UMEÅ UNIVERSITY MEDICAL DISSERTATIONS New Series No 1174 ISSN 0346-6612 ISBN 978-91-7264-555-4 Department of Public Health and Clinical Medicine, Dermatology and Venereology, Umeå University, Umeå, Sweden

Kallikrein-related Peptidases in Human -Studies on Activity, Regulation, and Function

Kristina Stefansson

Umeå 2008

Copyright © Kristina Stefansson New Series No 1174 ISSN 0346-6612 ISBN 978-91-7264-555-4 Printed by Print & Media, Umeå University, 2008:2004585

To Mikael and Edwin

TABLE OF CONTENTS

ABSTRACT 3 ORIGINAL ARTICLES 5 ABBREVIATIONS 6 POPULÄRVETENSKAPLIG SAMMANFATTNING 7 INTRODUCTION 9 The 9 Evolutionary Aspects of the Skin 9 Basal Structure of the Skin 9 Epidermal Turnover and Desquamation 12 The Corneodesmosome 13 The pH Gradient 13 Skin Immunology and Inflammation 14 14 Interleukin -1 15 -related Peptidases (KLKs) 15 Nomenclature 15 Genomic Organization 16 Structure 17 Substrate Specificity 17 KLK Function in Various Organs 18 Kallikrein-related Peptidases in Skin Physiology 20 KLKs in Desquamation 20 KLKs in Skin Immunology and Inflammation 21 Inhibitors of Kallikrein-related Peptidase Activity 22 Alpha-1 Antitrypsin 23 Skin-derived Antileukoproteinase (SKALP) 23 Secretory Leukocyte Protease Inhibitor (SLPI) 24 Lympho-epithelial Kazal-type Inhibitor (LEKTI) 24 Proteinase-activated Receptors (PARs) 26 PAR-2 Function in Various Organs 27 PAR-2 in Skin 27 Skin Diseases 29 29 30 Rosacea 31

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Ichthyosis 32 33 Short Summary 35 AIMS 36 MATERIALS AND METHODS 37 Protein Extraction 37 Extracts of Plantar Stratum Corneum 37 Tape Stripping 38 Recombinant 38 Polymerase Chain Reaction (PCR) 40 Analytical Methods 42 Electrophoresis and Immunoblotting 42 Zymography 42 Chromogenic Peptide Substrates 42 Immunohistochemistry 43 Tissues 43 Staining Procedures 44 Surface Plasmon Resonance (SPR) 45 Immunofluorescence 45 Intracellular [Ca2+] Measurements 47 RESULTS AND DISCUSSION 48 Paper I 48 Paper II 50 Paper III 53 Paper IV 55 Paper V 57 GENERAL DISCUSSION AND FUTURE PERSPECTIVES 59 CONCLUSIONS 61 TACK / ACKNOWLEGEMENTS 62 REFERENCES 65

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ABSTRACT

Introduction. The outermost layer of the epidermis, the stratum corneum (SC), plays a fundamental role in our defense against microorganisms, chemicals, and dehydration. The SC is composed of tightly packed keratinized skin cells, . For a functioning skin it is essential that corneocytes are constantly shed (desquamated). Kallikrein-related peptidase (KLK) 5 and KLK7 may be important in the desquamation process through degradation of desmosomal proteins. Severe hereditary diseases, where inhibition of KLK5 and/or KLK7 is missing, points to the importance of regulation of protease activity. KLKs may be regulated in various ways: tissue expression, activation of proforms, specific inhibitors, and physico- chemical properties like pH. Besides their involvement in desquamation, KLKs may also be important in immune defense and inflammation by processing of mediators and via activation of proteinase-activated receptors (PARs).

Aims. 1. To identify and characterize previously unknown proteases in the SC. 2. To further characterize KLK5 and KLK7 with special focus on activation mechanisms. 3. To identify new inhibitors of KLKs in human SC. 4. To further characterize KLKs regarding effects of various inhibitors and substrates. 5. To study possible functions of KLKs in inflammation, in particular via activation of PAR-2.

Methods. Plantar SC was used as a source for purification of proteins. Recombinant proteins were produced in different expression systems (insect cells, yeast cells, and bacteria). Different activity assays and kinetic studies were performed. Tissue expression was studied by immunohistochemistry, immunoblot and PCR. PAR-2 activation was studied by measurement of intracellular [Ca2+] and immunofluorescense in KNRK-PAR2 cells.

Results. Active KLK14 was purified from extracts of plantar SC. KLK14 showed a superior catalytic efficiency as compared to KLK5 when measuring -like activity. This indicated that KLK14, despite being present in low amounts in skin, may have great relevance for skin physiology. Among tested only KLK5 showed autocatalytic activity and is so far the only found in SC that can activate proKLK7. KLK5 could also activate proKLK14. This together with studies

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of pH dependence on activation placed KLK5 as a possible key activating enzyme in a proposed proteolytic cascade in the SC. In plantar SC extracts we have also identified the novel Kazal-type inhibitor 9 (SPINK9). Our results indicate that SPINK9 is preferentially expressed in palmo-plantar skin and specific for KLK5. Differences found regarding substrate specificity and inhibition profile can be useful in evaluating the contribution of individual KLKs to the proteolytic activity in crude SC extracts. One interesting finding was that KLK8, present at high protein levels in the epidermis, could not be inhibited by any protease inhibitor found in the extracts. PAR-2 activation studies showed that KLK5 and 14 but neither KLK7 nor 8 can activate PAR-2. Immunohistochemistry preferentially detected KLK14 in intraepidermal parts of the sweat ducts and in dermal sweat glands but we could also show coexpression of KLK14 and PAR-2 in the SC and stratum granulosum of the epidermis in inflammatory skin disorders. To summarize, KLK involvement in desquamation may be dependent on a proteolytic activation cascade regulated by an intrinsic pH gradient and specific inhibitors present in SC. Another possible function of KLKs is as mediators of inflammation through activation of PAR-2.

Keywords: Desquamation, epidermis, stratum corneum, KLK, kallikrein- related peptidase, serine protease inhibitors

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ORIGINAL ARTICLES

This thesis is based on the following articles referred to in the text by their Roman numerals.

I. Stefansson K, Brattsand M, Ny A, Glas B, and Egelrud T. Kallikrein-related Peptidase 14 may be a Major Contributor to Trypsin-like Proteolytic Activity in Human Stratum Corneum. Biol. Chem. 387:761-768, 2006

II. Brattsand M, Stefansson K, Lundh C, Haasum Y, and Egelrud T. A Proteolytic Cascade of in the Stratum Corneum. J. Invest. Dermatol. 124:198-203, 2005

III. Brattsand M, Stefansson K, Hubiche T, Nilsson SK, and Egelrud T. SPINK9: A Selective, Skin-specific Kazal Type Serine Protease Inhibitor. Submitted, 2008

IV. Stefansson K, Hubiche T, Brattsand M, and Egelrud T. Serine Protease Activity and Serine Protease Inhibitors in Plantar Stratum Corneum Extracts. Manuscript, 2008

V. Stefansson K, Brattsand M, Roosterman D, Kempkes C, Bocheva G, Steinhoff M, and Egelrud T. Activation of Proteinase-activated Receptor-2 by Human Kallikrein-related Peptidases. J. Invest. Dermatol. 128:18-25, 2008

The original articles were reprinted with permission from the publishers: Walter de Gruyter Scientific Publishers (Paper I) and Nature Publishing Group (Papers II and V).

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ABBREVIATIONS

α-1AT Alpha-1 antitrypsin AD Atopic dermatitis CDSN DSC1 Desmocollin 1 DSG1 Desmoglein 1 ELISA Enzyme-linked immunosorbent assay FPLC Fast protein liquid chromatography hCAP18 Human 18-kDa cationic antimicrobial protein ICE Interleukin-1β-converting enzyme IL Interleukin ILC Ichthyosis linearis circumflexa IV Ichthyosis vulgaris KLK Kallikrein/Kallikrein-related peptidase NS Netherton syndrome LEKTI Lympho-epithelial Kazal-type inhibitor PAGE Polyacrylamide gel electrophoresis PAR Proteinase-activated receptor PAR-AP PAR-activating peptide RPC Reversed phase chromatography RT Room temperature SBTI Soybean SC Stratum corneum SCCE Stratum corneum chymotryptic enzyme SCTE Stratum corneum tryptic enzyme SDS Sodium dodecyl sulphate SEMG Semenogelin SG Stratum granulosum SKALP Skin-derived antileukoproteinase SLPI Secretory leukocyte protease inhibitor SPI Serine protease inhibitor SPINK Serine protease inhibitor Kazal-type XRI X-linked recessive ichthyosis

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POPULÄRVETENSKAPLIG SAMMANFATTNING

Vår hud är ett oerhört viktigt organ. Alla levande organismer behöver någon sorts ‘hud’; en barriär som skyddar mot bland annat uttorkning och sjukdomsframkallande mikroorganismer (bakterier, virus och parasiter). Särskilt hudens yttersta lager, hornlagret, är viktigt för hudens barriär. Hornlagret har en hög kemisk och mekanisk motståndskraft. Detta beror på cellernas så kallade förhorning, de är tätt packade och fyllda med hållbara keratinfilament (proteiner sammankopplade till trådar). Runtomkring cellerna finns även fetter som är viktiga, särskilt som ett skydd mot vattenpassage. Cellerna hålls ihop av desmosomer. De ser ut som små tryckknappar, som går mellan ena cellens cellvägg till nästa. Men huden behöver inte bara hålla ihop, det är också viktigt att celler lossnar från hudytan i en normal takt, allteftersom de byts ut mot nya celler underifrån. Cellavstötningen från hudytan kallas med ett annat ord för deskvamation. Deskvamationen är beroende av proteaser. Proteaser är proteiner som klipper sönder andra proteiner. Kallikrein-relaterade proteaser (vi kallar dem för enkelhetens skull kallikreiner) är sådana proteaser och de finns i huden där deras uppgift är att klippa sönder desmosomer vilket leder till att cellerna lossnar från varandra. Det är även viktigt att kallikreiner inte är aktiva för långt ner i huden för då lossnar för mycket celler och hudens barriär fungerar inte. Det finns femton stycken kallikreiner, som förkortat kallas KLK1-15. Kallikreinerna kan vara inaktiva eller aktiva. De aktiveras genom att en liten del klipps bort. Aktiviteten kan även regleras genom omgivningens pH eller genom särskilda hämmare, så kallade inhibitorer. Den här avhandlingen handlar om några kallikreiner som finns i huden och hur de där kan påverka vissa processer. Innan studierna påbörjades visste man att KLK5 och KLK7 finns i huden och tros vara viktiga för deskvamationen. Man hade också vissa bevis som pekade på att kallikreiner kan vara inblandade i inflammatoriska sjukdomstillstånd.

I den första av de fem artiklar som den här avhandlingen bygger på beskrivs reningen av ett tredje kallikrein, KLK14, från huden. KLK14 visade sig vara ett väldigt aktivt protein. Trots att det finns lite av KLK14 i huden kan proteinet därför ändå ha stor betydelse för hudens funktioner. Nästa artikel handlar om hur kallikreiner samverkar för att aktivera varandra. Ett kallikrein kan aktivera ett annat som aktiverar ett tredje och så vidare i en kaskad som leder till fler och fler aktiva kallikreiner. Denna kaskad visade

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sig vara pH beroende. Aktiveringen av inaktivt KLK7 och KLK14 med redan aktivt KLK5 var till exempel snabbast vid ett surt pH. Det stämmer bra ihop med vetskapen att hudytan har ett surt pH. Ju längre ut mot ytan, desto surare pH har huden, och desto mer kallikrein-aktivitet behövs. Studierna visade också att KLK5 kan ha en central betydelse i kaskaden. KLK5 är lite av en ‘spindeln i nätet’ genom att den kan aktivera sig själv vid ett neutralt pH och därigenom sätta igång hela kaskaden.

Tredje och fjärde artikeln handlar om inhibitorer till kallikreiner. Inhibitorer är proteiner (eller ämnen som till exempel zink) som hämmar aktiviteten hos andra ämnen, i detta fall proteaser. Från ett extrakt av torkade hudflagor, tagna från fötterna på personer som varit till fotvårdsspecialister, kunde flera inhibitorer renas. När man blandade aktiva kallikreiner med de renade inhibitorerna från extraktet såg man att kallikreinernas aktivitet hämmades. En av de renade inhibitorerna var sedan tidigare okänd. Eftersom människans alla gener är sekvenserade visste man att genen för inhibitorn fanns men ingen har tidigare skrivit om den. Nu har vi visat att den hämmar KLK5 väldigt bra och att det finns mycket mer av den i huden i handflator och fotsulor än i övrig hud. Inhibitorn kallas SPINK9 och är ’släkt’ med inhibitorn SPINK5. SPINK5 finns i normal hud över hela kroppen och är välkänd eftersom personer som saknar SPINK5 drabbas av en mycket allvarlig ärftlig sjukdom kallad Nethertons syndrom (NS). NS är ett bra exempel på hur viktig huden och kallikreiner är. Barn som föds med NS har mörkt röd, inflammerad hud. För mycket kallikrein-aktivitet har skadat hornlagret. Barnen förlorar därför mycket vätska genom huden och behöver intensiv omvårdnad på sjukhus för att överleva.

Sista artikeln länkar samman kallikreiner med inflammatoriska och immunologiska funktioner i huden. Den visar att KLK5 och KLK14 men varken KLK7 eller KLK8 kan aktivera en cellyte-receptor som heter PAR-2 (proteas-aktiverad receptor-2). Andra studier på PAR-2 har visat att sådan aktivering leder till en rad händelser som har med inflammation och immunförsvar att göra. Sammanfattningsvis kan man säga att studierna i den här avhandlingen har lagt några fler pusselbitar till det invecklade samspelet mellan proteaser, inhibitorer och celler i huden. I framtiden kan resultaten få betydelse för behandlingen av exempelvis inflammatoriska hudsjukdomar som eksem, psoriasis och iktyos samt NS.

8 Introduction

INTRODUCTION

The Skin The skin as an organ is essential for a persons wellbeing. Among its many functions is the protection against harmful influence from the outside world. The skin provides a barrier, keeping pathogenic microorganisms and chemicals out and the vital water in. The skin is also important as a way of communication between individuals. It gives away our anger or embarrassment when we blush, or communicates feelings like friendship or love when we touch.

Evolutionary Aspects of the Skin All organisms are dependent on an outer layer defining the body, upholding the balance between incoming and outgoing substances. Some of the simplest forms of skin once arose in sea-living organisms. Corals and jellyfish have only two layers of cells, the ectoderm facing out towards the environment and the endoderm lining the gut. However, the ectoderm may contain specialized cells like glandular cells, pigment cells, and sensory or stinging cells. Insects on the other hand have a tough exoskeleton made of chitin which almost resembles a natural plastic. A drawback of the exoskeleton is that it has to be periodically shed to allow for growth of the organism. This leaves the organism unprotected and vulnerable. Vertebrates, to which we belong, represent another step in the evolution of skin. Their vast diversity of skin-derived structures like fur, feathers and scales, have aided them in the colonization of land [1].

Basal Structure of the Skin The skin is composed of three main layers, the underlying subcutis, the dermis, and the superficial layer epidermis (Figure 1). Subcutis is mainly composed of adipose tissue with fat cells (adipocytes) as the dominating cell-type. It serves to protect from trauma and functions as a temperature isolator. The thickness of the subcutis is dependent on heredity, hormone balance, and nutrition. The dermis is a 1-10 mm thick layer composed of cells, collagen fibers, and a matrix of glucosaminoglycans and hyaluronic

9 Introduction

Figure 1. Schematic cross section of the skin. Schematic picture showing the three main layers of the skin, epidermis, dermis, and subcutis. The stratum basale is shown with its characteristic downward folds (rete ridges), which prevents separation of the epidermis from the dermis. Picture also shows the principal skin appendages; hairs, sebaceous glands, and sweat glands.

acid. The dermis provides the vascular supply for the avascular epidermis and a base for the skin appendages. Blood vessels and nerves, present in the dermis, have a great importance on body temperature regulation. The most common cell type in the dermis is the fibroblast. There are also inflammatory and immune cells including mast cells, macrophages, and ‘patrolling’ lymphocytes. The epidermis is a keratinized stratified squamous epithelium (Figure 2). The main cell type of the epidermis is the . However, there are also melanocytes, pigment cells responsible for skin coloration, and Langerhans cells, which are immunological cells, scattered throughout the epidermis. The epidermis also shelters patrolling lymphocytes. Merkel cells are specialized touch receptors found among the basal cells in the epidermis [2].

10 Introduction

Stratum basale is the lowermost layer of the epidermis. It consists of cuboidal or low columnar cells forming a single layer, separated from the dermis by a basement membrane. Cells in this layer have the ability to divide. The next layer, the stratum spinosum, consists of relatively large, polyhedral cells. Cytokeratin, which aggregates to form intracellular fibrils known as tonofibrils, is the predominant product of these cells. Many desmosomal junctions are seen between the cells of the stratum spinosum. The stratum granulosum (SG) has two to three layers of flattened cells with dense basophilic, keratohyalin, granules in the cytoplasm. In the upper cell layer of the SG, cells ‘die’ and loose most of their organelles. Enzymes released from the lysosomes are thought to be important in the final stages of keratinization. The stratum corneum (SC) is the most superficial layer. In the SC the cells (corneocytes) are flattened, devoid of nuclei and other organelles, and filled with mature keratin. The thick keratin filaments in the corneocytes and the so called cornified cell envelopes give the SC a high mechanical and chemical strength. The cornified cell envelope is the modified cell membrane, composed of strong crosslinked proteins and lipids. Other important features of the SC are the intercellular lamellar lipids. These are mainly important as a barrier towards water passage. The SC is about 15 µm thick on most locations, except in palms and soles where it is much thicker. Individual corneocytes are about 1 µm thick [2-4].

Figure 2. Epidermis. a) Normal non palmo-plantar skin from the lower back. b) Palmar skin. c) Schematic representation of the epidermis. SB, stratum basale, SS, stratum spinosum, SG, stratum granulosum, SC, stratum corneum. Note the different scales and the spiral formed sweat duct in b.

11 Introduction

Epidermal Turnover and Desquamation The skin is exposed to mechanical wearing on a daily basis. This calls for a constant renewal of the skin. In the stratum basale, there is a continuous de novo production of , balancing the rate of cell shedding. Another name for this cell shedding is desquamation. It takes about five to six weeks for one cell to journey from the stratum basale to the most superficial layer of the SC where it is desquamated. Around three of these weeks are spent in the living parts of the epidermis and two in the SC. During this time the cell goes through a series of maturation steps, forming the above mentioned layers, in a process known as keratinization [4].

Desquamation is needed to maintain the thickness of the SC, and the process continues constantly even independent of mechanical wearing [5, 6]. However, this process has to be tightly regulated. Desquamation must not occur too soon, in the barrier forming parts of the epidermis, as this would be detrimental. It has been speculated on the existence of a ‘programming’ of the SC before cells leave the upper living layers of the epidermis [7]. To understand the process of desquamation we need first to understand the nature of the cell cohesion and barrier function in the SC. When looked upon by routine light microscopy, the SC has the appearance of a loose ‘basket weave’ with loosely attached, disorganized, corneocytes with no apparent functional significance. However, this is merely an artifact owing to the processing of the tissue. It is now commonly accepted that the corneocytes provide a strong physico-chemical resistance whereas lipids are mainly important for the barrier function against for example water loss [1, 8, 9].

Over the years, the theories behind the SC cohesion and resistance have changed. From the mid 1960:s it was believed that lipids were the main mediators of both cohesion and epidermal permeability barrier. The SC was then thought of as a ‘plastic wrap’, encompassing the body. In the 1970:s the concepts changed to the belief that the barrier is mediated through a two- compartment ‘bricks and mortar model’. The bricks would be the corneocytes, forming a backbone for the intercellular lipids (mortar). Today the concept has again shifted towards the ‘living stratum corneum’ model [8]. This is explained by the many examples of metabolic activity still presiding in the otherwise dead SC. One such example is the proteolysis of

12 Introduction filaggrin to amino acids [10]. Not the least is the example of the proteolysis of extracellular parts of corneodesmosomes [11-16].

In 1988, Egelrud and Lundström developed a simple model system to study desquamation in human plantar skin. Through use of protease inhibitors they could prove that desquamation is dependent upon proteolysis and that desquamation only occurs from the outer surface of the SC [12]. Later it was shown that the cell shedding involved endogenous proteolysis of the desmosomal protein desmoglein 1 [13]. Much evidence now pointed to the importance of proteolytic degradation of corneodesmosomes for desquamation to occur [11, 13-16].

The Corneodesmosome The desmosome is a kind of anchoring junction which mechanically attaches cells and their cytoskeleton. It can be described as a tiny button holding cells together. The intracellular part of the desmosome is made up of a dense cytoplasmic plaque composed of plakoglobin and desmoplakin which are intracellular anchoring proteins. Via plakoglobin and desmoplakin the cytoskeleton is connected to the transmembrane adhesion proteins desmoglein (DSG) and desmocollin (DSC). The transmembrane adhesion proteins on adjacent cells interact via their extracellular domains [17]. Desmosomes found in the SC have been altered during the keratinization process. During the last stages of keratinization, corneodesmosin (CDSN) is synthesized. CDSN is present in the extracellular part of the altered desmosome called corneodesmosome [14, 18-21].

The Stratum Corneum pH Gradient There is a pH gradient ranging from a neutral pH in the living cells just below the SC, to an acidic pH around 4.5 to 5 on the skin surface [22]. Acidifying factors are fatty acids from sebum, lactic and amino acids from sweat, and free fatty acids processed from epidermal phospholipids [23, 24]. This gradient may be very important. Even minor alterations in the pH due to use of synthetic detergents lead to increased counts of bacteria on the skin surface [25]. SC pH may have impact on antimicrobial activity through pH dependent activation of cytokines [9, 26]. The significance of the so called ‘acid mantle’ of the skin is implied by certain subpopulations being more

13 Introduction prone to infections; those include atopic dermatitis (AD) patients and newborns who display a less acidic pH on the skin surface [24, 27]. In newborn skin the pH gradient has yet to form, a process which takes several weeks to months [27].

Skin Immunology and Inflammation Although important as such, the barrier provided by the SC is much more than just a mechanical barrier. The SC confers resistance to microbial threats in several ways; low water content, acidic pH, existing normal micro flora, and presence of antimicrobial lipids and peptides [9, 26]. In the epidermis, keratinocytes produce a wide range of chemical agents influencing inflammatory and immunological reactions in both healthy and diseased skin. Upon stimulation they produce cytokines, chemokines, proteases, neuropeptides, non-peptide neurotransmitters, and defensins. Such stimulation can be exerted through ultraviolet radiation, contact allergens, irritants, microbiological agents, or trauma. Due to its production of immunological agents, the keratinocyte has been described as a ‘cytokine factory’ [28]. Langerhans cells, which are professional antigen presenting cells in the epidermis, also produce certain cytokines [29]. Another way to mediate inflammatory and immunological reactions in skin is through proteolytic stimulation of receptors. PAR-2 (proteinase-activated receptor-2) is a receptor present on keratinocytes which has been highly implicated in immunological reactions of the skin (see chapter on PAR-2 below) [30].

Cathelicidin One example of antimicrobial peptide produced in the epidermis is cathelicidin. Human cathelicidin is synthesized as a precursor protein named hCAP18 (human 18-kDa cationic antimicrobial protein). hCAP18 is processed through serine protease cleavage into the broad spectrum antimicrobial peptide LL-37 [31, 32] which kills human pathogens such as Escherichia coli, [33] and group A Streptococcus [34]. Additionally, hCAP18 is induced upon cutaneous [34] and stimulates cytokine release [35], chemotaxis [36], angiogenesis [37], and wound repair [38].

14 Introduction

Interleukin -1 Interleukin -1α (IL-1α) and IL-1β are cytokines produced by for instance macrophages, endothelial cells, and keratinocytes [39-41]. They act on the receptors CD121a (IL-1RI) and CD121b (IL-1RII) to initiate a wide variety of pro-inflammatory effects [41]. Among other cytokines, IL-1α and IL-1β also have a role in promoting the migration of Langerhans cells to lymph nodes where these can present antigens to naïve T cells [29]. IL-1α and IL- 1β are both produced as 31-kDa precursors. As both interleukins bind to the same receptor, they exert the same biological actions. However, there are many differences between them. For example IL-1α precursor is fully active whereas IL-1β needs to be activated by proteolytic cleavage into the mature 18-kDa form by the interleukin-1β-converting enzyme (ICE) [42-44]. IL-1β has been purified from human plantar SC where there is evidence for an alternative activation mechanism. The majority of active IL-1β in the SC exists in a variant with an N-terminal sequence suggesting processing by a protease different from ICE [45].

Kallikrein-related Peptidases (KLKs) Tissue kallikreins (KLKs) comprise a large family of serine-proteases present at many sites of the body. The first human KLK was found in the late 1920:s and named kallikrein. The name kallikrein comes from the Greek word for pancreas, kallikreas, where KLKs were found and thought to originate from [46, 47]. For a long period of time, only three human KLKs were known, these were KLK1 (tissue kallikrein, pancreatic/renal kallikrein, hPRK) [48], KLK2 (human glandular kallikrein -1, hGK-1) [49] and KLK3 (human prostate-specific antigen, PSA) [50]. With the advances of the Project, the KLK family was extended as twelve additional KLKs were discovered. This led to an explosion of publications in the field of KLKs.

Nomenclature The naming of the many KLKs has long been a problematic issue but the scientific field has now united in a common nomenclature. According to the new nomenclature, all KLKs except the first are named kallikrein-related peptidase followed by the number 2-15. KLK1 still goes by the name

15 Introduction kallikrein 1. However, the abbreviation of all members of the family is KLK. To distinguish between protein and name, gene name is written in italics. When talking about gene transcripts, this is made clear by using a prefix within parenthesis; for instance (mRNA)KLK2. Abbreviations are always written in capital letters except in mouse and rat where only the first letter is capital. If needed, species specifying prefixes are used according to the codes established by SWISS-PROT (http://www.expasy.ch/cgi- bin/speclist); for instance (HUMAN)KLK2 and (MOUSE)Klk4 [51].

Genomic Organization The human kallikrein gene locus is located on the long arm of 19, in the cytogenic region q13.3-q13.4 and contains at least fifteen [52-54]. All genes have five coding exons and the coding exon sizes are similar or identical. DNA and amino acid sequences are conserved between different KLKs with about 36-80% sequence homologies [55, 56]. KLK2 and KLK3 are transcribed in the same direction, from centromere to telomere, whereas the other genes are transcribed in the reverse direction [52]. The kallikrein gene locus also includes pseudogenes. There are conflicting data as to how many they are, there may be as many as five [57], but according to recent reviews, they may be regarded as two short pseudogenes with homology to exon 2 of KLK1-3 (Figure 3) [56].

Figure 3. Schematic representation of the human kallikrein gene locus. Arrows depict approximate location and orientation of genes. Numbers refer to KLK number. Pseudogenes are situated between KLK2 and KLK4.

16 Introduction

Protein Structure Besides their localization to the same chromosomal region, the members of the kallikrein gene family share certain similarities. They all encode for putative serine proteases with a conserved . KLKs are expressed as preproproteins having a 16-34 amino acid prepeptide and a 3- 37 amino acid propeptide as N-terminal extensions (Figure 4). The task of the prepeptide is to direct the protein to the extracellular space. Upon removal of the propeptide through proteolytic processing, a conformational change leads to the opening of the catalytic cleft rendering an active enzyme. The kallikrein loop is a structure of eleven amino acids which is conserved in KLKs 1-3 but shorter or absent in the other KLKs. This loop flanks and partly covers the catalytic cleft, thereby probably affecting substrate access [55, 56].

Figure 4. Structure of a KLK preproenzyme. Numbers refer to amino acid numbering according to the consensus.

Substrate Specificity KLKs may be chymotrypsin- or trypsin-like in their substrate specificity. Trypsin-like enzymes preferably cleave their substrate after the positively charged lysine (K) or arginine (R) residues whereas chymotrypsin-like enzymes prefer the aromatic amino acids tryptophan (W), (Y), or phenylalanine (F) [58]. KLKs themselves are most likely activated by enzymes with trypsin-like activity as evidenced by their amino acid sequences. All except KLK4 have propeptide sequences ending with either an arginine or a lysine residue [56].

The serine protease activity of the mature KLK is defined by the presence of an active serine residue in the catalytic cleft. Almost one-third of all

17 Introduction proteases belong to the serine protease class. Serine proteases are divided into clans depending on the amino acids of their catalytic triads or dyads. KLKs belong to the PA(S) subclan according to the MEROPS database (http://merops.sanger.ac.uk/). This clan has a catalytic triad consisting of histidine (H), aspartic acid (D), and serine (S) in the appropriate positions (amino acids 57, 102, and 195 respectively using chymotrypsin numbering). Further specificity is usually determined by the residues at positions 189, 216, and 226 [59]. Most KLKs have trypsin-like specificity as they have an aspartic acid at position 189. KLK3 has a serine residue at position 189, corresponding to chymotryptic specificity. KLK7, 9, and 15 have alternative residues at this position (asparagine (N), glycine (G), and glutamic acid (E) respectively [56, 60]). KLK7 is a chymotrypsin-like enzyme [61] whereas KLK15 shows trypsin-like activity [62, 63].

KLK Function in Various Organs Members of the KLK family are widespread in the body and may participate in a variety of physiological and pathophysiological processes. According to mRNA patterns, many of the KLKs are highly expressed in the pancreas (KLKs 1, 6-8, and 10-13) and prostate (KLKs 2-4). Moreover, KLKs are expressed at various levels in a variety of other tissues including kidney, salivary gland, testis, esophagus, mammary gland, brain, gastro intestinal tract, liver, heart, skin, skeletal muscle, and fetal organs [54, 57, 64]. Below follows a few examples of KLK involvement in various organs.

KLK1 is believed to have an important role in the regulation of hypertension, and cardiovascular and renal diseases. KLK1 exerts its function through processing of low-molecular-weight kininogen to Lys- bradykinin (kallidin). Binding of kinins to the kinin B2 receptor triggers effects such as vasodilatation, smooth muscle contraction and regulation, inflammation, and pain. Several studies have shown a relationship between low levels of KLK1 in urine and hypertension. KLK1, which belongs to the tissue kallikrein family, should not be confused with , which share no other resemblance to KLK1 than its ability to produce kinin peptides. Plasma kallikrein is found in circulating blood and is involved in the blood cascade [65].

18 Introduction

KLK3 or PSA is present in prostatic fluid and cleaves the predominant seminal vesicle protein. Hence its function is to carry out the liquefaction of the semen coagulum [66, 67]. KLK2, 5, and 15 (also known as prostin) are possible activators of proKLK3 in this system, giving rise to speculations on a proteolytic cascade of KLKs in seminal clot liquefaction [63, 68, 69]. A somewhat different theory is presented in a recent review. According to this, KLK3 in prostatic fluid is proteolytically processed into the mature form but inhibited by Zn2+. Upon ejaculatory mixing, Zn2+ is sequestered by seminal vesicle proteins semenogelin-1 and -2 (SEMG-1 and -2). Removal of inhibition allows KLK3 to cleave SEMG-1 and -2, leading to seminal clot liquefaction. As the presence of Zn2+ in prostatic fluid would inactivate other KLKs as well, activation of KLK3 in prostatic fluid ought to be executed by another, Zn2+ resistant protease [56, 70]. KLK3 levels in serum can be used as a biomarker for prostate [71]. However, in Sweden it is debated whether a population based screening would have the desired positive effect. Scientific documentation is not satisfactory and points to a high risk for negative effects due to falsely positive tests, overtreatment, and extensive costs [72].

KLK4 (enamel matrix serine proteinase 1, EMSP1) has been recognized as a tooth enzyme with a role in the proteolysis of enamel proteins [73-75]. Normal KLK4 function is critical for enamel mineralization as evidenced by the finding of a mutation in the KLK4 gene causing the autosomal recessive hypomaturation defect amelogenesis imperfecta [76].

As implied by their alternative names, neurosin and neuropsin, KLK6 and KLK8 are expressed in brain where also KLK11 (hippostasin) and KLK14 are relatively abundant [54, 57, 77, 78]. At least KLK6 and KLK8 seem to be important in central nervous system biology. KLK6 has been implicated in amyloid metabolism and the pathogenesis of Alzheimer’s disease [79, 80] whereas KLK8 may have a role in brain plasticity and development [81, 82].

Apart from their functions in various organs, many KLKs besides the above mentioned KLK3, have been intensely investigated regarding their potential utility as cancer biomarkers as reviewed by Paliouras et al. [83].

19 Introduction

Kallikrein-related Peptidases in Skin Physiology Much experimental data points to an involvement of KLKs in skin physiology. KLKs 1, 4-11, and 13-14 are expressed in epidermal tissue as supported by RT-PCR, in situ hybridization, and immunohistochemical methodologies [84-90]. The most extensively studied KLKs in skin are KLK5, originally named stratum corneum tryptic enzyme and KLK7, stratum corneum chymotryptic enzyme. Both have been purified from plantar SC extracts in their catalytically active form [64, 91]. The localization of KLK5 and KLK7 in the uppermost layers of the living part of epidermis, the SG and the SC [86, 87], and the notion of their activity there have lead to further studies concerning their possible involvement in desquamation and inflammatory skin diseases such as psoriasis [92].

KLKs in Desquamation As explained above, desquamation is the shedding of corneocytes from the skin surface, a process which is dependent on proteolysis of corneodesmosomes. Both chymotrypsin- and trypsin-like enzymes are involved in this process [13, 15, 16, 93, 94]. Experiments have shown that the trypsin-like activity may be due to KLK5 activity as this enzyme has the capacity to cleave the corneodesmosomal proteins CDSN, DSC1, DSG1, DSG2, and plakoglobin [95, 96]. KLK5 was found not to cleave DSG1 and DSG2 at pH 7.2 [95]. Nevertheless, at pH 5.6 both these proteins were degraded by KLK5 [96]. The chymotryptic activity may derive from KLK7 which degrades CDSN, DSC1, and plakoglobin but neither DSG1 nor DSG2 regardless of pH [95, 96]. Other KLKs may also have impact on desquamation. It has been shown that KLKs 1, 6, and 14 have the ability to degrade DSG1. Among these KLKs, KLK1 is the less and KLK14 the most efficient DSG1-degrading enzyme [97]. According to immunoreactive quantification, KLK8 is present in higher amounts in the SC than KLK5 and KLK14 [98, 99]. However, when evaluating number of transcripts, KLK5 and KLK7 seem to be the major KLKs in skin [56]. Regardless, KLK8 may also be involved in desquamation as suggested by the phenotype of Klk8(-/-) knock-out mice. These mice show a higher number of cell layers in the SC when compared to wild type mice and a greater thickening of the SC following UV-radiation. Authors of the studies on KLK8 knock-out mice speculate on a role for KLK8 in both desquamation and epidermal proliferation [100, 101].

20 Introduction

KLKs in Skin Immunology and Inflammation The functions of KLKs in skin may include inflammatory and immunological reactions. Research has shown that in skin, the cathelicidin hCAP18 is processed by KLK5 into the active form LL-37. KLK5 further processes LL-37 into the smaller peptides KS-29, KS-30, and KS-22 all having antimicrobial activity. KLK7 cleavage of LL-37 gives rise to the antimicrobial peptides RK-31 and KR-20. However, peptides generated by KLK7 cleavage are short-lived and KLK7 may thus function as an inactivator of LL-37. All this suggests that KLKs are important in the formation and regulation of the anti-microbic defense system in skin [32]. In the inflammatory skin disease rosacea, there are abnormally high levels of cathelicidin-derived peptides such as LL-37 in the facial skin. There are also unique peptides, not present in normal healthy skin, indicating an altered processing of hCAP18 in rosacea. Data strongly points to an involvement of KLK5 in the pathological changes seen in rosacea as there is an increase in the amounts of KLK5 as well as an abnormal expression pattern. Injection of KLK5 in amounts mimicking those observed in rosacea, into mouse skin, also increases cathelicidin processing and induces skin inflammation [102].

KLK7 may also be implicated in rosacea due to its processing of cathelicidin but has been more extensively studied in two other inflammatory skin diseases. This KLK may be implicated in the pathogenesis of inflammatory skin diseases such as AD and psoriasis through activation of pro-inflammatory cytokines such as proIL-1β. KLK7 can convert proIL-1β into active IL-1β and may thus be involved in the alternative activation route of proIL-1β mentioned above (see chapter on interleukins) [103]. However, the N-terminal sequence of the main part of mature IL-1β found in plantar SC extracts corresponds neither to cleavage through ICE nor KLK7 [45, 103]. Furthermore, KLK7 may be upregulated in psoriatic skin as indicated by immunohistochemical findings. In lesional psoriatic epidermis, a greater number of cell layers are positive for KLK7 as compared to normal skin [92]. The upregulation of KLK7 in psoriasis is supported by additional studies. According to ELISA (Enzyme-linked immunosorbent assay) and enzymatic assays, lesional SC show significantly increased levels of KLKs 5-8, 10-11, and 13-14. Levels of KLK6, 8, 10, and 13 are elevated also in the sera of psoriasis patients where levels correlate significantly with PASI (Psoriasis Area and Severity Index) score [104].

21 Introduction

Likewise, in the SC of AD patients, the same KLKs, with the exception of KLK11, were significantly increased [105].

Studies on KLK7 transgenic mice provide further evidence of a role for KLK7 in the pathophysiology of inflammatory skin diseases. Epidermal overexpression of human KLK7 gives rise to a chronic itchy dermatitis. In other words, it leads to epidermal hyperproliferation, decreased skin barrier function as well as severe pruritus (itch) [106, 107]. The pruritus observed may be due to malfunctions in epidermal barrier giving proteolytic enzymes from the skin surface access to pruritic nerves. The PAR-2 receptor is present on afferent nerve fibers and implicated in nociceptive and pruritogenic nerve signaling [30, 108, 109]. Steinhoff et al. suggested already in 1999 that KLKs or other trypsin-like proteases in the epidermis could be implicated in inflammatory reactions through the activation of PAR-2 [110].

Inhibitors of Kallikrein-related Peptidase Activity KLK activity can be regulated in various ways. The most obvious regulation is inactivity owing to the propeptide. However, once the propeptide has been cleaved off, regulation can be accomplished through other mechanisms. Metal ions such as Zn2+ may function as inhibitors. Interestingly, Zn2+ inhibits KLK5 very efficiently at neutral pH but has little effect at pH 5.5 [111]. Other possible regulatory mechanisms suggested are substrate modifications via glycosylation, pH, water content or lipids [112, 113]. Cholesterol sulphate deserves a special note as this lipid was found to inhibit both trypsin- and chymotrypsin-like activities in SC thereby affecting desquamation (see also section on ichthyosis) [114]. Endogenous serine- protease inhibitors (SPI), of the protein-class, in skin include α−1ΑΤ, SKALP, SLPI, and LEKTI. The definition of an inhibitor is “any compound that decreases the measured rate of hydrolysis of a given substrate”. This means that one competing substrate in a reaction may function as an inhibitor of the hydrolysis of another substrate. Thus, some inhibitors can be described as substrates forming very tight but sometimes reversible complexes [58]. This is true for the inhibitors mentioned below.

22 Introduction

Alpha-1 Antitrypsin Alpha-1 antitrypsin (α-1AT) belongs to the SERPIN (serine protease inhibitor) superfamily. These are large proteins (350-500 amino acid residues in size) which use a unique suicide substrate-like inhibitory mechanism. Upon the encounter of a protease, the SERPIN changes conformation to irreversibly trap the protease [115]. α-1AT is a 52 kDa inhibitor also known as α1-proteinase inhibitor. Because of its stability and its one to one (equimolar) reaction with trypsin-like proteases, it is useful as an active-site titrant [58]. In plantar skin, α-1AT has been found in complex with KLK7 (Egelrud, unpublished observation). However, this inhibitor seems most important in the function of lungs and liver, as the inherited disease α-1AT deficiency is mainly manifested by severe lung and liver malfunctions [116].

Skin-derived Antileukoproteinase (SKALP) Skin-derived antileukoproteinase (SKALP) also known as was first purified from psoriatic scales. SKALP is an inhibitor that can be found in the epidermis in several inflammatory skin diseases [117-119]. SKALP expression in normal human epidermis is controversial. In a study by Pfundt et al., SKALP could not be found in normal epidermis with the exception of keratinizing cells lining the sweat gland duct [120]. Nara et al., on the other hand, showed an intense SKALP immunostaining in the SG [121]. In psoriatic epidermis, SKALP was found in suprabasal keratinocytes [122]. According to Molhuizen et al. the inhibitor is synthesized as a larger precursor molecule but the mature inhibitor exists in two forms, a free 6 kDa form and an immobilized 9.9 kDa form. The transglutaminase substrate motif of SKALP crosslinks it to the cornified envelope [123]. The relevance of SKALP in the inhibition of KLKs is uncertain considering its elastase-like inhibitory profile. Nevertheless, SKALP does show inhibition, but only weak such, towards KLK7 [124]. Furthermore, SKALP shows partial amino acid homology to SLPI, a more effective inhibitor of KLK7 [118, 124].

23 Introduction

Secretory Leukocyte Protease Inhibitor (SLPI) Secretory leukocyte protease inhibitor (SLPI) (antileukoprotease (ALP), human seminal inhibitor-1 (HUSI-1)) is an inhibitor that targets elastase, trypsin, chymotrypsin, and G. Its calculated molecular weight is 11.7 kDa [125-127]. Similar to SKALP, SLPI is upregulated in psoriatic epidermis, though it can also be detected in the SG of normal human epidermis [128]. Franzke et al. concluded in 1996 that SLPI may be the most important KLK7 inhibitor in the epidermis. This was based on the potent inhibition of KLK7 seen, together with the finding that SLPI could completely inhibit the shedding of corneocytes from human plantar skin in an in vitro model based on the one developed by Lundström and Egelrud in 1988 [93, 124]. Yet, one should keep in mind that at this time, LEKTI (see below) was still not discovered.

Apart from its inhibitory function, SLPI also confers antimicrobial activity. As shown by Wiedow et al. in 1998, SLPI kills the bacteria Pseudomonas aeruginosa, Staphylococcus aureus, and Staphylococcus epidermidis as well as the fungi Candida albicans [129]. SLPI also shows antiretroviral activity as it inhibits human immunodeficiency virus -1 (HIV-1) activity. The presence of SLPI in saliva might therefore contribute to the protection against oral HIV-1 infection [130].

Lympho-epithelial Kazal-type Inhibitor (LEKTI) Lympho-epithelial Kazal-type inhibitor (LEKTI) is encoded by the gene SPINK5 (serine-protease inhibitor Kazal-type 5). It was discovered by Mägert et al. in 1999. Their results showed that LEKTI is a 15-domain serine protease inhibitor. Two of the domains were isolated from human blood filtrates and shown to inhibit trypsin [131]. Since then additional studies have shown that LEKTI domains inhibit various proteases such as trypsin, A, , and with different efficiencies [132, 133].

In normal , LEKTI is localized to the SG and inner root sheath of the hair follicle. In psoriatic skin LEKTI can be detected in both SG and stratum spinosum, thereby showing a broader expression pattern [134-136]. Furthermore, all stratified epithelia are positive for LEKTI staining [136]. In Netherton syndrome (NS) (see section on Netherton syndrome below)

24 Introduction

LEKTI is lacking due to mutations in the SPINK5 gene [135, 137]. NS patients show no or very faint immunostaining of LEKTI [134-136].

LEKTI fragments of various sizes can be detected in skin-derived cells. Bitoun et al. detected LEKTI as two N-glycosylated precursor proteins of 145 and 125 kDa in differentiated normal human keratinocytes (NHK). Three additional C-terminal fragments of 68, 65, and 42 kDa could also be seen on western blot. These were suggested to be a result of intracellular processing of LEKTI [136]. Further processing of LEKTI could give rise to additional forms. Ahmed et al. purified a 30 kDa LEKTI fragment from cell media of differentiated keratinocytes [138]. Although LEKTI could theoretically be cleaved into at least 14 fragments, no evidence of single- domain fragments were detected in cultured keratinocytes by Tartaglia- Polcini et al. [139, 140]. Contrary to this, human epidermis contains no detectable 125 and 145 kDa precursor forms. Instead there are multiple LEKTI fragments of 10, 13, 15, 20, 31, 37, 42, and 65 kDa. This was shown by Deraison et al. using three different antibodies against LEKTI [141]. Noteworthy is also that although named ‘Lympho-epithelial Kazal-type inhibitor’, only domains 2 and 15 are true Kazal-type inhibitors as defined by the spacing between six cysteine residues in the amino acid sequence. Domains 1 and 3-14 lack two cysteines and thus one of the three conserved disulfide bridges [131].

LEKTI is highly involved in the process of desquamation as revealed by many studies. For instance, SPINK5 knockout mice show a lethal phenotype; mice die within hours after birth as a consequence of severe skin blistering and water loss. Skin of LEKTI deficient mice show abnormally high proteolytic activity, loss of cell adhesion, and premature proteolysis of CDSN [142]. These defects can most likely be attributed to the loss of inhibition of KLK5 and KLK7. LEKTI domain 6 has the ability to inhibit KLK5 and KLK7 as shown by Egelrud et al. [143]. Further studies by Schechter et al. showed that LEKTI fragments consisting of domain 6-9 and 9-12 both could inhibit KLK5 whereas only fragment 6-9 inhibited KLK7. This pointed to differences in specificities among various forms of LEKTI. The inhibition was shown to be pH dependent [144]. In a recent study by Deraison et al., other LEKTI fragments were shown to inhibit KLK5, KLK7, and also KLK14 in a pH dependent manner. According to the authors of this study, KLK5 is the major target of LEKTI. A LEKTI

25 Introduction fragment consisting of domains 8-11 showed highest inhibitory activity. Inhibition was mediated through a strong complex formation which dissociated at pH 5 as shown by Surface Plasmon Resonance (SPR) analysis on a Biacore instrument [141].

Proteinase-activated Receptors (PARs) Proteinase-activated receptors (PARs) are G-protein coupled seven transmembrane receptors which are activated by a unique mechanism [145- 148]. This mechanism involves the proteolytic cleavage of the N-terminal extracellular part of the receptor. Cleavage leads to the release of a tethered ligand which can fold back and interact with the second extracellular loop of the receptor. This is followed by a conformational change in the receptor. Activation of PARs leads to the release of calcium ions and further downstream signaling events (Figure 5). This mode of activation is unique in the sense that the activator is not a circulating ligand but a protease and the ligand is a part of the receptor itself [149-151].

The PAR family encompasses four different receptors designated PAR1-4 [145-148]. Activation is mediated via serine proteases. In vitro, PARs 1, 3, and 4 are all activated by [145, 147, 148, 152]. PAR4 is also activated by trypsin and cathepsin G [145, 152, 153]. Henceforth this work will focus on the activation and function of PAR-2. PAR-2 has a somewhat different activation pattern than the other PARs, as it is not activated by thrombin [146, 154]. Trypsin [146], mast cell [155], coagulation factors VIIa and Xa [156], [157], the bacterial protease gingipain [158, 159], and membrane-type serine protease [160] are known to activate PAR-2. Activation is accomplished through a cleavage between arginine and serine at positions 36 and 37. This exposes the tethered ligand SLIGKV [146, 154]. PARs can also be activated by synthetic peptides (PAR activating peptides, PAR-AP) resembling the tethered ligand sequence [146, 161].

26 Introduction

Figure 5. Activation mechanism of PARs. Picture shows the tethered ligand sequence of human PAR-2. Adopted from Steinhoff et al. 2005 [151]

PAR-2 Function in Various Organs PAR-2 and the other PARs are expressed in many organs, such as brain, kidney, liver, lung, ovary, pancreas, skin, stomach, testes, and uterus [162] where they exert various biological effects. Examples of the diverse functions of PAR-2 are bronchoconstriction in human airways [163], angiogenesis [164], proliferation of astrocytes in rat brain [165], pancreatic juice secretion in murine pancreas [166], and remodeling of human endometrium during the menstrual cycle [167]. PAR-2 has also recently been implicated in the generation of nociceptive (pain) signals in patients with irritable bowel syndrome [168]. For a full overview, the reader is referred to detailed reviews in the field [149-151].

PAR-2 in Skin PAR-2 is expressed by keratinocytes, dermal endothelial cells, dermal immunocompetent cells, dermal mast cells, as well as by dermal sensory neurons in human skin [108, 110, 162, 169, 170]. As shown by immunohistochemistry and immunofluorescence, PAR-2 localizes to the SG (similar to KLK5 and KLK7), endothelial cells, hair follicles, and myoepithelial cells of sweat glands. When using sensitive methods or higher

27 Introduction antibody concentrations PAR-2 can also be detected in the stratum spinosum and stratum basale [110].

PAR-2 is implicated in skin physiology in several ways. Hachem et al. claims that PAR-2 is involved in epidermal permeability barrier through inhibition of lamellar body secretion [171]. Seiberg et al. and Paine et al. have studied PAR-2 involvement in pigmentation. According to their studies, SBTI in soy-milk induces depigmentation of skin via inhibition of PAR-2 activation [172, 173]. Other studies have indicated that PAR-2 is expressed in cells involved in the scar formation process and may play a role in the modulation of wound healing [174]. These findings may prove important in the beauty-industry.

An important function of PAR-2 is its involvement in inflammation and immune response. The examples of this are many. PAR-2 agonists lead to upregulation of cytokines such as IL-1β, IL-6, and IL-8, in many cell types including keratinocytes [175-179]. Studies on PAR-2 deficient knock-out mice strengthen the role for PAR-2 in inflammation and immune reactions. Upon injection of trypsin IV into the paw, PAR-2 deficient mice do not show and granulocyte infiltration as do the wild type mice. Trypsin IV also induces and mechanical allodynia in wild type mice, but not in PAR-2 -/- mice [180]. As indicated by other PAR-2 knock-out mice studies, PAR-2 has a crucial role in type IV allergic dermatitis [181].

Inflammatory reactions dependent on PAR-2 activation may be, in part, induced by a neurogenic mechanism. PAR-2 on the cell membrane of sensory nerve endings may be activated through tryptase from degranulated mast cells (or other serine proteases). Activation stimulates release of calcitonin gene-related peptide (CGPR) and substance P (SP) which in turn induce inflammation. For instance, SP may lead to degranulation of mast cells providing a positive feedback [30]. PAR-2 has also been described as a link between inflammatory and sensory phenomena in AD patients. In lesional AD skin, the numbers of PAR-2 positive nerve fibers are increased as compared to normal skin. AD patients also show enhanced immunoreactivity of PAR-2 in their nonlesional skin as compared to healthy controls. When injected into the skin of AD patients and healthy volunteers PAR-2-AP provoked a dose-dependent pain, followed by itch [108]. Conclusions of the study were that PAR-2 is an important mediator of

28 Introduction pruritus and inflammation in human skin. Consequently, so are the proteinases responsible for the activation of PAR-2.

The endogenous activators of PAR-2 in skin are not known. It has been speculated that mast cell tryptase could activate PAR-2 during inflammatory conditions upon mast cell infiltration and degranulation [110]. Trypsin IV which can activate PAR-2 on KNRK-PAR2 cells as well as epithelial- derived cell lines, was another possible activator of PAR-2 in epidermis [182]. It had also been suggested that other trypsin-like serine proteases such as KLKs could activate PAR-2 during normal conditions [110].

Skin Diseases The incentive to study proteases in skin physiology and pathophysiology is of course the wish to find better treatments for skin diseases. To treat a disease it is important to know the underlying mechanisms behind both the specific ailment and the processes in healthy skin. Often treatments do not deal with the real cause of the disease; they are only symptomatic. New insights into the mechanisms of the SC barrier and turnover as well as the genetics behind makes way for future specific therapies [183].

Development of better treatments is of utmost importance for the individuals afflicted with a severe skin disease. For example, Uttjek et al. have investigated life quality in a psoriasis population in Västerbotten, Sweden. Findings show that many patients feel marked by their disease in different social situations, sometimes causing depression. Especially childhood is a problematic period for patients. This together with the knowledge that the disease is chronic and cannot be cured cause an impact on their quality of life [184, 185]. Similar results have been obtained for other skin diseases [186]. Below follows short descriptions of a selection of common or severe skin diseases where KLKs may be involved in the pathogenesis.

Atopic Dermatitis Atopic dermatitis (AD) is also known as atopic eczema. The term atopic means having a genetic predisposition to form antibodies of the IgE-class against proteins in the surrounding milieu. However, only 70-80% of AD

29 Introduction patients have elevated IgE levels. Allergic manifestations such as allergic asthma and hay fever are common in AD patients. Dermatitis is the term for an inflammation in the skin. Patients suffer from long-term, itching dermatitis and dry skin. AD is most common among children. Approximately 10-20% of children worldwide suffer from the disease. The prevalence has increased by two to three fold during the last three decades in industrialized countries [187].

The cause of AD seems to be multifactorial. Genetics has a strong role in the pathogenesis of AD [188-190]. Filaggrin mutations have been strongly linked to AD [191, 192]. Filaggrin is important for the aggregation of keratin filaments into tight bundles. Lack of filaggrin leads to defects in differentiation and cornification of the keratinocytes. The defects in the skin barrier may facilitate sensitization to allergens and thus explain development of allergies in AD patients [193]. Other factors influencing the disease are breast feeding, food, and airborne allergens [194-197].

AD skin is easily colonized with bacteria like Staphylococcus aureus, and yeast fungi (Candida and Malassezia) [24, 198]. This may be in part due to the lack of acidity in the SC and the shortage of antimicrobial peptides [24, 199]. Infections by viruses such as herpes simplex virus and molluscum contagiosum virus is also more common in AD skin [200]. Moreover, AD skin show a skin barrier defect which is aggravated by the itch-induced scratching of the skin [201]. All this leads to a vicious cycle of itching, scratching, and inflammation.

Psoriasis Psoriasis affects between 1.5 and 3% of the Scandinavian population. This disease is uncommon in children but the incidence increases with age. In its most common form, psoriasis vulgaris, the disease manifestations are sharply demarcated plaques with thick silver-white scaling. Plaques may vary in size from one to several centimeters in diameter and are often found on the trunk or limbs. Underneath the scaling is an erythema; when peeling off scales, bleeding can occur. However, there are many clinically different forms of psoriasis and the severity ranges. The disease may present or worsen following skin damage, stress, lack of sun exposure, infections, or use of certain medical drugs. Numbers vary in different studies, but up to

30 Introduction

30% of psoriasis patients also develop psoriatic arthritis [202]. The etiology of the disease includes genetic factors. Many chromosomal loci have been implicated in psoriasis, and the disease has a complex mode of inheritance [203-206].

Histopathological changes found in the psoriatic plaque include hyperproliferation and parakeratosis (dysfunction of the keratinization process; nucleus containing cells are still present in the SC). The SG is virtually absent and there is a pronounced infiltration of leukocytes [202]. The turnover time of the epidermis is considerably shortened; a basal cell reaches the skin surface in only four days, as opposed to a couple of weeks in normal epidermis [202, 207].

Sabat et al. suggested in 2007 a model for the pathogenesis of psoriasis. This model describes the onset of psoriasis as a three-phase immune reaction. The ‘sensitization phase’ involves antigen processing and inflammation leading to generation of T-cells. Upon this follows the “silent phase’. This phase can be of variable length. In the ‘effector phase’, T-cells and other immune cells infiltrate the skin. Immune cell activation then leads to a keratinocyte response and the above mentioned epidermal changes. After treatment, the disease goes back to the silent phase but can be reactivated and enter the effector phase again [208].

In an effort to improve treatment and allow the best individualized therapy, the PsoReg has been created. PsoReg is a web-based registry of systemic psoriasis treatment in Sweden. It aims to analyze safety and effectiveness of different systemic treatments of psoriasis [209].

Rosacea Rosacea is a common skin complaint, especially among older individuals. The lesions are concentrated to the area around nose, cheek, chin, and forehead. The symptoms vary in severity and may include -resembling papules and pustules, vascular erythemas, and . Rosacea is often associated with eye-symptoms such as conjunctivitis and blepharitis. Moreover, the disease may be hereditary but the genetic factors are largely unknown. Although many therapies exist, the pathogenesis of rosacea is unclear and the disease has been described as an enigma for physicians.

31 Introduction

Triggering factors include sun exposure, emotional stress, cold weather, spicy foods or other alimentary factors, indoor heat, cosmetics, and medications. Treatments used are anti-inflammatory and antibacterial emulsions, azaleic acid, and oral antibiotics [210, 211]. As mentioned above, one possible etiologic factor suggested for rosacea is increased serine protease activity and cathelicidin expression [102].

Ichthyosis Ichthyotic diseases are a heterogeneous group of skin diseases, most of which are inherited. Skin changes may be present at birth, or appear later in life. In the so called primary ichthyoses, only the skin is affected. There are also several rare ichthyosiform or ichthyotic syndromes. Netherton syndrome (see below) belongs to these. Ichthyotic diseases may also be aquired and can occur as a result of malabsorption, hepatic and renal disease, and malignancies. The clinical symptoms of ichthyoses are dryness of skin, , and scaling. One difference separating ichthyoses from other chronic scaly skin diseases is the diffuse, uniform, and generally fixed pattern of scaling [202]. Sections below further describe some primary ichthyoses.

Ichthyosis vulgaris (IV) is the most common form of ichthyosis. It is also considered a mild form of ichthyosis. Around 1 in 250 are affected by this disease [202]. It is an autosomal dominant condition and similar to AD, the defective gene product is filaggrin. There is a close association between IV and atopic diseases. 37-50% of IV patients also show atopic features [191, 202, 212].

X-linked recessive ichthyosis (XRI) only affects men. The prevalence has been estimated to 1 in 2000 [202]. The underlying gene defect affects the enzyme steroid sulphatase [213, 214]. The function of steroid sulphatase is to cleave cholesterol sulphate and sulphated steroid hormones. Without it, cholesterol sulphate accumulates in skin. Cholesterol sulphate composes 12- 30% of lipids in the SC of XRI skin as opposed to 3% in healthy skin. The hyperkeratosis seen in XRI has been attributed to a defect in desquamation. The hypothesis is that cholesterol sulphate gives rise to an elevated pH in the SC, leading to decreased proteolytic activity. Cholesterol sulphate also has inhibitory effect on trypsin- and chymotryspin-like proteases, which

32 Introduction may directly affect desquamation. XRI is classified as a typical ‘retention ichthyosis’ with a delayed degradation of desmosomes [114, 202, 215].

Lamellar ichthyosis, is a very uncommon, often severe, autosomal recessive disorder. The incidence is about 1 in 500 000. It is characterized by large, pigmented scales in the absence of erythroderma [202]. The defect gene or gene product can be transglutaminase-1 [216], lipoxygenase [217], ichthyin [218, 219] or ABCA12 [220]. For instance, in a recent paper, a strong correlation was found between the mutation of the ichthyin gene and specific ultrastructural changes in the SG [218]. Ichthyin is a several transmembrane-spanning receptor, with homologies to G-protein coupled receptors. Although ichthyin is highly expressed in keratinocytes, much is still unknown about its role in skin and elsewhere [219].

Netherton Syndrome Netherton syndrome (NS), also known as Comel Netherton syndrome, is a very rare autosomal recessive genetic skin disorder. The incidence worldwide is 1 in 100 000. In Sweden, approximately 10-15 people live with this disease. The skin of the newborn NS patient is red (erythroderma), thin, and scaly. Water is lost through the skin due to barrier defects. To survive, the newborn requires intense care. Topical moisturizing treatment of the skin is needed several times per day to prevent transepidermal water- loss. The newborn also suffers from difficulties to retain food, and impaired uptake of nutrients. Another important concern is the impaired temperature regulation in the patients. It is important to avoid overheating as well as cold.

Hair defects described as ‘bamboo hair’ (trichorrhexis invaginata) are diagnostic in NS; the hair shaft displays bulbs which break easily. Other symptoms are itching eczema and atopic features such as food, pollen, and animal dander allergies. Presently, there is no effective treatment for the disease but the symptoms usually ameliorate with age. The skin symptoms gradually fade and another state called ichthyosis linearis circumflexa (ILC) arises. As indicated by the name, ILC is an ichthyotic symptom with circular lesions of dry scaly skin. Sometimes lesions have bleeding edges. Flare-ups of red skin may appear, probably due to for instance stress or infections [202, 221].

33 Introduction

As discussed above (see section on LEKTI) NS is caused by mutations in the SPINK5 gene which encodes for the serine protease inhibitor LEKTI. Several mutations, most leading to premature termination codons, have been found in different families [135, 137]. It is now generally believed that the lack of functional LEKTI causes loss of inhibition of proteolytic enzymes in skin. An excessive amount of desmosome-degrading proteolytic activity leads to the premature desquamation of the barrier forming layers of the epidermis. In particular KLK5 and KLK7 are suggested to be involved in this process [140, 142, 143, 222]. Coding gene polymorphisms of LEKTI have been linked to atopic diseases such as AD and asthma [223]. A suggested explanation for this is that LEKTI is required for the differentiation of immune cells in the thymus [224]. Severity of NS has been correlated to serine protease activity and residual LEKTI expression [140, 225].

34 Introduction

Short Summary The skin is an important, multilayered, organ which confers protection against microorganisms, chemicals, trauma, temperature, and water loss. Several of the protective properties of the skin are dependent on a working balance of proteolytic enzyme activity and inhibition. KLKs are serine proteases expressed in skin, suggested to be important for skin physiology. Picture below (Figure 6) summarizes possible functions and utilizations of KLKs described in the text.

Figure 6. Summary. Possible KLK functions and utilizations in different organs (black wavy arrow), processes (black straight arrows), or skin diseases (grey arrows).

35 Aims

AIMS

The general aim of the investigations presented in this thesis was to expand our knowledge of KLKs and especially their involvement in skin physiology. Major concerns were the possibility of an activation cascade of KLKs in the SC and how this cascade might be regulated. It was also of interest to find other possible functions of KLKs besides their involvement in the desquamation process.

Specific aims were as follows:

I. To identify and characterize previously unknown proteases in the SC.

II. To further characterize KLK5 and KLK7 with special focus on activation mechanisms.

III. To identify new proteinase inhibitors active against KLKs.

IV. To further characterize KLKs in the SC regarding effects of various inhibitors and substrates.

V. To study possible functions of KLKs in inflammation, in particular via activation of PAR-2.

36 Materials and Methods

MATERIALS AND METHODS

Protein Extraction

Extracts of Plantar Stratum Corneum Papers I, III, and IV are based on extracts of plantar SC. The SC material used was collected by Egelrud and coworkers in cooperation with the Society for Swedish Pedicurists from the early 1990:s and forward [91]. Clipped or cut scales of hyperplastic but otherwise normal plantar SC was air dried and stored at -20ºC until used. For one batch of protein extraction, 10 grams of dry scales were homogenized by grinding in a mortar or by use of an electronic mixer. If using a mixer, scales were first allowed to swell in 0.1 M acetic acid. Homogenized SC was suspended in 100 ml 0.1 M acetic acid. The pH was adjusted to 2.5 with glacial acetic acid and the suspension was left over night at 4ºC. The slurry was cleared by centrifugation and the pellet was washed with another 100 ml of 0.1 M acetic acid. Supernatants were pooled and filtered, first through filter paper (OOH-quality, Munktell filter AB, Grycksbo, Sweden), and then through 0.45 µm Minisart N filters (Sartorius, Göttingen, Germany). The filtrate was subjected to reversed phase chromatography (RPC) on a Fast Protein Liquid Chromatography (FPLC) apparatus (Amersham Biosciences, Uppsala, Sweden). In Papers I and III gel exclusion chromatography was also performed.

Chromatography methods are used to separate proteins depending on their different properties. In RPC, the column matrix used has hydrophobic properties. Proteins in aqueous solution loaded onto the column adhere to the hydrophobic phase. During gradient elution, proteins are separated, based on their hydrophobicity, and collected into concentrated fractions. Smaller molecules are often found in the earlier fractions and larger molecules in the later fractions. In gel exclusion chromatography, proteins are passed through a porous, semisolid substance. Smaller molecules tend to diffuse into the interior of the porous particles whereas larger molecules flow unhindered right through. Thus, proteins are separated depending on size and the largest ones are found in the earliest fractions [226, 227].

37 Materials and Methods

Tape Stripping Tape stripping is a commonly used method within skin-research. It is a method to remove the whole or only the outermost parts of the SC. If desired, soluble corneocyte-proteins can be extracted from the tape-strips. In this thesis (Paper II), tape stripping was performed in the following manner: A 15 centimeter piece of scotch tape (Polyester Tape Trans. 12.7 mm, No. 021200-07410, 3M, St Paul, MN) was applied on the forearm of a human volunteer. The tape strip was carefully rubbed to ensure attachment of corneocytes. After removal, the tape strip was sealed into a plastic bag containing 1 ml of 1 M acetic acid, with non-adhesive sides in contact, and left over night at RT on a rocking platform. After extraction, the bag was washed with 1 ml of 0.1 M acetic acid. Extracts and washings were pooled and freeze-dried. Freeze-dried material from one single tape strip was mixed with 30 µl of electrophoresis sample buffer (SB) (62.5 mM TRIS-HCl pH 6.8/10% glycerol/0.001% bromophenoleblue/1.75% SDS/2% β-mercapto- ethanol). Of this, 10 µl were loaded on a 12% SDS-PAGE gel and subjected to electrophoresis and immunoblotting [228, 229]. Remaining proteins were extracted by addition of 0.5 ml of SB to the bag which was again sealed and incubated over night at 37ºC. This second extract was subjected to SDS- PAGE and coomassie-staining and used as a normalization of the amount of corneocytes trapped on each tape strip.

Recombinant Proteins Various methods have been employed to produce the recombinant proteins used in this thesis. The first systems to be used were insect cell systems. Proteins have been produced in both High Five insect cells and Drosophila Schneider 2 cells. Eukaryotic insect cells, as compared to prokaryotic bacteria, allow for a more accurate folding and posttranslational modification of the protein produced. The subsequently used yeast cell system (Pichia pastoris Expression System), combines the advantage of eukaryotic cells with an easy mode of growth resembling that of Escherichia coli [230]. Modifications were made in the sequences of some proteins. These include N- or C-terminal tags to facilitate purification and mutated propeptide sequences with cleavage site for recombinant enterokinase to facilitate activation. No differences in activity or function of the various variants of the same enzyme have been detected. The following

38 Materials and Methods sections aim to clarify what constructs have been made. For primer pairs used, see Table 1. For primer sequences, see Table 2.

Paper II describes the production of wild type proKLK5 (pro-hK5) and proEKKLK5 (proEKhK5) in High Five insect cells. proEKKLK5 is a mutated form with the activation cleavage site replaced by an enterokinase cleavage site. In Paper III, KLK5 is produced in Pichia pastoris strain X-33 as a fusion protein with the propeptide of KLK7 mutated with an enterokinase cleavage site (proEK7KLK5). Production of wild type proKLK7 used in Papers I and II is described by Hansson et al. [231]. This protein was expressed in murine C127 cells and could be activated by agarose-bound trypsin. Paper V describes the production of proEKKLK7 in Pichia pastoris strain X-33 and the subsequent activation with recombinant enterokinase. This KLK7 variant was also used in Paper III. KLK8 production is described in Paper V. This protein, containing C-terminal His tag, was produced as proform in Pichia pastoris strain KM71H. Although it contains the native propeptide sequence, proKLK8 was easily activated with recombinant enterokinase. In Paper II, proKLK14 was produced in Drosophila Schneider 2 cells. Two variants were made, one containing C- terminal His and V5 tags. Although no mutation of the propeptide had been made, proKLK14 was readily activated by recombinant enterokinase treatment. KLK14 was also produced in Pichia pastoris strains GS115 and KM71H. As expected, strain GS115 yielded the proform of KLK14 (proKLK14P) used in Paper I. Surprisingly, strain KM71H, transfected with the same plasmid, yielded a catalytically activated form (referred to as KLK14P in Paper II).

Production of SPINK9 in Escherichia coli is described in Paper III. This protein was constructed with N-terminal thioredoxin-, His-, and S-tags for purification purposes and an enterokinase cleavage site. After purification, tags were cleaved off by incubation with recombinant enterokinase. Recombinant enterokinase used for processing of SPINK9 in Paper IV was produced at the lab in Pichia pastoris strain X-33, principally according to the methods described by LaVallie et al. and Vozza et al. and purified using a protocol modified from Peng et al. [232-234]

39 Materials and Methods

Table 1. Primer pairs used for production of recombinant proteins. Recombinant protein Forward primer(s) Reverse primer(s) Protein tag proKLK5 P12 rLJ4 No tags proEKKLK5 P12 rLJ4 No tags proEK7KLK5 KLK5EK7F1 rJ9x No tags proEKKLK7 KLK7FD4K KLK7R2 No tags proKLK8 KLK8F1 KLK8R1 C-terminal His tag proKLK14 KLK14-F1g KLK14-R1 No tags proKLK14V5HIS KLK14-F1g KLK14-as C-terminal His and V5 tags (pro)KLK14P KLK14-F1 KLK14-R1 No tags SPINK9 SPINK9F1 SPINK9R1 N-terminal TRX-, SPINK9LICs2 SPINK9LICas His- and S-tags* *All tags can be removed by enterokinase cleavage

Polymerase Chain Reaction (PCR) The polymerase chain reaction (PCR) technique was used to amplify cDNA for cloning purposes (Papers II, III, and V) and to quantitate the SPINK9 expression at the mRNA level using a human tissue cDNA panel (Clontech laboratories, Mountain View, CA) (Paper III). Table 2 shows sequences for primers used for cloning; Table 3 shows primers used for semiquantitative PCR.

Table 2. Primers used for cloning. Primer F/R Sequence Comment KLK14-as R 5’-GTC CCG CAT CGT TTC No stop codon included CTC AAT CC-3’ KLK14-F1 F 5’-CAA GAG GAT GAG AAC Starts at KLK14 propeptide AAG ATA ATT GG-3’ KLK14-F1g F 5’-AGC CCC TAA AAT GGT Starts before KLK14 CCT CCT GCT-3’ prepeptide, includes improved Kozac sequence KLK14-R1 R 5' TCA TTT GTC CCG CAT Stop codon included CGT TTC CTC 3' KLK5EK7F1 F 5’-CAC GTG GGA AGA AGA For KLK5 with KLK7 CGA TGA TGA CAA GAT CAT propeptide and rEK cleavage CAA TGG ATC CGA CTG CGA site TA-3’ KLK7EKas R 5’-GGC GCC ATC AAT AAT Mutagenesis primer, gives CTT GTC ATC ATC GGC TTC incomplete rEK cleavage site TTC TCC TGC AG-3’ (DDDK)

40 Materials and Methods

KLK7EKs F 5’-CTG CAG GAG AAG AAG Mutagenesis primer, gives CCG ATG ATG ACA AGA TTA incomplete rEK cleavage site TTG ATG GCG CC-3’ (DDDK) KLK7FD4K F 5’-GAA GAA GAC GAT GAT Combined cloning and GAC AAG ATT A-3’ mutagenesis primer, inserts D to give complete rEK cleavage site (DDDDK) KLK7R2 R 5’-TCT AGA TTA GCG ATG Includes stop codon and XbaI CTT TTT CAT GGT GTC AT-3’ site KLK8F1 F 5’-CAG GAG GAC AAG GTG Starts at propeptide CTG GGG-3’ KLK8R1 R 5’-CCC TTG CTG CCT ATG Does not include stop codon ATC TTC TTG-3’ P12 F 5’-GTC TCA GCG CAG TGC Upstream of initiating CGA TGG T-3’ methionine (KLK5 primer) rEKas R 5’-GTC GGA TCC ATT GAT Mutagenesis primer, gives GAT CTT GTC GTC GTC GTC rEK cleavage site (DDDK) in ATC CGA CCG GGC-3’ KLK5 propeptide rEKs F 5’-GCC CGG TCG GAT GAC Mutagenesis primer, gives GAC GAC GAC AAG ATC ATC rEK cleavage site (DDDK) in AAT GGA TCC GAC-3’ KLK5 propeptide rJ9x R 5’-TCC CCC CGG GTC AGG Includes stop codon and AGT TGG CCT GGA TGG TTT XmaI site (KLK5 primer) C-3’ rLJ4 R 5’-GAG GAG AAG CCC GGT Includes stop codon (KLK5 TCA GGA GTT GGC CTG GAT primer) GGT TT-3’ SPINK9F1 F 5’-CTG ACT GCC TTG GCC Upstream of initiating ACT TCA GT-3’ methionine SPINK9LICas R 5’-GAG GAG AAG CCC GGT Includes stop codon in TAA CAT TTT CCA AAA TGT published SPINK9 sequence. ACA AAT TTA AG-3’ For cloning into LIC vector SPINK9LICs2 F 5’-GAC GAC GAC AAG ATA Starts at proposed cleavage GAA TGT GCC AAA CAG ACG site. For cloning into LIC AAA CA-3’ vector SPINK9R1 R 5’-ATG TGA CTC TTG TGA Downstream of stop codon ATA GAT GCA TTA-3’ F = forward primer, R = reverse primer.

Table 3. Primers used for semiquantitative PCR. Primer F/R Sequence SPINK9as1 R 5’-GAT GAG TAG GCA ATG TGG CAT AGA T-3’ SPINK9s1 F 5’-AAA GTT ACC ACC AGG ACA ACA GAG A-3’ F = forward primer, R = reverse primer.

41 Materials and Methods

Analytical Methods

Electrophoresis and Immunoblotting Recombinant proteins and plantar SC extracts were analyzed through electrophoresis and immunoblotting [228, 229]. 12% SDS-PAGE gels were used for separation of enzymes around 30 kDa in size whereas 15% gels were used for the smaller inhibitors. Gels were either directly stained with coomassie brilliant blue or proteins were transferred to polyvinylidene difluoride (PVDF) membranes for immunoblotting. Table 4 shows primary antibodies used in immunoblotting.

Table 4. Primary antibodies used in immunoblotting. Antigen Species Immunogen Antibody Company/reference name KLK5 Rabbit Synthetic peptides Cotton/Co-5 Own production (Paper II) KLK7 Rabbit rKLK7 RASP Symbicom AB [235] KLK8 Mouse rKLK8 MAB1719 R&D systems KLK11 Goat rKLK11 AF1595 R&D systems KLK14 Rabbit Synthetic peptides Al-14 Own production (Papers I and II) LEKTI Mouse rLEKTI protein 1C11G6 Zymed laboratories SKALP Goat Unknown HP9025 Hycult biotechnology SLPI Goat rSLPI AB-260-NA R&D systems SPINK9 Rabbit rSPINK9 and synthetic Ce-SPINK9 Own production peptides (Paper III) r = recombinant. All antibodies are directed against human proteins.

Zymography Zymography is a method that allows detection of protease activity on a SDS-PAGE gel. Unreduced proteins are separated in a gel containing 0.1% denatured casein. After electrophoresis, the SDS is washed away and the gel is incubated over night at 37ºC to allow for degradation of casein. Gels are counterstained with coomassie brilliant blue. Appearance of light bands indicates caseinolytic activity.

Chromogenic Peptide Substrates Chromogenic peptide substrates were used in all of the papers in this thesis. These are small, 3-5 amino acid residues long, peptides, linked to a

42 Materials and Methods chromophore. The chromophore is in this case p-nitroaniline (pNA). The residues mimic the sequence of the natural substrates of various enzymes. Upon cleavage of the substrate, pNA is released giving rise to a color reaction which can be detected at 405 nm in a photometer. The rate of pNA formation is proportional to the proteolytic activity. With the aid of chromogenic peptide substrates, KLK activity could be determined and quantitated. Table 5 shows substrate names, structures, and examples of proteases preferentially cleaving each substrate. Only substrates used in this thesis are included. Substrates were purchased from Haemochrom Diagnostica AB (Mölndal, Sweden).

Table 5. Chromogenic peptide substrates. Substrate Structure Substrate for* S-2251 H-D-Val-Leu-Lys-pNA·2HCl Plasmin S-2288 H-D-Ile-Pro-Arg-pNA·2HCl Broad spectrum of serine proteases S-2302 H-D-Pro-Phe-Arg-pNA·2HCl Plasma kallikrein S-2586 MeO-Suc-Arg-Pro-Tyr-pNA·HCl Chymotrypsin *According to manufacturer.

Immunohistochemistry Immunohistochemistry is a method to examine the localization of specific proteins in tissues. This is accomplished through cutting tiny sections of paraffin-embedded or frozen tissues and staining them with protein-specific antibodies.

Tissues For immunohistochemical staining in Papers I and III, four mm punch biopsies were obtained from the lower back or palms of healthy human volunteers. Figures in papers show representative images, usually from four individuals (females and males included). For Paper V, punch biopsies were obtained from patients with AD and rosacea. AD and rosacea tissues were supplied by Dr Martin Steinhoff at the Department of Dermatology, University of Münster, Münster, Germany. All tissues were obtained after informed consent from human volunteers according to the declaration of Helsinki principles. The studies were approved by the Medical Ethical Committee of Umeå University.

43 Materials and Methods

Staining Procedures Skin biopsies were fixed in buffered formaldehyde and embedded in paraffin according to routine protocols. Four µm sections were cut and placed on superfrost plus glass slides. Deparaffinization and rehydration was performed according to routine protocols. Endogenous peroxidase activity was quenched through hydrogen peroxide treatment. Sections were rinsed three times in phosphate buffered saline between each of the following steps: Ten minutes incubation at RT in 0.1 M citric acid preheated to boiling point; fifteen minutes incubation at RT in 0.04 mg/ml pepsin diluted in 0.2 M HCl; blocking in 20% goat serum for thirty minutes at RT; over night incubation at 4ºC with primary antibody diluted in 20% goat serum; thirty minutes incubation at RT with biotinylated secondary antibody. Sections were developed with the VECTASTAIN Elite ABC kit (Vector Laboratories, Burlingame, CA) and the AEC+ substrate chromogen (DakoCytomation, Solna, Sweden). The above protocol was used in Papers I and III. Antibodies were diluted to concentrations ranging from 0.4 to 1.6 µg/ml. Negative controls were prepared by omission of the primary antibody, replacement with normal rabbit IgG or through adsorption experiments. Table 6 summarizes primary antibodies used in this thesis.

Table 6. Primary antibodies used for immunohistochemistry. Antigen Species Immunogen Antibody Company/reference name KLK5 Rabbit rKLK5 Pe-C1 Own production [86] KLK7 Rabbit rKLK7 RASP Symbicom AB [235] KLK14 Rabbit rKLK14 Ra-14 Own production (Paper I) KLK14 Rabbit Synthetic peptides Em-14 Own production (Papers I and II) LEKTI Mouse rLEKTI protein 1C11G6 Zymed laboratories PAR-2 Rabbit Synthetic peptides H-99 Santa Cruz biotechnology SPINK9 Rabbit rSPINK9 and synthetic Ce-SPINK9 Own production peptides (Paper III) r = recombinant. All antibodies are directed against human proteins or peptides.

44 Materials and Methods

Surface Plasmon Resonance (SPR) Surface Plasmon Resonance (SPR) technologies can be used to detect and quantify molecular interactions in real time. In Biacore instruments, one interaction partner is immobilized on the lower surface of a sensor chip whereas the second, solution-borne, partner is passed over it in a continuous flow. Light of the right wavelength, coming from the right angle (the SPR angle) excites plasmons (electron charge density waves) in the gold film of the sensor ship. SPR is then seen as a dip in the intensity of the reflected light at the SPR angle. Binding of a biomolecule to its immobilized partner, results in a change of the refractive index of the medium near to the sensor chip surface, and consequently a change of the SPR angle. This change can be monitored over time and is displayed as a sensorgram. By following the association and dissociation of biomolecules over time, interaction strength and kinetics can be calculated. The SPR signal is expressed in resonance units (RU). One RU generally corresponds to 1 picogram of material bound per square millimeter of surface area [236].

In Paper III, protein interaction studies were performed on a Biacore 2000 (Biacore, Uppsala, Sweden). Recombinant SPINK9 was immobilized on CM5 sensor chips. Solution-borne recombinant KLK5, 7, 8, and 14, were investigated for possible binding to SPINK9. For calculation of affinity and kinetic parameters, eleven different concentrations were used. Sensorgrams were evaluated using the BIAevaluation software version 3.2 (Biacore).

Immunofluorescence Paper V describes the immunofluorescence of KNRK-PAR2 cells as a means to evaluate KLK mediated PAR-2 cleavage. KNRK-PAR2 cells stably express the human PAR-2 receptor. The PAR-2 receptor in these cells has been genetically modified to include an N-terminal Flag epitope and a C-terminal HA11 (12CA5) haemagglutinin epitope [237-239]. Cells, which had been grown on glass coverslips, were subjected to KLK or control to allow for receptor cleavage. Staining was performed with primary mouse- anti FLAG (F3040, Sigma, St Louis, MO) and rabbit anti-HA (H6908, Sigma) antibodies. Secondary antibodies were FITC-conjugated goat anti- rabbit (111-095-003, Jackson Immuno Research, Camebridgeshire, UK) and Cy3 anti-mouse antibody (715-166-151, Jackson Immuno Research).

45 Materials and Methods

Coverslips were mounted on superfrost slides and observed in a confocal microscope.

Color of stained KNRK-PAR2 cells allowed for discrimination between intact and cleaved PAR-2. The secondary antibody to anti-FLAG was conjugated to Cy3, a red fluorophore, and the secondary antibody to anti- HA was conjugated to FITC, a green fluorophore. As proteolytic cleavage of the receptor removes the N-terminal FLAG epitope, the resulting merged confocal image would display only green color around the cell-membrane. If no cleavage had occurred, both the FLAG and HA11 epitopes would give rise to fluorescent staining and hence the confocal image would show yellow color (red and green together). Figure 7 explains the principle of this antibody staining.

Figure 7. Principle of KNRK-PAR2 immunofluorescence staining. The FLAG and HA11 epitopes of the PAR-2 receptor are visualized through staining in two steps to yield red and green fluorescence respectively. Panel to the right shows confocal images, using different filters, of stained KNRK- PAR2 cells. Red color from the FLAG-epitope and yellow color in the merged image indicates intact receptor around the cell membrane.

46 Materials and Methods

Intracellular [Ca2+] Measurements Since immunofluorescence (described above) only gives information concerning cleavage of the PAR-2 receptor, not activation, intracellular Ca2+ measurements were performed. Activation of PAR-2 leads to the release of Ca2+ from intracellular pools and an increase in cytosolic Ca2+ concentration [161]. Cytosolic Ca2+ concentration can be measured by spectrofluorometry with the fluorescent calcium indicator fura2/AM (Molecular Probes, Eugene, OR). KNRK-PAR2 cells (described above) were grown on glass coverslips and loaded with fura2/AM. Coverslips were inserted into a cuvette in a spectrofluorometer and KLK was injected. Fluorescence was immediately measured at 340 and 380 nm excitation wavelengths. Intracellular Ca2+ concentration was calculated from the ratio of the fluorescence signal at the two wavelengths. Trypsin was used as a positive control.

47 Results and Discussion

RESULTS AND DISCUSSION

Paper I Paper I describes the purification of KLK14 from human plantar SC extracts. KLK5 and KLK7 had previously been purified from the same material [64, 91] but the action of KLK5 alone could not explain the total trypsin-like activity found in the extracts. At least 40% of the trypsin-like activity had to be the result of another or several other proteases. Fortunately, some characteristic within the KLK14 protein made it separate into later chromatography fractions as compared to KLK5 and KLK7 when using gel exclusion chromatography columns. This protein was first detected as a band slightly below KLK7 on a casein zymography gel of cromatography fractions, and it migrated with an apparent molecular mass of 25 kDa on SDS-PAGE. Amino acid sequencing of the protein separated by SDS-PAGE and transferred to a PVDF filter finally identified it as KLK14. An explanation to why KLK14 had not been previously detected in SC extracts is that when using whole extracts, the KLK7 and KLK14 proteins did not separate enough on zymography gels but was considered one single band (KLK7).

The subsequent production of specific antibodies allowed the further characterization of KLK14. Immunoblotting of tape strip extracts revealed that KLK14 is present in the superficial SC of normal non palmo-plantar skin of all subjects tested so far. However, semi-quantitative analysis comparing staining intensities of KLK14 in SC and amounts of recombinant protein made clear that KLK14 is present in lower levels than KLK5 and KLK7 (1/10 to 1/100). These results are strengthened by conclusions from other researchers. Komatsu et al. finds KLK14 to be one of the minor KLKs in the SC. Their ELISA shows that KLK14 is present in amounts 10 times less than KLK5 and 50-100 times less than KLK7 [99]. The proportion of active versus proform of KLK14 was evaluated with an α-1AT assay. α- 1AT reacts with active serine-proteases forming a covalent complex which is possible to discern on an immunoblot. Results showed that the major part of immunoreactive KLK14 in the plantar SC extract is present in its catalytically active form.

48 Results and Discussion

The next question to be addressed was the localization of KLK14. Many attempts were made to answer this question. First, peptide antibodies Al-14 and Em-14 were used. However, these were considered unreliable as they stained the whole epidermis but the staining could not be abolished by adsorption control experiments. When antibodies with recombinant KLK14 used as immunogen had been developed, a specific pattern of KLK14 localization began to unfold. At first it was hard to obtain a reproducible staining but sometimes a strong staining was seen in specific skin structures. These were sweat gland ducts which were scarce in the sections of normal skin. To be certain of having sweat gland ducts on the sections, we began using sections of palmar skin, a tissue rich in sweat glands. Strong, apparently specific, staining was observed in intraepidermal parts of sweat gland ducts and the myoepithelial cells of sweat glands. When staining with peptide antibodies, this staining could be abolished with a ten times excess of the KLK14-peptides use for immunization. Unfortunately, recombinant KLK14 could not be used in adsorption experiments as the protein showed a tendency to adhere to the glass slides. This stickiness may be a property of KLK14 also reflected in the gel exclusion chromatography, which made its purification possible.

The observed localization of KLK14 differed from that of KLK5 and KLK7 [86, 87]. KLK5 and KLK7 are mainly detected in the SG and SC of both normal non palmo-plantar and palmo-plantar skin. KLK5 and KLK7 antibodies also stain cornified parts of intraepidermal sweat gland ducts but not dermal parts of sweat glands (results not shown). The low total levels of KLK14 as detected by immunoblotting of SC extracts may explain why KLK14 could not be found in the SG or the SC by immunohistochemistry. The levels might have been below the detection limit for the method used. However, there is also the possibility that KLK14 is truly a sweat-related protein. The presence of proteolytic enzymes in sweat has been investigated by Horie et al. who found that clean eccrine sweat (without epidermal contamination) contains a variety of proteases of which one has a molecular weight of 25 kDa. The 25 kDa protease was one of two proteases also found in an extract of isolated human sweat glands [240]. Poblete et al. and Hibino et al. later found that sweat contains KLK1 [241, 242]. However, KLK1 was detected as a 40 kDa band and the majority of S-2302 hydrolytic activity in sweat did not coelute with KLK1 on FPLC [242]. The sweat- related protein found by Horie et al. might as well have been some other

49 Results and Discussion protease, like KLK6, or KLK8, both of which are found in eccrine sweat glands through immunohistochemistry [89].

KLK14 is a very potent enzyme. As shown in Paper II and Paper V, KLK14 has a higher potential than KLK5 to cleave the chromogenic peptide substrates S-2288 and S-2302, and to cleave and activate the PAR-2 receptor. In theory, it could be beneficial, not to have this enzyme activated until it reaches the skin surface where it can contribute to the desquamatory processes. It is known that sweating is one of the triggering factors of itch in AD patients [243]. Moreover, itch can be induced experimentally by injection of trypsin- or chymotrypsin-like proteases [244, 245]. We therefore hypothesized that when sweating, KLK14 is released upon the skin surface, from where it is allowed to enter the viable part of the epidermis because of the impaired barrier function of AD skin. KLK14 mediated PAR-2 activation would then trigger pruritogenic nerve fibers resulting in itch. A later used (see Paper V), slightly modified staining technique revealed that KLK14 can in fact also be found in the SG of normal skin (results not shown), however, the strong staining in sweat glands and ducts should not be ignored. KLK14 delivered via the sweat may contribute to itch as well as other inflammatory responses in AD skin.

Paper II The presence in the SC of multiple KLKs, with the presumed ability to activate each other, led to the question of the existence of a proteolytic cascade; a cascade eventually ending in the breakdown of corneodesmosomes and hence desquamation. Similar cascades have been thoroughly studied in other physiological processes such as blood clotting [246]. The endogenous pH gradient over the SC [22] implied a potentially important regulatory mechanism.

Recombinant KLK5, KLK7, and KLK14 proforms were activated with recombinant preparations of their active forms at pH ranging from 4 to 11. Although both KLK5 and KLK14 displayed maximum trypsin-like activity against chromogenic peptide substrates at neutral to alkaline pH (pH 8.5-9), their ability to activate other KLKs differed both regarding substrate specificity and pH maxima. Results showed that KLK5 activates KLK7 best at an acidic pH, activation peaked at pH 5.5. KLK5 is also, to the best of our

50 Results and Discussion knowledge, the only enzyme found in the epidermis, which can activate KLK7. KLK5 activation of KLK14 peaked at pH 6.5. KLK5 was also shown to have autocatalytic properties. Active KLK5 activates proKLK5 with activation optima around pH 8-9. Similar to KLK5, KLK14 also activates proKLK5 at neutral to basic pH. No significant activation took place below pH 6 and the peak of activation was seen at pH 9. KLK14, in contrast to KLK5, cannot autoactivate. KLK7, with its chymotryptic substrate specificity, can activate neither KLK5 nor KLK14 proforms. Figure 8 shows a summary of the activation pattern within KLKs 5, 7, and 14. Results shown in Paper II have been strengthened by recent research by Yoon and colleagues. By using fusion proteins with the propeptide of KLKs fused to a soluble carrier protein they studied the ‘human KLK activome’. They confirmed for instance that KLK5 can autoactivate, preferentially at an neutral pH, and that KLK5 can activate KLK7 and KLK14 [247].

Figure 8. pH dependent activation pattern of the KLK activation cascade. Thick arrows show conversion from proform to catalytically active form via removal of the propeptide. Thin arrows show activating enzyme affecting conversion. pH numbers refer to approximate pH value at which activation reaches significant velocity.

51 Results and Discussion

Based on the results described above, we propose a model for the alleged activation cascade of KLKs in epidermis. We believe that KLK5 may act as a key enzyme due to its autocatalytic ability. KLK5 may autoactivate in the SG or in the deeper cell layers of the SC, just above the transition from the living SG, where the pH is close to neutral. As active KLK5 is pushed upwards, moving with the cell-layers towards the surface, more and more active KLK5 is accumulated. So is KLK14, which after being activated by KLK5 can activate more proKLK5. KLK14 on the other hand may also act as a negative regulator of KLK5, as it can degrade and thus inactivate KLK5. At the skin surface, KLK5 activation of KLK7 performs at its fastest rate. KLK5, KLK7, and KLK14 there act concerted on the corneodesmosome, breaking it apart, allowing the shedding of cells. The acidic pH of the skin surface may be important for this last step in several ways. As mentioned before, KLK5 degradation of the corneodesmosomal proteins DSG1 and DSG2 is only effective at an acidic pH [96]. Moreover, regulation of KLK activity through SPI can be pH dependent. LEKTI domains inhibit KLK5, KLK7, and KLK14 in a pH dependent manner. At an acidic pH, LEKTI is released from the of KLK5, bringing the inhibition to an end [141]. Figure 9 shows a schematic representation of the proteolytic cascade theory presented herein.

Figure 9. Model of the proteolytic KLK cascade in the SC. Thick arrows represent conversion of proform into active KLK via removal of the propeptide. Thin arrows represent activation (+) or inactivation (-) pathways.

52 Results and Discussion

Paper II also describes kinetic parameters of KLK5 and KLK14 compared to pancreatic trypsin, assayed with chromogenic peptide substrates. Results show that although inferior to trypsin, KLK14 is superior to KLK5 as regards maximum catalytic efficiency as well as substrate affinity. This should be taken into account when analyzing trypsin-like enzymes in skin. Although KLK14 is present in lower levels than KLK5, the total trypsin-like activity of KLK14 may be as great as, or greater than, that ascribed to KLK5. This, together with the different substrate specificities of individual KLKs implies they may all be important in skin physiology.

Paper III In our efforts to characterize the trypsin- and chymotrypsin-like activities in SC extracts, the presence and importance of endogenous inhibitors of KLK activity naturally came into focus. Previously known SPI in the epidermis were LEKTI, SKALP, SLPI, and α-1AT [117-119, 125, 127, 131, 248]. Analyses of partly purified endogenous inhibitors separated through RPC (Paper IV), lead us to suspect there were other, hitherto unknown, inhibitors present. RPC fractions of an acetic acid extract of plantar SC, showing inhibitory activity against KLK5, were pooled and subjected to further purification and concentration steps. Separation on SDS-PAGE finally revealed a protein-band which was analyzed through amino acid sequencing and mass spectrometry. Database searches identified this protein as the serine protease inhibitor Kazal-type 9 (SPINK9). We had thus identified a previously unexplored SPI in skin tissue.

Wu, Bartels, and Schroeder, who deposited the SPINK9 mRNA sequence in 2003, suggested the name LEKTI2 for the resulting protein. No doubt, being a Kazal-type inhibitor, SPINK9 shares great resemblance to LEKTI, encoded by the gene SPINK5. LEKTI though, is a multidomain inhibitor, having fifteen inhibitor domains, of which only two are true Kazal-type inhibitors. SPINK9 is a Kazal-type inhibitor, as defined by the spacing between six cystein residues in the amino acid sequence. In the Laskowski database (http://www.chem.purdue.edu/LASKOWSKI), 471 Kazal-type inhibitor sequences are deposited. According to this database, the reactive cleavage site of SPINK9 appears unique. There are no other reports of a H residue in position P2 (two amino acids N-terminal of the active site of the

53 Results and Discussion inhibitor). This unusual amino acid sequence may be reflected in the restrictive inhibitory pattern which we found for SPINK9.

Inhibitory effects of recombinant SPINK9 on recombinant KLK5, 7, 8, and 14 activity were investigated by use of chromogenic peptide substrates, fluorescent casein substrate (all except KLK8 which could not cleave this substrate), the macromolecular substrate fibrinogen, and through Surface Plasmon Resonance (Biacore) analysis. SPINK9 inhibitory capacity towards human thrombin, bovine trypsin, and bovine chymotrypsin was also investigated using chromogenic peptide substrates. Results showed that of the enzymes tested, SPINK9 efficiently inhibited only KLK5. This inhibition had a Ki of 30 nM. This is high compared to the Ki of SBTI on KLK5, which is 2.3 nM. However, we propose that the inhibition of KLK5 by SPINK9 might still be physiologically relevant. SBTI is not an endogenous inhibitor, and there are possibilities that the Ki of SPINK9 is in fact higher than that observed. Our recombinant SPINK9 preparations are made in Escherichia coli and parts of the preparation may contain proteins not correctly folded. It should be added that the SPINK9 preparation used differs from the SPINK9 species found in the SC extracts. The recombinant protein bears six additional amino acid residues in the N-terminal part as compared to the endogenous SPINK9. Among those six amino acid residues there is one cystein that is lacking in the native SPINK9. This unpaired cystein may cause dimerization and increase the risk for missfolding of the protein.

Although inhibition assays using small molecular weight chromogenic peptide substrates show inhibition by SPINK9 only towards KLK5, SPR (Biacore) analysis showed binding between SPINK9 and both KLK5 and KLK8. The binding properties revealed two types of binding reactions, one strong and one weak binding. This might be explained by a heterogeneous SPINK9 preparation (both correctly and incorrectly folded protein) or by binding of the inhibitor to other sites of the enzyme than the active site. To shed further light on the possible interaction between SPINK9 and KLK8, a high molecular weight substrate was used. It was shown that KLK8 mediated degradation of fibrinogen can only be partially inhibited by SPINK9 at high concentrations. KLK5 mediated degradation of fibrinogen can on the other hand be totally abolished by low concentrations of SPINK9. We interpreted these findings as strengthening the hypothesis that

54 Results and Discussion binding between KLK8 and SPINK9 takes place not at the reactive center but somewhere else on the enzyme molecule. The inhibition observed for KLK8 when using a high molecular weight substrate may thus be due to steric hindrance. Important to note is that very high concentrations of inhibitor had to be used in order to detect any inhibition of KLK8 activity, further implicating that SPINK9 is probably not a physiologic inhibitor of KLK8.

The tissue expression and localization of SPINK9 was analyzed through PCR and immunohistochemistry. According to PCR, using a human tissue cDNA panel, highest expression of SPINK9 can be observed in skin. Palmar skin is the only tissue for which a band appears after only twenty-five PCR cycles. After thirty cycles, thymus, brain, liver, and pancreas are also positive for SPINK9 cDNA. After thirty-five cycles, all tested tissues except leucocytes are positive. Thus, SPINK9 seems to be preferentially expressed in skin tissue. There are, however, differences depending on the type of skin. Results from immunoblottig and immunohistochemical analyses, showing that high SPINK9 expression is limited to palmo-plantar epidermis was confirmed by PCR. At low numbers of PCR cycles (twenty-five cycles), only palmar skin was positive for SPINK9. It should be noted that palmo-plantar skin has another physiology than the skin on the rest of the body. Palmo-plantar skin is thicker, have a high density of sweat glands but no hairs. The thickness of the palmo-plantar skin and the high resistance to mechanical trauma displayed may be reflected in the tissue specificity of SPINK9.

Paper IV Paper IV describes the characterization of the plantar SC extracts on which much of this thesis (Paper I and Paper III) is based. An acetic acid extract made from 10 grams of dry plantar SC was subjected to reversed phase chromatography. Proteins were thus separated and fractions were analyzed in various ways. Results from immunoblots showed the presence of KLKs 5, 7, 8, 11, and 14 in the fractions and how these enzymes chromatograph. In a typical FPLC chromatogram of thirty 1-ml fractions, KLKs elute mainly in fractions 15-18. Small inhibitors of the protein-class, LEKTI, SKALP, SLPI, and SPINK9, eluted in earlier fractions; mainly in fractions 9-14. Thus, fractions 9-14 were termed ‘inhibitor fractions’, and pooled

55 Results and Discussion fractions 15-18 were termed ‘enzyme pool’ in this paper. These fraction numbers are valid in general but may be slightly displaced in different FPLC runs.

Fractions were also analyzed for activity towards chromogenic peptide substrates. As expected, activity coeluted with KLKs. ‘Inhibitor fractions’ were analyzed for inhibitory capacity against both ‘enzyme pool’, considered to contain a representative mix of proteolytic activity, and recombinant KLKs. The partial separation of inhibitors gave some clues as to how each inhibitor contributes to KLK inhibition in the SC but the reaction is complex and fractions may include other, unknown inhibitors. The most intriguing result of this assay was that KLK8 could not be specifically inhibited by any inhibitor present in the ‘inhibitor fractions’.

Further analyses were made using recombinant SKALP, SLPI, and SPINK9 as well as pure soybean trypsin inhibitor (SBTI) and aprotinin. Noticeable results were that only KLK5 was inhibited by SPINK9, and only KLK7 was efficiently inhibited by SLPI. Furthermore, KLK8 was strongly inhibited by aprotinin but not by SBTI whereas KLK14 was strongly inhibited by SBTI. Based on these inhibition patterns combined with special substrate specificities, KLK activities in the extract were classified into four distinguishable groups: KLK5-like activity responsible for 33% of trypsin- like activity in extracts, KLK7-like activity responsible for 76% of chymotrypsin-like activity, KLK8-like activity responsible for 46% of trypsin-like activity and finally, KLK14-like activity responsible for 21% of trypsin-like activity. This refers to trypsin-like activity as assessed with chromogenic substrate S-2288 and chymotrypsin-like activity as assessed with S-2586.

Additionally, an assay showing that the combination of SLPI and SPINK9 abolishes all fibrinogen degrading activity in the ‘enzyme pool’ suggests that KLK5 and KLK7 are important as general degraders of macromolecular substrates in the SC. However, conclusions should be considered tentative as many parameters affect the results. For once, experiments were performed in a highly artificial milieu considering for instance water content, ionic strength, and pH. Both activity and inhibition of KLKs are pH dependent [141, 249]. Moreover, the harsh processing of the extract may have led to denaturation of important enzymes and inhibitors, and only

56 Results and Discussion proteins extractable by acetic acid were at all present in the extract. Results in Paper IV contribute to the understanding of the complex interplay between KLKs and their inhibitors in the SC. It should also be kept in mind that physiological substrates in the skin for by instance KLK8 remain to be identified.

Paper V The new concept of proteases as modulators of inflammatory and pruritogenic processes through activation of PARs [30, 108, 151], pointed out other possible functions of KLKs in skin, besides regulation of desquamation. To test the hypothesis that KLKs can cleave and activate PAR-2, immunofluorescence and Ca2+ assays were performed (see Materials and Methods). Results indicated that KLK5 and KLK14 but neither KLK7 nor KLK8 can cleave the N-terminal part of PAR-2. This, however, does not prove that the PAR-2 receptor is activated. Cleavage may also serve to inactivate the receptor. Therefore, Ca2+ responses in KNRK- PAR2 cells, stably expressing PAR-2 were evaluated after treatment with KLK5, 7, 8, or 14. These experiments strengthened the prior results. Treatment of cells with KLK5 and KLK14 led to mobilization of Ca2+ from intracellular stores indicating activation. We had thus identified KLK5 and KLK14 as two endogenous epidermal proteins with PAR-2 activating ability that could possibly activate human PAR-2 in vivo. Our results confirm previously published studies by Oikonomopoulou and colleagues. They found that human KLKs 5, 6, and 14 activate murine PAR-2 but they did not evaluate KLK7 and KLK8 [250]. Other proteases in the epidermis proposed to activate PAR-2 are mast cell tryptase [110] and trypsin IV [182], both are, like KLK5 and 14 able to activate PAR-2 in vitro. In AD and psoriasis, the number of mast cells in the dermis is increased. In AD skin, tryptase-containing mast cells are found close to the dermal-epidermal border and in psoriatic skin, they may even invade the epidermis [110]. However, KLKs seem to be more likely candidates to activate PAR-2 based on their respective location in the skin.

Under normal conditions, highest expression of PAR-2 is observed in the SG whereas during inflammation, PAR-2 is expressed also in lower layers of the epidermis [108, 110]. KLK5 is also expressed in the SG whereas KLK14, as discussed above (Paper I), may be more of a sweat-related

57 Results and Discussion protein present on the skin surface. Paper V, however, includes an immunohistochemical study showing that KLK14 and PAR-2 are colocalized in the SG and the SC of inflamed skin tissue from AD and rosacea patients. This colocalization strengthens a role for KLK5 and KLK14 in inflammatory and pruritogenic reactions in inflamed skin tissue.

58 General Discussion and Future Perspectives

GENERAL DISCUSSION AND FUTURE PERSPECTIVES

After the purification of KLK5 and KLK7 from human plantar SC extracts, these two enzymes were considered as important proteolytic enzymes in epidermal tissue. Results presented herein strengthen these assumptions and adds KLK14 as a possible important factor in skin physiology. In this work, mainly KLKs 5, 7, 8, and 14 were studied. However, additional KLKs may prove important in skin function as many KLKs are expressed in skin tissue and may be involved in a complex network of proteolytic activation and degradation.

Despite great homology, individual KLKs show diverse substrate specificities and consequently various inhibitor specificities. One noteworthy finding is that KLK8 cannot be inhibited by any endogenous inhibitor in our SC extracts. KLK8 is also the only one of our studied KLKs which can cleave S-2251, indicating specificity clearly different from that of KLK5 and KLK14. Results by Komatsu et al., pointing out KLK8 as one of the most abundant KLKs in the SC and in sweat [99], makes the function of KLK8 an interesting question. Which are the true substrates in skin for this enzyme and which are the inhibitors of KLK8? Yoon et al. partly answers this question. In their study of the human KLK activome, KLK8 cleaves the propeptides of KLK1-3, 5-6, 9, and 11-12 at varying degrees. But the activation rate is slow when compared to KLK5 and KLK14 mediated activation of the same KLKs [247]. Nevertheless, KLK8 is recognized as an enzyme involved in desquamation and epidermal proliferation based on studies of knock-out mice [100, 101].

The difference between KLK8 on the one side and KLK5 and KLK14 on the other may be reflected in the apparent inability of KLK8 to activate PAR-2. Through activation of PAR-2, KLK5 and KLK14 are both likely to be involved in inflammatory and pruritogenic processes in skin. This renders them possible therapeutic targets for a range of inflammatory skin diseases. Regulation of KLKs in diseased skin may be accomplished through application of specific inhibitors. If KLK5 is truly the key enzyme we speculate it to be, KLK5 may be an interesting target. The herein described discovery of the KLK5 inhibitor SPINK9 in human palmo-plantar

59 General Discussion and Future Perspectives skin may thus be an important finding. However, many questions remain regarding the function of this inhibitor. Is it really specific for KLK5, and if not, which other proteases does it inhibit? What is the physiological role of SPINK9 and what are the implications of its restrictive expression pattern?

A conference abstract (35th Annual Meeting of the ADF, February 28 – March 1, 2008) by Wu and colleagues has recently come to our attention. Wu and colleagues have purified SPINK9 from heel SC extracts and conclude that the major SPINK9 species is the 61-amino acid residue variant. This is the same variant which we have found in plantar SC extracts. They also found 62- and 63- residue variants of SPINK9 to be major contaminants. Analyses of recombinant SPINK9 preparations revealed that the 62- and 63-residue variants have antimicrobial activity. This activity is lacking in the 61-residue variant and shorter (59-, 58-, and 57-residue) variants. As the authors conclude, this places SPINK9 as a novel skin-derived antimicrobial peptide [251].

There are as yet no formally proved functions of KLKs but there are many results indicative for their involvement in the physiology of skin as well as in other organs. This thesis adds a few pieces to the puzzle but clearly many interesting questions remain for the elucidation of the intricate play between KLKs, PARs, and inhibitors. These questions may be the scope of future studies within the field of dermatological research and other disciplines.

60 Conclusions

CONCLUSIONS

I. KLK14, like KLK5 and KLK7, is an endogenous proteolytic enzyme in human epidermis. The major part of KLK14 in plantar SC is present in a catalytically active form and may contribute substantially to total trypsin-like activity. Strong specific immunohistochemical staining of KLK14 can be observed in dermal sweat glands and epidermal sweat gland ducts. Thus KLK14 may be, at least partly, a sweat related protein.

II. The activation patterns of KLKs are consistent with the existence of a proteolytic cascade of KLKs in the epidermis, ending in the breakdown of corneodesmosomal proteins and desquamation. KLK5 may be a key activating enzyme in this cascade based on its autocatalytic properties and pH-dependent activation pattern and specificity.

III. SPINK9 is a previously not described serine protease inhibitor, with a restricted tissue expression pattern. It is found in normal palmo-plantar but not non palmo-plantar epidermis. Moreover, SPINK9 is an efficient inhibitor of KLK5 only, among the enzymes tested so far.

IV. KLK5 and KLK14 may contribute to as much as 33 and 21% respectively of the total trypsin-like activity in plantar SC. KLK7 may represent 76% of the total chymotrypsin-like activity. KLK8 may contribute with as much as 46% of trypsin-like activity but unlike the others it is not inhibited by any endogenous inhibitor found so far in plantar SC extracts.

V. KLK5 and KLK14 may be implicated in inflammatory and pruritogenic processes through activation of the PAR-2 receptor.

61 Tack / Acknowledgements

TACK / ACKNOWLEGEMENTS

Jag vill rikta några STORA VARMA TACK till de personer som hjälpt och stöttat mig under mina doktorand-år. Vad skulle jag ha gjort utan ER!?

Torbjörn, min huvudhandledare, många tack för ditt stora engagemang. Ditt till synes outtömliga dermatologiska kunnande har verkligen inspirerat mig och jag har uppskattat våra många och långa diskussioner.

Maria, min förträffliga, biträdande handledare. Tack för att du svarat på tusen dumma frågor på labbet med samma tålamod. Du har varit min levande uppslagsbok, full av förklaringar och nya idéer.

Tack till alla övriga medförfattare från Umeå: Annelii Ny, du är bara sååå duktig!, Bo Glas, alltid lika trevligt att prata några ord med dig, Christine Lundh, mer arbetsvillig student får man leta efter, Stefan Nilsson, vår Biacore-master, och så även den medförfattare jag aldrig träffat, Ylva Haasum.

Thomas Hubiche, our french co-author; you came to us with millions of great ideas! It was fun to have you in the lab and I will never forget your opinion on Swedish feet! I quote: “Swedish feet smell bad”.

BIG THANKS to Martin Steinhoff and co-workers in Münster, Germany, for the nice treatment I got during my stays in your lab. Dirk (funny guy) Roosterman, thanks for introducing me to the immunofluorescence and Ca2+ techniques, Georgeta (salsa-lover) Bocheva, thanks for the nice stainings and Cordula Kempkes thanks for being such a nice, kind, person, you really made me feel welcome at the lab. You are welcome back to visit us anytime!

Tack till alla studenter som har gett värdefulla bidrag till mitt arbete: Isabelle Mellqvist, Christine Lundh, Maria Berglund, Heidi Kokkonen, Astrid Lundberg, Frida Jonsson, Åsa Tengö, Farah Shakir och Anneli Lambertsson. Jag har verkligen uppskattat ert sällskap på labbet.

62 Tack / Acknowledgements

Enheten för dermatologi och venereologi, särskilt våra förra och nuvarande sekreterare Cecilia Elofsson, Ulla Westermark och Karin Norberg, ni var/är oumbärliga. Astrid Lundgren ska ha stort tack för teknisk hjälp och många trevliga små pratstunder.

Alla på institutionen för medicinsk biovetenskap och särskilt sekreterarna som alltid ställer upp när man har frågor, vill boka lokal med mera. Många tack till Åsa Lundsten för hjälp med beställningar och annat praktiskt. Tjejerna på patologen, Pernilla Andersson, Elisabeth Dahlberg och Birgitta Ekblom, tack för all hjälp jag fått med immuno-färgningarna, har verkligen frågat er om ALLT, typ 100 gånger! Tack även till alla andra som jag har plågat med stora och små frågor och till torsdagsfika-gänget för allt gott fika och trevligt sällskap!

Jag vill även tacka alla som lämnat prover, tejp strippar, hudflagor, svett prover med mera och särskilt tack till Göran Brattsand som bokstavligen offrat sin rumpa för min forskning!

Och vad skulle man göra utan alla vänner som förgyller vardagen? Både inom forskar-världen och utanför, långt borta och nära. Särskilt tack till Anna Johansson (fattar inte att du ska flytta till andra sidan jorden!), Maria Henriksson (bästa sommar-stipendie-handledaren, ever!), Anna Åhsgren, Therese Norrsken, Linn Sohl, Heli Rasinperä, Camilla och Jonas och Lilla och Tintin. Olov Pettersson, det började med en joggingtur och fortsatte med många tidiga morgnar på IKSU. Utan dig vore jag en (ännu mer) otränad doktorand. Johan Lidman, du var väldigt viktig under min första doktorand tid, tack för att du alltid ställde upp för mig.

Slutligen vill jag tacka mina familjer; Familjen Strömsöe, tack för att ni från första stund så totalt inkluderade mig i er varma gemenskap. Familjen Stefansson, dvs Mamma och Pappa, mina syskon Lena (med familj), Tomas, Maria och Johan, ni har gjort mig till den jag är. Jag älskar er alla!!!

Slutligen, de viktigaste människorna i mitt liv, Mikael och Edwin, jag kan aldrig få nog av att pussa dina fötter (det gäller alltså Edwin, INTE Mikael), älskar er för alltid!

63 Tack / Acknowledgements

This work was supported by: The Swedish Medical Research Council, Lions Cancer Research Foundation, Umeå University, the Welander-Finsen Foundation, Arexis AB, the French Academy of Medicine, the VINNOVA Foundation, Arners Foundation, the Kempe Foundation, Galderma and Rosacea Foundation.

64 References

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