in the upper stratum granulosum of the day 5 and

day 9 HSE. Together these data confirmed that the expression and localisation of KLK7,

LEKTI, SKALP, CDSN and DSC1 was aberrant in HSE models from 5 days onwards

compared to native skin.

KLK7 activity is attenuated in the HSE model compared to native skin

To address whether the enhanced KLK7 protein expression observed in HSE models (Fig. 4)

translated to observable changes in proteolytic activity, in situ zymography was performed

(Fig. 5a). Fresh, frozen skin sections were overlayed with quenched FITC-labelled casein,

which releases a fluorescent product upon proteolytic cleavage.25 This fluorescent product

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allowed for the localisation of proteolytic activity within the tissue. Native skin had a high

level of caseinolytic activity throughout the entirety of the epidermis, with the greatest level

of activity present in the upper stratum granulosum (Fig. 5a). Caseinolytic activity was also

detectable throughout the epidermis in the day 0, 3 and 5 HSEs, but was primarily localised

to the suprabasal epidermal layers in the day 9 HSE. At each time point investigated,

caseinolytic activity was much weaker in the HSE models than was observed in native human

skin (Fig. 5a), which indicated that proteolytic activity in the HSE model was attenuated.

In order to further investigate proteolytic activity in the HSE model and native skin,

epidermal proteins were extracted and analysed by zymography with casein as the in-gel

substrate (Fig. 5b). Several bands were visible that corresponded to KLK5 (31 kDa), and

KLK7 and/or KLK14, which co-migrate (20-23 kDa), according to previous studies.32,33 The

highest level of overall proteolytic activity was observed in the day 0 HSE model and native

skin (Fig. 5b). While KLK5 activity was more pronounced in native skin than in the HSE

model, it was unclear whether the enzymatic activity observed at 20-23 kDa was attributable

to KLK7 or KLK14 in these extracts (Fig. 5b). To confirm the presence of KLK7 protein in

the epidermis of the HSE and native skin, epidermal extracts were probed for KLK7 using

Western immunoblotting (Fig. 5c). Bands correlating to KLK7 were observed in HSE models

which had been cultured at the air-liquid interface for 3, 5 and 9 days, respectively, in

addition to native skin. In accordance with immunohistochemistry results, KLK7 (20-23 kDa)

was not detected in the extracts from the day 0 HSE model epidermis (Fig. 5c). Higher

molecular weight bands ranging between 45 kDa to 70 kDa present in extracts from the HSE

models at days 3, 5 and 9 suggested that a portion of the epidermal KLK7 may have been

covalently bound to an inhibitor protein (Fig. 5c). Together, this suggests that protease

activity was decreased and that KLK7 was not active in HSE models, as compared to native

human skin.

Diffuse epidermal localisation of protease inhibitors LEKTI and SKALP was observed using

IHC (Fig. 4b, c). To confirm this, Western immunoblotting was performed on HSE and

native skin epidermal extracts (Fig. 5d, e). SKALP protein was identified as a

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S1). Furthermore, LEKTI was detected as multiple bands in the protein extracts, including the

full-length LEKTI precursor proteins (145 kDa and 125 kDa) in the day 5 and 9 HSE and in

native skin, in addition to the previously reported 42 kDa, 65 kDa and 68 kDa biologically-

active inhibitory LEKTI polypeptides which were highly expressed in the day 9 HSE (Fig.

5e).34 Lower molecular weight LEKTI fragments were also detected.10 These results suggest

that LEKTI is present in both HSE and native skin samples predominantly in the form of

inhibitory LEKTI polypeptide fragments (Fig. 5e). Together, these data suggests that there is

an over-abundance of LEKTI and particularly SKALP protease inhibitors in the HSE model

as compared to native skin, which may contribute to attenuated KLK7 activity in HSE

models.

Discussion

HSEs are commonly employed as pre-clinical models to study, for example, the mechanistic

and biological aspects of wound healing,35 skin sensitisers,36 and the role of ultraviolet (UV)

light in DNA damage.37 However, it has previously been reported that HSE models develop

an overly thick stratum corneum,15,16 which has important implications for skin research. For

example, increased thickness of the stratum corneum can affect the penetration of UV light

and consequently its biological effects.38 Therefore in this study we investigated the

mechanisms preventing desquamation, which we anticipated underlined the excessive

thickening of the stratum corneum observed in HSE models.

In accordance with previous reports,15,16 we observed stratum corneum thickening in the HSE

compared to normal skin (Fig. 2a, e). In addition, Ki-67 and K1 data (Fig. 2b, c, f, g)

indicated that a greater proportion of the epidermis was mitotically active in the HSE than

was observed in native skin. Loricrin immunoreactivity revealed that a higher proportion of

keratinocytes in the epidermis had committed to terminal differentiation in HSEs compared to

native skin (Fig. 2d, h), leading to a thicker stratum corneum as observed with H&E staining

(Fig. 2a). Acanthosis (diffuse epidermal thickening) and hyperkeratosis (thickening of the

stratum corneum) are characteristics also present in epidermal hyper-proliferative diseases

such as psoriasis, squamous cell carcinoma, actinic keratosis and keratoacanthoma.39 A

histological feature of these disorders is the detection of SKALP,39 a protein which confers

protection from epidermal damage by infiltrating neutrophils.40 SKALP is typically

undetectable in adult inter-follicular epidermis, but is induced following injury and in

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regenerating skin.12,41 The studies reported herein indicated that both SKALP gene expression

(Fig. 3d) and SKALP protein (Fig. 4c, Fig. 5d) were located throughout all suprabasal layers

of the epidermis in the HSE. This data indicated that the HSEs were in an active, hyper-

proliferative state and suggested that homeostasis has been disrupted compared to native skin.

Cleavage of corneodesmosomes leads to the shedding of corneocytes and thus

desquamation.2,5,6 Previously, HSE models have been reported to contain a higher density of

corneodesmosomes than native skin, contributing to the formation of a dense and compact

stratum corneum.15 In light of this, we used an immunohistochemistry (IHC) approach to

investigate the localisation of CDSN and DSC1, which together with DSG1 comprise the

extracellular portion of the corneodesmosome.2,3 In the epidermis KLK5 degrades CDSN,

DSC1 and DSG1, whereas KLK7 can degrade CDSN and DSC1, but not DSG1.6 However, it

is unclear whether KLK5 acts indirectly via the activation pro-KLK7, to degrade CDSN.6 We

observed a higher expression of CDSN in the stratum granulosum and stratum corneum of the

HSE model, compared to native human skin (Fig. 4d). Furthermore, in accordance with

Wang et al. (2014), DSC1 was present in the stratum spinosum and lower stratum

granulosum in the HSE model,42 but was predominantly localised in the upper stratum

granulosum in native skin (Fig. 4e). Our results established that CDSN and DSC1 expression

and localisation differ between the HSE and native skin and led us to further investigate these

extracellular corneodesmosomal components. Previous studies have described decreased

CDSN immunoreactivity as an indicator of corneodesmosome degradation in epidermal

extracts.6,43 Since we reported a persistence of CDSN immunoreactivity throughout the

stratum corneum of HSEs, but not native skin (Fig. 4d), we suggest that it may have been

degraded in the stratum corneum of native skin, but not in the HSE model (Fig. 4d). While

the DSC1 gene was highly expressed (Fig. 3f), decreased DSC1 immunoreactivity in the

upper stratum granulosum in HSE models suggests that it may have been degraded (Fig. 4e).

Together, this suggests that a lack of CDSN degradation may have prevented corneocyte

sloughing and led to excessive stratum corneum accumulation in the HSE model.

Attenuated CDSN degradation indicates that desquamation is not functioning effectively in

our model system. The two major epidermal proteases responsible for desquamation in

human skin are KLK5 and KLK7,4-6 both of which were more highly expressed in the HSE

than native skin at the gene level (Fig. 3a, b) and with KLK7 at the protein level (Fig. 4a, Fig.

5c). However, it is the actual activity of KLKs that are an important consideration. For

example, Komatsu et al. (2007) reported that although KLK7 protein levels were

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significantly higher in psoriatic lesions compared to non-lesional and normal skin,

chymotrypsin-like activity was similar between all three tissues.44 Since KLK7 is currently

thought to represent the majority of chymotryptic activity in the skin,45 it is possible that

KLK7 activity was unchanged in psoriasis compared to unaffected and normal skin, despite

differences in overall KLK7 protein expression. The authors instead proposed that the results

could be explained by LEKTI inhibition of excess KLK7 activity.44 In view of this, we used

casein zymography to establish that caseinolytic activity was noticeably lower in HSEs

compared to native skin (Fig. 5a, b). Furthermore, comparison of casein gel zymography and

Western blot results suggested that KLK7 may not be active in HSE models, and that the

proteolytic activity observed in the day 0 HSE was possibly due to the activity of KLK14,

since KLK7 was not detected by Western immunoblotting at this time point (Fig. 5b, c).

Interestingly, the more diffusely distributed proteolytic activity observed throughout all

suprabasal layers in both the HSE and native skin (Fig. 5a), did not correlate with the IHC-

based detection and localisation of KLK7 in native skin (Fig. 4a). This further suggests that

other proteases, such as matriptase,46 or KLK14,33 which also may have been detected with

the in situ zymography used, may contribute to desquamation in human skin. The proteolytic

activity may also correspond to KLK8, which was detected in the gene microarray analysis

(Table 1). However, the functional role of KLK8 in desquamation is less well known.43

The presence of known desquamation inhibitors in the HSE model may have an influence on

proteolytic activity and desquamation in vitro. While we found PI3 gene expression in both

HSE models and native skin (Fig. 3d), the encoded protease inhibitor protein SKALP was

only detectable in the HSE models using IHC (Fig. 4c), but low levels were also detected in

one of three native skin samples using Western blot analysis (Fig. 5d). Furthermore, Western

blot analysis revealed that SKALP protein was highly over-expressed in the HSE model

compared to native skin, and that this over-expression increased over time (Fig. 5d,f). In

addition, secreted leukocyte protease inhibitor (SLPI) is expressed in normal skin, and similar

to SKALP, is up-regulated in psoriatic lesions and atopic dermatitis.47 Both SLPI and SKALP

inhibit KLK7 activity, with SLPI being more potent than SKALP,48 which may have

contributed to attenuated KLK7 activity in this model (Fig. 5b). Furthermore, SLPI and

SKALP appear to specifically inhibit KLK7 activity, and not the activity of any other KLK

found in human epidermis.4 However, SLPI expression was not investigated in our study, but

would be an interesting target to investigate in future studies. Moreover, Western blot

analysis of KLK7 in epidermal extracts identified high molecular weight bands ranging from

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approximately 45 kDa to 70 kDa in HSE models at days 3, 5 and 9 (Fig. 5a). This suggests a

possible covalent association between KLK7 and an inhibitor. Previously, in normal

epidermis KLK7 was reported to co-localise with serine protease inhibitor A12 (also known

as vaspin), but this co-localisation was reduced in non-lesional and absent in psoriatic skin

sections.49 Furthermore, vaspin was found to specifically inhibit epidermal KLK7, but not

KLK5.50 The interactions between vaspin and KLK14, however, have not yet been

investigated. Together, this suggests that KLK7 activity may be suppressed in HSE models

by additional protease inhibitors such as vaspin, thereby contributing to attenuated

desquamation in HSE models.

Western blot analysis revealed that LEKTI was present predominantly as inhibitory

polypeptides,10,34 and was highly expressed in both the HSE model and in native skin (Fig.

5e,f). Such high expression of LEKTI suggests that the attenuated KLK7 activity we have

observed in the HSE model (Fig. 5b) may predominantly be attributed to inhibition by

SKALP, rather than LEKTI. Interestingly, LEKTI accumulation may have occurred in HSE

models due to a lack of KLK7-mediated degradation. Furio and Hovnian (2011) have

previously suggested that LEKTI is a more potent inhibitor of KLK5 and KLK14, than

KLK7, because KLK7 is able to degrade LEKTI.51 The absence of LEKTI can have

destructive consequences on the skin; Netherton syndrome is characterised by mutations in

the SPINK5 gene, resulting in the absence of functional LEKTI. The uninhibited KLK5, 7

and 14 protease activity causes excessive corneodesmosome degradation and subsequent

detachment of the stratum corneum from the underlying epidermis and loss of barrier

function.52,53 Therefore the inhibitory effect of LEKTI on KLK7 may be attributed to the

ability of LEKTI to inhibit KLK5, thereby attenuating activation of pro-KLK7 by KLK5.51

Furthermore, the pH gradient present throughout the epidermis plays an important role in

KLK5 and KLK7 activity. Inhibition of KLK activity via LEKTI fragments is pH-dependent,

with the inhibitory function of LEKTI only efficient at neutral pH.10 We did not investigate

the epidermal pH gradient in our HSE models as its presence and similarity to native skin has

been reported elsewhere.54,55 In contrast with these reports, a recent study on epidermal-only

models, which lack a dermal substrate, suggests that the pH in these constructs was neutral

throughout the entirety of the epidermis.56 It is possible that interactions between the

epidermis and the underlying dermal component, which was lacking in the study by Sun et

al.56, is a crucial regulator of epidermal pH gradient formation. Therefore in future studies it

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may be important to consider the recapitulation of the epidermal pH gradient in HSE models

to ensure efficient desquamation and epidermal homeostasis.

Previously, SKALP was reported to be induced by FCS and EGF present in HSE culture

media.11 The inclusion of fibroblasts in HSE culture systems has also been proposed to assist

in normalising SKALP expression,33 although this has been contested.32 Fibroblasts are a key

component of native skin and assist in regulation of epidermal morphogenesis and

enhancement of basement membrane formation through cross-talk with epidermal

keratinocytes. The result of this cross-talk is a stratum corneum which is less compact and

maintains the normal basket weave structure. 57-59 A dense and compact stratum corneum and

a lack of desquamation in HSEs has long been recognised as a limitation of HSE model

systems. In this study we report that HSE models are characterised by hyper-proliferation and

disproportionate terminal differentiation resulting in the development of an excessively thick

and compact stratum corneum compared to native human skin. In the HSE model system we

studied here, stratum corneum thickening seems to be attributed to: a) higher

corneodesmosome frequency (particularly CDSN) within the epidermis; b) increased

expression of the protease inhibitors, in particular SKALP, but also LEKTI and potentially

other unidentified inhibitors such as vaspin; and c) reduced activation and subsequent activity

of KLK7. In order to generate HSE models with physiological expression, localisation and

activity of desquamation-associated proteins, restoration of epidermal homeostasis is

required.

Acknowledgements

We wish to acknowledge Dr A. Kane and his patients, who donated their surgical discards for

this research. Furthermore, we would like to thank Ms Cindy Philipp for assistance with

discard tissue collection and preparation, and the staff at the Brisbane Private Hospital and St

Andrews War Memorial Hospital (both located in Brisbane, Queensland, Australia). We

would also like to thank Dr Katia Nones and Dr Daniel Haustead for their assistance with the

gene microarray studies, and Dr Daniela Loessner and Ms Lipsa Mohanty (both from the

Institute of Health and Biomedical Innovation, Queensland University of Technology) for

donation of the KLK7 and RPL32 primers, respectively.

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49 Schultz S, Saalbach A, Heiker JT et al. Proteolytic activation of prochemerin by kallikrein 7 breaks an ionic linkage and results in C-terminal rearrangement. The Biochemical journal 2013; 452: 271-80.

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Table 1. Summary of terminal differentiation and desquamation gene expression from

gene microarray data in the developing human skin equivalent model

Gene symbol Encoded protein Relative fold change compared to Day 0

Day 3

Day 5

Day 9 Epidermal Structural Proteins

FLG Filaggrin 2·06 3·53 3·77 FLG2 Filaggrin 2 3·87 6·87 10·32 HRNR Hornerin 2·95 3·37 10·85 RPTN Repetin 7·01 10·97 10·99 EVPL Envoplakin 1·49 2·47 2·47 PPL Periplakin 1·71 2·60 2·92

Cornification Enzymes TGM1 Transglutaminase 1 1·40 1·98 2·31 TGM3 Transglutaminase 3 2·33 7·27 13·80 TGM5 Transglutaminase 5 1·97a 2·55a 3·59a

1·79b 2·49b 4·06b Corneodesmosomes

CDSN Corneodesmosin 1·70 3·79 5·18 DSC1 Desmocollin 1 4·95 17·87 29·54 DSG1 Desmoglein 1 2·80 3·73 4·71

Desquamation Enzymes and Inhibitors KLK5 Kallikrein-related peptidase 5 2·16 1·83 1·67 KLK7 Kallikrein-related peptidase 7 1·86 1·67 1·53 KLK8 Kallikrein-related peptidase 8 1·47 1·96 2·04 CTSD Cathepsin D 1·64 2·56 3·06 CST6 Cystatin E/M 1·62 1·79 1·55

SPINK5 Lympho-epithelial Kazal-type-related inhibitor

3·62 4·10 3·96

SPINK6 Serine peptidase inhibitor, Kazal type 6

-7·89 -7·90 -4·12

aTGM5 transcript variant 1 bTGM5 transcript variant 2

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Documents

c 2016 British Association of Dermatologists · 2020. 6. 18. · SKALP is expressed by hyper-proliferative keratinocytes, such as in psoriasis, wound healing and cell culture conditions,