Characterization of Kallikrein-Related Peptidase-8 in Normal Human Epidermis and Psoriasis

Characterization of Kallikrein-related Peptidase-8 in Normal Human Epidermis and Psoriasis

Azza Eissa

A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Laboratory Medicine and Pathobiology University of Toronto

Characterization of Kallikrein-related Paptidase-8 in Normal Human Skin Epidermis and Psoriasis

Azza Eissa

Doctor of Philosophy

Department of Laboratory Medicine and Pathobiology

University of Toronto

2013 Abstract

Kallikrein-8 (KLK8) is a relatively-uncharacterized epidermal protease. Although proposed to regulate wound-healing and barrier repair in KLK8-deficient mouse skin, KLK8-catalytic activity was never demonstrated in human epidermis and its regulators and targets remain largely unknown. KLK8 overexpression was reported in inflammatory skin diseases, but the underlying mechanisms are poorly understood. In this thesis, we elucidated for the first time KLK8-specific activity in normal human non-palmoplantar stratum corneum and sweat, and identified epidermal regulators and targets that augment its involvement in a skin-barrier proteolytic cascade. Given that inflammatory skin diseases have interlinked immune and epidermal roots, we hypothesized that epidermal KLK8 expression is distinctly regulated by the aberrant T-cell immunity implicated in the two common skin diseases, psoriasis and atopic dermatitis, independent of skin- barrier insults. We profiled secretion of KLK8 by normal human keratinocytes post-treatment with T-helper (Th1, Th17 and Th2) cell-derived cytokines, and investigated the effect of KLK8 overexpression on terminal keratinocyte differentiation and innate immunity gene expression.

Our results show that TNFα and IL-17A synergistically induce potent KLK8 hyper-secretion, while IL4 and IL13 reduce its expression. TNFα and IL-17A overexpression and KLK8

ii hyperactivity resulted in hyperkeratosis and upregulation of keratinocyte innate defense genes’ expression mimicking psoriatic lesions. Consistently, KLK8 expression was reduced in lesional skin of atopic dermatitis patients and significantly elevated in lesional skin and sera of psoriatic patients. KLK8 levels correlated with psoriasis skin severity and were significantly reduced by effective treatment with biologic TNFα-blockers, correlating positively with psoriasis clearance.

Thus, KLK8 is a new epidermal psoriasis therapeutic target. We performed high throughput screens of small molecule compound libraries to identify KLK8-specific inhibitors and discovered promising KLK8 small molecule inhibitors with IC50s in the nanomolar range. This thesis provides original findings corroborating KLK8 as an active serine protease in normal human skin and a down-stream epidermal respondent to TNFα and IL17A overexpression in psoriatic skin. Our novel KLK8-specific inhibitors may have future potential as topical barrier- enhancing agents in psoriasis.

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Acknowledgments

Though my name appears on the cover of this dissertation, it would not have come into existence without the following remarkable individuals:

My supervisor, Dr. Eleftherios P. Diamandis – Thank you for providing me with exceptional professional opportunities and teachable moments. I joined the lab as a naïve and enthusiastic young student and will be graduating as a mature professional. Thank you for pushing me to work independently and supporting me continuously. I learned valuable skills from you and I feel fortunate to have you as my supervisor and mentor.

Our LMP graduate department coordinator, Dr. Harry Elsholtz – Thank you for being so approachable and resourceful. I appreciate your guidance and open door policy over the years.

My thesis advisory committee, Dr. David Irwin, Dr. Sylvia Asa, Dr. Nades Palaniyar and Dr.Herman Yeger – I appreciate all your valued contributions! Thank you for your insightful feedback and advice. Your supervision and thoughtful review of my thesis was most helpful. My sincere appreciation to my external thesis advisor, Dr. Alain Hovnanian, for taking the time to contribute to my thesis and traveling long distance to be present during my thesis defense.

Last but not least, the ACDC lab members of the near past and present, my work family – Thank you all for your valuable friendships. Your presence and support in the lab was a blessing.

Special thanks to Ferzeen Sammy, Rama Ponda, Denitza Roudeva, Yiannis Prassas, Yijin Yu, Connie Zao, Vanessa Amodeo, Daniela Cretu, Antoninus Scoospialli, Dr. Gennady Poda, Dr. Martin Steinhoff, Dr. Ulf Meyer-Hoffert, Dr. Vinod Chandran, and Dr. Morely Hollenberg for helping me maneuver around some unexpected bumps in my research path and for being great friends, mentors and collaborators.

Also, a big thank you to the Natural Science and Engineering Research Council (NSERC), Canada Graduate Scholarship, Ontario Graduate Scholarship (OGS), Helen Marion Walker- Soroptimist Women’s Health Research scholarship and LMP department for funding my research.

Dedication

To My Wonderful Family! To Mama & Baba – for all the sacrifices you made and your endless love and support. Thank you for encouraging me to be fearless in seeking insight and facing challenges. You taught me how to cultivate the right attitude and learn from every single life lesson. You had blind faith in me and supported my aspirations even when you were not sure where they were taking me. Thank you! I am who I am today because of you.

To Omer ‘amoory’ – I couldn’t have asked for a better brother & best friend! I am very proud of you. You’ve been our rock. Thank you for being you!

To ‘bit al siroor al azama’ Fatma, my extraordinary grandmother who raised six children on her own and faced tough challenges with unequivocal strength, and to our family guru, my dear uncle, ‘amo’ Abdel Salam– If you ask me about superheroes: “Superwoman” and “Superman”, I would say: I know them! Despite the oceans and lands separating us, your superpowers reach and move me. Your leadership, intellect, empathy and wisdom are legendary. They transcend time and place to inspire generations to care and do more. I plan to share your life stories with the world one day! For now, this thesis dedication will serve as a small token of my love and admiration. Thank you for being my inspiring role models!

To ‘Denitza’ & ‘Puneet’ – my amazing girlfriends, my sisters! Living together and sharing over 10 years of friendships and laughs was a blast! Our incredible journey as young best friends from completely different backgrounds made me a better person. Thank you for always being there and for cheering me on through my entire undergrad and graduate programs. I couldn’t have done it without you. I love you both.

Thank you all for allowing me to live “like a river flows, carried by the surprise of its own unfolding” ~ John O’Donahue.

Table of Contents

Chapter 1 ...... 1 1 Introduction ...... 2 1.1 Serine Proteases: Digesting the Basics ...... 2 1.1.1 General protease classification ...... 2 1.1.2 Serine proteases catalytic mechanism ...... 4 1.1.3 Human trypsin-like serine proteases of family S1 clan PA ...... 7 1.2 Kallikrein-related Peptidase-8 At a Glance ...... 7 1.2.1 Discovery of the Kallikrein-related peptidases ...... 7 1.2.2 Genomic and proteomic structure of Kallikrein-related peptidases...... 10 1.2.3 Molecular properties of Kallikrein-related peptidase-8 ...... 12 1.2.4 Kallikrein-related peptidase-8 knock out mouse ...... 14 1.3 Kallikrein-related peptidases in Normal Human Epidermis ...... 15 1.3.1 Normal skin structure and function ...... 15 1.3.2 Kallikrein expression in the skin ...... 18 1.3.3 Dermatological roles of Kallikrein-related peptidases ...... 21 1.3.4 Regulation of epidermal Kallikrein-related peptidases ...... 23 1.4 Kallikrein-related peptidases in Skin Diseases ...... 28 1.5 Psoriasis and Atopic Dermatitis ...... 33 1.6 Kallikrein-related peptidase-8 in normal and inflamed skin 1.5 Psoriasis and Atopic Dermatitis ...... 36 1.7 Rationale, Hypotheses and Objectives ...... 37 1.7.1 Rationale ...... 37 1.7.2 Hypotheses ...... 38 1.7.3 Objectives ...... 38

Chapter 2 ...... 40 2 Kallikrein-related peptidase-8 (KLK8) is an active serine protease in human epidermis and sweat and is involved in a skin barrier proteolytic cascade ...... 41 2.1 Introduction ...... 41

2.2 Materials and Methods ...... 42 2.2.1 Cloning, expression, and purification of recombinant human KLK8 proteins ...... 43 2.2.2 Detection of recombinant mat-KLK8 and pro-KLK8 protein expression ...... 44 2.2.3 Gelatin-Zymography ...... 45 2.2.4 AMC substrate profiling and kinetics constant determination ...... 45 2.2.5 Cleavage of a positional-scanning library of FRET-quenched peptides ...... 46 2.2.6 pH profiling, divalent cation, and glycolsylation effect on KLK8 activity……... .. 46 2.2.7 Mat-KLK8 autodegradation assays ...... 47 2.2.8 Pro-KLK8 zymogen activation by KLK5, KLK1, and lysyl-endopeptidase ...... 47 2.2.9 Inhibition of KLK8 by epidermal inhibitors and general serpins ...... 48 2.2.10 Activation of Pro-KLK1, pro-KLK11, and pro-KLK5 by KLK8 ...... 48 2.2.11 Proteolytic processing of LL-37cathelicidin antimicrobial peptide ...... 49 2.2.12 Calcium induction of keratinocyte differentiation and KLK8 expression ...... 50 2.2.13 Collection and preparation of sweat and stratum corneum (SC) extracts ...... 50 2.2.14 KLK8 expression in sweat, stratum corneum extracts, and skin cell cultures ...... 51 2.2.15 Immunocapture of KLK8 activity in sweat and SC epidermal extracts...… ...... 52 2.3 Results ...... 52 2.3.1 Recombinant mat-KLK8 and pro-KLK8 protein characterization ...... 52 2.3.2 Pro-KLK8 zymogen activation in an epidermal cascade ...... 57 2.3.3 Effect of cations on KLK8 activity 1.3.2 Kallikrein expression in the skin ...... 59 2.3.4 Differential inhibition by skin specific inhibitors and general serpins ...... 59 2.3.5 KLK8 AMC substrate profiling and steady-state kinetics… ...... 61 2.3.6. Rapid endopeptidase library screening of KLK8 P2-P2’substrate specificity ...... 61 2.3.7 Activation of co-localized epidermal pro-KLKs by active KLK8 n ...... 64 2.3.8 KLK8 processing of LL-37 antimicrobial peptide ...... 66 2.3.9 KLK8 is a keratinocyte-specific protease induced during terminal keratinocyte differentiation… ...... 68 2.3.10 KLK8 is expressed in a free form in human sweat and non-palmoplantar stratum corneum ...... 68 2.3.11 KLK8 is catalytically active in normal human sweat and non-palmoplantar stratum corneum ...... 70 2.4 Discussion ...... 74

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Chapter 3 ...... 79 3 Kallikrein-related peptidase-8 is upregulated by TNFα and IL17A resulting in epidermal hyperplasia and elevation of psoriasis-related innate immunity gene expression ...... 80 3.1 Introduction ...... 80 3.2 Materials & Methods ...... 81 3.2.1 HaCat keratinocyte cell differentiation model and cytokine treatment ...... 82 3.2.2 Enzyme-linked immunosorbent assay (ELISA) and LDH assays ...... 82 3.2.3 BrdU cell proliferation assay ...... 82 3.2.3 KLK8 treatment of full thickness 3D skin equivalents ...... 82 3.2.5 Immunocytochemistry and immuohistochemistry ...... 83 3.2.6 Reverse Transcription and quantitative PCR ...... 84 3.2.7 Clinical samples from patients ...... 84 3.3 Results ...... 85 3.3.1 Keratinocyte secretion of KLK8 is differentially regulated by Th1, Th17 and Th2 cytokines ...... 85 3.3.2 TNFα and IL17A-treated keratinocytes have an altered differentiation program and mimic lesional psoriatic skin ...... 89 3.3.3 Overexpression of KLK8 alters keratinocyte differentiation program, induces epidermal hyperplasia and up-regulates innate immunity gene expression ...... 91 3.3.4 KLK8 is significantly elevated in lesional psoriatic skin and reduced in lesional acute atopic dermatitis skin………………………………………………………97 3.3.5 KLK8 elevation in psoriatic patients’ lesional skin and sera is significantly reduced after effective treatment with the TNFα blockers...... 101 3.4 Discussion ...... 105

Chapter 4 ...... 110 4 Serum kallikrein-related peptidase-8 levels correlate with skin activity, but not psoriatic arthritis, in patients with psoriatic disease………………………………..… ...... 111 4.1 Introduction ...... 111 4.2 Materials & Methods ...... 112 4.2.1 Collection of synovial fluids (SF) from PsA and control patients ...... 112

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4.2.2 Immunohistochemistry ...... 112 4.2.3 Setting and participants ...... 113 4.2.4 Enzyme-linked immunosorbent assays (ELISAs)...... 114 4.2.5 Statistical Analysis...... 114 4.3 Results ...... 114 4.3.1 KLK6 and KLK8 are elevated in PsA synovial fluids and lesional psoriatic skin 114 4.3.2 PsA and PsC patients...... 115 4.3.3 KLK8 is independently elevated in sera of patients with psoriatic disease… ...... 120 4.3.4 KLK8 serum levels in PsA correlate with the PASI score, but not inflamed joint counts … ...... 122 4.4 Discussion ...... 127

Chapter 5 ...... 130 5 Ongoing studies, general discussion and future directions…………………..… ...... 131 5.1 Introduction ...... 131 5.2 Materials and methods ...... 131 5.2.1 KLK8-mediated PAR2 signaling by cell-based assays 131 5.2.2 Search for KLK8 inhibitors by high throughput screens of small molecule compounds...... 132 5.3 Results ...... 131 5.3.1 KLK8 displays differential PAR2 signaling compared to KLK14……………….135 5.3.2 Identification of KLK8-specific inhibitors by high throughput screens of small molecule compounds...... 139 5.4 General discussion ...... 143 5.5 Future directions ...... 148 References ...... 150 Appendices ...... 172

List of Abbreviations

Acronym Actual word/phrase

AD Atopic dermatitis

AP-2 Activator protein-2

CDSN Corneodesmosin

DSC Desmocollin

DSG Desmoglein

ELISA Enzyme-linked immunosorbent assay

FDA Food and drug administration

FPLC Fast protein liquid chromatography hBD4 Human beta-defensin-4

HPLC High pressure liquid chromatography

HTS High throughput screen

IL Interleukin

KLK Kallikrein/Kallikrein-related peptidase

LG Lamellar granules

LEKTI Lympho-epithelial Kazal-type inhibitor

NS Netherton syndrome

PAGE Polyacrylamide gel electrophoresis

PAR Proteinase-activated receptor

PASI Psoriasis and area and severity index

Ps Psoriasis

PsA Psoriatic Arthritis

RPC Reversed phase chromatography

RT Room temperature

RT-PCR Reverse- transcription-polymerase chain reaction

S100A7 Psroiasin member of the S100 family

SBTI Soybean trypsin inhibitor

SC Stratum corneum

SCCE Stratum corneum chymotryptic enzyme

SCTE Stratum corneum tryptic enzyme

SG Stratum granulosum

SKALP Skin-derived antileukoproteinase

SLPI Secretory leukocyte protease inhibitor

SLS Sodium Lauryl Sulfate

SPI Serine protease inhibitor

SPINK Serine protease inhibitor Kazal-type

Th T Helper cells

TPA 12-O-tetradecanoylphorbol-13-acetate

xi List of Tables

Table Title Page

1.1 Relative trypsin-like KLK levels measured by enzyme-linked

immunosorbent assay (ELISA) of stratum corneum tissue extracts and sweat of normal human epidermis 20

1.2 Summary of KLK involvement in skin disease pathologies 31

1.3 Differences between atopic dermatitis and psoriasis 35

2.1 Divalent ion effect on mat-KLK8 activity 60

3.1 KLK8 serum levels predict positive response to TNFα-blockers 104

4.1 Demographics and clinical characteristics of psoriatic disease patients 118

4.2 KLK levels in the serum of plaque-type cutaneous psoriasis (PsC) and

psoriasis arthritis (PsA) patients 121

4.3 Polychotomous logistic regression analysis to identify biomarkers

associated with patients having psoriasis alone and psoriatic arthritis 123

5.1 Libraries selected for KLK-inhibitor screening 134

5.2 Primary HTS assays identify potential KLK5, KLK8 and KLK14-specific 140 small molecule inhibitors

5.3 KLK-specific inhibitors IC50’s 141

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List of Figures

Figure Title Page

1.1 Endoproteases and exproteases 3

1.2 The Schechter and Berger enzyme-substrate binding scheme 6

1.3 Schematic representation of serine protease catalytic mechanism 7

1.4 Kallikrein-related peptidase-8 in the protease family tree 9

1.5 Genomic and proteomic structure overview of Kallikrein-related 11 peptidases

1.6 Splice isoforms of the KLK8 gene 13

1.7 Stratified human epidermis 17

1.8 Kallikrein skin barrier proteolytic cascade 27

2.1 Activity and autodegradation of recombinant mat-KLK8 54

2.2 Pro-KLK8 activation by KLK5 54

2.3 KLK8 displays restricted substrate specificity based on cleavage of FRET 60 peptides

2.4 KLK8 activation of pro-KLK1 and pro-KLK11 62

2.5 Proteolytic processing of the LL-37 antimicrobial peptide by KLKs 64

2.6 KLK8 is a skin barrier protease 66

2.7 Immunocapture of KLK8 activity in normal human sweat and non- 71 palmoplantar stratum corneum ex vivo

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2.8 The majority of sweat and SC KLK8 is catalytically active 73

3.1 Differential Kallikrein-8 secretion by differentiating keratinocytes in 87 response to Th1 (TNFα and IFNγ), Th17 (IL17A, IL22) and Th2 (IL4,

IL13, IL25) cytokines

3.2 TNFα+IL-17A treatment induces changes in HaCat keratinocytes that 90 mimic psoriatic skin.

3.3 KLK8 treatment enhances differentiation and induces desquamation of 92 stratification domes in HaCat keratinocytes monolayers.

3.4 KLK8 treatment enhances differentiation of normal full thickness human 94 epidermis model and alters innate immunity gene expression.

3.5 Alterations in proliferation and differentiation markers in KLK8-treated 95 full thickness epidermis model and psoriatic skin.

3.6 KLK8 overexpression induces drastic changes in full thickness epidermis 96 model

KLK8 is significantly overexpressed in lesional psoriatic skin washes 3.7 97 only, unlike other KLKs

KLK8 epidermal expression is elevated in lesional psoriasis and reduced

3.8 in lesional atopic dermatitis skin, compare to respective non-lesional 98 counterparts.

3.9 KLK8 overexpression in lesional psoriatic skin, is not restricted to the 100 epidermis, but is also seen in dermis immune infiltrate near the epidermis, unlike atopic dermatitis skin.

3.10 Expression of KLK8 and other innate immunity genes in lesional psoriatic 102 skin pre and post-treatment with the TNFα-blocker, etanercept

4.1 Expression of KLK proteases in PsA inflamed joint synovial fluids and 116 control (osteoarthritis) synovial fluids.

4.2 Immunohistochemical expression of KLK6 and KLK8 in lesional psoriatic skin. 117

KLK6 and KLK8 cannot function as screening biomarkers for arthritis in 4.3 psoriasis patients 125

4.4 KLK8 correlates positicely with PASI scores in psoriatic disease 126

5.1 KLK8 does not cause calcium signalling via either human PAR1 or 136 PAR2, but disarms thrombin-mediated human PAR1 signalling

5.2 KLK8 does not trigger human PAR2 and -arrestins interaction nor PAR2 137 internalization, unlike KLK14

5.3 Unlike KLK14, KLK8 does not activate P42/44 MAP kinase-signalling in 138 human PAR2-expressing cells

5.4 An example of KLK8-sepcific inhibitor identified from the high 136 throughput screen

5.5 KLK8 in normal and psoriatic skin 147

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List of Appendices

Label Title Page

2.1 Steady-state kinetic parameters for the hydrolysis of synthetic AMC 172

substrates by mat-KLK8 in optimal activity buffer

3.1 Demographics and disease characteristics of psoriasis patients pre and 173 post-treatment

3.2 KLK serum levels pre and post psoriasis treatment with TNFα-blockers 174

5.1 Differences in KLK5 and KLK8 active site pockets 175

xvii 1

Chapter 1

Introduction

Sections of this chapter were reproduced from the following published manuscripts:

Eissa A, and Diamandis E.P. Tissue Kallikrein-related peptidases as promiscuous modulators of homeostatic barrier functions. Biol Chem. 2008; 286: 687-706

Eissa, A. and Diamandis, E. P. Kallikrein protease involvement in skin pathologies supports a new view of the origin of inflamed itchy skin in Proteases and Their Receptors in Inflammation, N. Vergnolle and M. Chignard Editors. Springer Basel. 2011

Eissa, A. and Diamandis, E. P. Kallikrein-related peptidase-8 (KLK8) in Handbook of Proteolytic Enzymes. Third Edition. Neil Rawlings and Guy Salvesen Editors, Elsevier. 2013

1 Introduction 1.1 Proteases: Digesting the Basics

Various types of proteases are working diligently inside and outside our human cells and many of them are incredibly fascinating. Proteases and peptidases (also known as proteolytic enzymes or proteinases) are enzymes that breakdown peptide bonds linking amino acids together in proteins or polypeptides, via a process known as proteolysis. Proteases are often depicted as “mother nature’s swiss army knives” (Seife, 1997) due to their ability to cut proteins at specific sites. Peptides can withstand hours of boiling heat in an acid, but they cannot endure more than a few microseconds in the presence of a protease. Hence, protease activity must be tightly regulated and blunted with endogenous inhibitors until physiologically required. Proteases were primarily known for their roles as digestive enzymes, since early studies of their roles date back to the 19th century with the characterization of pepsin and trypsin in 1836 and 1856 (Drag and Salvesen, 2010 ). Our understanding of proteases since then has outgrown digestion and degradation. Scientists are becoming increasingly more aware of protease roles as important regulatory and signaling molecules with hormone-like and innate immune-like properties in several tissues. Dysregulated protease activities impact various pathways and can lead to devastating outcomes including cardiovascular diseases, neurodegeneration, inflammation and cancer (Turk, 2006). Thus, proteases form up to 10% of currently approved FDA-drugs, and many more are in development as potential disease drug targets including proteases identified from the human genome project or from genomes of disease-causing organisms (Bachovchin and Cravatt, 2012).

1.1.1 General protease classification

Based on the location of their cleavage site on a protein or a polypeptide, proteases/peptidases are classified as endopeptidases or exopeptidases. Endopeptidases cleave their target protein or polypeptide internally, while exopeptidases cut at the polypeptide terminals, as depicted in Figure 1.1. Exopeptidases are further divided into aminopeptidases or carboxypeptidases depending whether they cleave their target peptide bond, known as the scissile bond, near the N- terminus or C-terminus, respectively, as shown in Figure 1.1.

Scissile peptide bond in the middle of polypeptide

N-terminal

C-terminal

Exopeptidase Endopeptidase

Figure 1.1. Endopeptidases and exopeptidases. (A) Protease/peptidase classification based on site of the scissile bond being cleaved by the protease (depicted as scissors in the Figure ). (B) Classification of proteases based on scissile bond cleavage site.

Over 600 human proteases are identified to date, accounting for 2-4% of the human genome. The availability of 3D structural information on these proteases facilitated their categorization into five distinct classes based on their catalytic mechanism. Approximately 200 metalloproteinases, 178 serine proteases, 160 cysteine proteases, 30 threonine proteases and 25 aspartic acid proteases have been identified, with the remaining proteases belonging to groups with an unknown or unclassified catalytic mechanism (Drag and Salvesen, 2010; Turk, 2006). A sixth class of proteases, known as glutamic proteases, exists but these proteases are not found in mammals. Metalloproteases, aspartic acid and glutamic proteases utilize an activated water molecule as a nucleophile to attack the peptide bond, whereas the nucleophile in serine, cysteine and threonine proteases is a key amino acid residue (Ser, Cys or Thr, respectively) located in the protease active site from which the class name is derived (Hartley, 1960; Turk, 2006). Differences in the mechanisms are also based on the presence or absence of a covalent acyl- enzyme intermediate in the reaction pathway. Serine and cysteine peptidases catalysis involves a covalent intermediate (ester and thiolester, respectively), whereas aspartic and metallopeptidase protease catalytic mechanisms do not. Proteases of the different catalytic types can be further grouped into families and clans. Barrett and coworkers’ protease classification system of protease families and clans forms the basis of the eminent peptidase database MEROPS (Rawlings et al., 2008). In this classification, proteases are divided into ‘clans’ based on their 3D structural homologies and into ‘families’ on the basis of common ancestry (Barrett and Rawlings, 1995). This thesis focuses on Kallikrein-related peptidase-8, which is a serine protease. Serine proteases are grouped into ~ 13 clans and 40 families, representing over one third of all known proteolytic enzymes (Di Cera, 2009).

1.1.2 Serine proteases catalytic mechanism

Serine proteases bind their substrates in a groove or cleft where the peptide amide bond gets hydrolyzed. According to the Schechter and Berger nomenclature, the substrate amino acid side chains occupy enzyme sub-sites in the cleft, designated as S3, S2, S1, S1', S2' and S3', which correspond with substrate residues P3, P2, P1, P1', P2' and P3' from the N-terminal to the C- terminal, as shown in Figure 1.2. Cleavage occurs between P1 and P1’ positions of the substrate. Thus, the S1 sub-site residue in the protease active-site determines the substrate specificity.

The mechanism of serine protease catalysis begins with the binding of the polypeptide or protein substrate target to the surface of the serine protease. The scissile bond gets inserted into the active site, with the carbonyl carbon of this bond positioned near the nucleophilic Ser 195. As mentioned above, scissile bond refers to the covalent chemical bond that gets cleaved by a protease. As shown in Figure 1.3, the mechanism involves aceylation and deacylation steps through which several intermediates are formed. Stabilized by Asp102, the nitrogen of His57 accepts a proton from Ser195, allowing the nucleophilic oxygen atom of the hydroxyl group of Ser195 to attack the carbonyl carbon of the scissile peptide bond, and a pair of electrons from the double bond of the carbonyl oxygen moves to the oxygen. As a result, the scissile bond gets broken, releasing the new amino-terminus and forming an ester bond between the enzyme and the substrate called acyl enzyme tetrahedral intermediate. In the second deacylation step, a water molecule hyrdolyzes the ester bond of the acyl-enzyme intermediate to liberate the protease and a peptide with a free carboxyl group (Polgar, 2005).

Figure 1.2. The Schechter and Berger enzyme-substrate binding scheme. Cleavage site is indicated with an arrow and substrate residues (P) binding the protease binding subsites (S) are shown. Prime and non-prime designations indicate the C-side and the N-side of the cleavage site, respectively.

Acylation step Enzyme

Acyl-enzyme intermediate Substrate scissile bond Amino-terminus Leaving group

Deacylation step

Water as a nucleophile Acyl-enzyme intermediate Peptide with a free carboxyl group

Figure 1.3. Schematic representation of serine proteases catalytic mechanism. The polypeptide R’-NH-CO-R scissile bond is cleaved by a serine protease in a sequential fashion involving two major steps: acylation in which the oxygen of the hydroxyl group of serine acts as a neucleophile resulting in formation of the acyl-enzyme intermediate and deacylation in which water acts as a nucleophile to release the shorter broken peptide with a free carboxyl group.

1.1.3 Serine proteases of family S1 clan PA(S)

Of the ~ 690 proteases described in man, 178 are serine proteases and 138 of them belong to the S1 family (Di Cera, 2009). Over two thirds of the PA clan is comprised of the S1 family of serine proteases, which bear the archetypal trypsin fold. These proteases have a two-domain structure, with each domain containing a β barrel, and the active site cleft lying in-between. Their catalytic triad is in the order Histidine, Aspartate, Serine, where the serine residue acts as a nucleophile, the histidine as a proton donor, and the aspartate for proper orientation of the imidazolium ring of the histidine, as shown in Figure 1.3. The trypsin-like peptidases of family S1 and clan PA are the most abundant serine peptidases and among the best studied (Laskar et al., 2012). Most clan PA(S) proteases are endopeptidases and have trypsin-like substrate specificity and prefer cleaving at the carboxyl side of arginine (Arg) or lysine (Lys) side chains at the P1 position. Some display chymotrypsin-like and elastase-like specificity. The majority of these proteases are secreted in inactive latent forms and are activated via proteolytic activation cascades to participate in important physiological processes, including digestion (trypsin, chymotrypsin), immune responses (complement factors B, C, D), blood coagulation (factors VIIa, IXa, Xa, XIIa), fibrinolysis (urokinase, tissue plasminogen activator, plasmin, kallikrein) and fertilization allowing the sperm to penetrate the egg (acrosin) (Page and Di Cera, 2008). Interestingly, the largest cluster of serine proteases is located on chromosome 19 and 16, encoding Kallikrein-related peptidase family and the Tryptase family, respectively, which belong

1.2 Kallikrein-related peptidase-8 at a glance

1.2.1 Discovery of the Kallikrein-related peptidase family

Kallikrein-related peptidases (KLKs) or tissue Kallikrein-related peptidases are a family of 15 members belonging to the chymotrypsin-like serine endopeptidase family S1, clan PA (Diamandis et al., 2000; Yousef and Diamandis, 2001). A list of the KLKs in the human protease family tree is shown in Figure 1.4, along with their assigned OMIM reference numbers and former alternate gene/protein names. The first member of this family, KLK1, was discovered in the 1930s in the pancreas, known as ‘kallikreas’ in Greek. This protease is expressed in multiple tissues and displays kinninogenase activity, whereby it cleaves kininogens to produce kinin peptides, which bind to kinin receptors, triggering inflammation and several biological effects. Another kinninogenase enzyme expressed solely in the liver and encoded by a single gene on

8 chromosome 4q35 was subsequently discovered and named a kallikrein as well, based on its kinnogenase activity. However, these two kallikrein proteases share no genomic or proteomic structural homologies. Hence, they were designated to two separate categories, whereby the kallikrein encoded by chromosome 19q13.4 was dubbed human tissue kallikrein (KLK1) and the one in chromosome 4q35 was dubbed plasma kallikrein (KLK1B) (Lundwall et al., 2006).

During the late 1980s, two additional tissue kallikrein genes (KLK) were discovered in the same genomic vicinity as KLK1; the human glandular kallikrein (KLK2) and the prostate specific antigen (PSA, KLK3) (Borgono and Diamandis, 2004). These two Kallikrein-related peptidases exhibited very little to no kinninogenase activity despite sharing genomic and proteomic homologies with KLK1. Accordingly, the traditional definition of a tissue kallikrein being a kinninogenase acting on high molecular weight substrates to produce bioactive kinins was modified. The term “tissue kallikrein” was then introduced to define the serine proteases encoded by genes on chromosome 19q13.4 sharing extensive structural homologies to KLK1 at the DNA and protein level, regardless of their enzymatic activities. KLK 1, 2, and 3 were referred to as “classical tissue Kallikrein-related peptidases” as they share a loop region found in rodent Kallikrein-related peptidases important for the enzyme’s substrate specificity (Borgono et al., 2007a). The remaining eleven “non-classical” KLKs, including KLK8, do not have this loop as a result of diverting further from rodent kallikrein genes during evolution. In addition to mouse and rat, kallikrein gene families have been identified in the chimpanzee, dog, pig, and opossum mammalian species (Elliott et al., 2006).

The last decade of the 20th century culminated with the full characterization of the KLK locus expanding the human tissue kallikrein family from three to fifteen genes and a pseudogene (KLK1), tandemly mapped to a contiguous cluster of ~ 400 kbp on chromosome 19q13.4, forming the largest protease gene cluster in the human genome (Clements et al., 2001; Diamandis et al., 2000; Yousef and Diamandis, 2001). The most recent nomenclature of the kallikrein family refers to KLK1 as “human tissue kallikrein”, while the remaining KLKs are dubbed “kallikrein-related peptidases” (Lundwall et al., 2006).

Figure 1.4. Kallikrein-related peptidase-8 in the human protease family tree

1.2.2 Genomic and proteomic structure of Kallikrein-related peptidases

Kallikrein-related peptidases share a high degree of genomic and proteomic homology as summarized in Figure 1.5. All KLK genes contain five coding exons of similar sizes and a conserved intron-phase pattern of I-II-I-0 (Yousef and Diamandis, 2001). About 82 KLK mRNA forms have been reported as each KLK gene has at least one alternative splice variant (Kurlender et al., 2005). KLK genes contain both 5’ and 3’ untranslated regions (UTRs) of varying lengths, except for the classical KLKs. Alternatively, kallikrein proteins (KLKs) have a characteristic multidomain single chain structure consisting of an amino terminal pre-peptide, a pro-peptide essential for maintaining the pro-KLK protein in a latent form, and a catalytic serine protease domain containing a highly conserved triad of histidine (H), aspartic acid (D), and serine (S) amino acids. All KLKs are secreted proteases, as shown in Figure 1.5. KLKs get secreted as pro- KLK zymogens upon removal of their pre-peptide signal. Cleavage of the pro-peptide induces a conformational change in the enzyme’s active site and substrate pocket, resulting in extracellular activation of the mature enzyme (Borgono and Diamandis, 2004). Pro-KLK activation is a key regulatory process postulated to occur via a proteolytic activation cascade similar to the coagulation, fibrinolysis, and complement system activation cascades (Yoon et al., 2007). Once active, Kallikreins employ a serine-directed nucleophilic attack mechanism to hydrolyze peptide bonds of target substrates, resulting in substrate activation, inactivation, or degradation. The majority of KLK proteins have acidic Asp residue at position 189, or Glu189 in the case of KLK15, in their substrate binding pocket allowing them to interact with basic arginine or lysine residues in their target substrates and rendering them to have trypsin-like substrate specificity. On the other hand, KLKs 3, 7, and 9 function as chymotrypsin-like serine proteases as they contain Ser189, Asn189, and Gly189 in their substrate binding pocket, respectively, accommodating bulky non polar amino acids such as tyrosine or phenylalanine.

Intracellular Chromosome 19

ACPT 1 15 3 2 1 4 5 6 7 8 9 10 11 12 13 14 SIGLEC-9 19q13.4 Centromere Coding exons 5’UTR 3’UTR I H II D I 0 S 5’ 1 2 3 4 5 3’

H57 D102 S195 KLK mRNA

N Pre Pro Serine protease domain C

KLK protein

Zymogen Secretion Pro-KLK H57 D102 S195 N Pro Serine protease domain C

Mat-KLK Enzyme H57 D102 S195 Activation N Pre NPro Serine protease domain C Extracellular

Figure 1.5. Genomic and proteomic structure overview of Kallikrein-related peptidases. KLK genes are localized on chromosome 19q13.4 flanked by the testicular acid phosphatase gene (ACPT) and the sialic acid–binding immunoglobulin-type lectin-type 9 (SIGLEC-9). Each arrow represents a certain KLK gene with its direction of its transcription. The 5’ untranslated region and 3’ untranslated region are shown in the primary mRNA transcript. H, D, and S represent the catalytic histidine, aspartic acid, and serine triad residues. In the KLK mRNA schematic, boxes indicate exons and lines indicate introns, whereby KLKs have 5 coding exons and an intron phase pattern of I,II,I,0. KLK mRNAs are translated as inactive pre-proenzymes which are directed to the endoplasmic reticulum for secretion via their prepeptide secretion signal. Extracellular cleavage of the propeptide by a trypsin-like protease is required for enzyme activation. Mat-KLK refers to the mature active form of the enzyme.

1.2.3 Molecular properties of Kallikerin-related peptidase-8

Kalikrein-related peptidase-8 gene (KLK8) is located in the KLK locus between KLK7 and KLK9 on the long arm of chromosome 19q13.4 and is transcribed from telomere to centromere, as shown in Figure 1.5. The KLK8 cDNA was initially cloned by Yoshida et.al (Yoshida et al., 1998) from human skin keratinocytes as a homologue of a mouse brain protease called neuropsin (Kishi et al., 1999). Its cDNA has a single open reading frame of 780 bp that encodes a 260 amino-acid protein. KLK8 gene consists of 6 exons and 5 introns, with the first exon being non- coding. The first intron is interrupting the 5’-untranslated region while the remaining 4 interrupt the coding sequence, as shown in Figure 1.6. Two repeat sequences are present in the promotor region of KLK8, but neither one is a TATA or a known transcription-factor binding sequence (Yoshida et al., 1998). Regulatory transcription factors of the KLK8 promoter are still unknown. Yet, the transcription site of KLK8 gene seems tissue-specific (Lu et al., 2007).

At the RNA level, KLK8 has been shown to have at least 5 splice variants (Magklara et al., 2001) forming KLK8 isoforms 1, 2 to 5. These isoforms have been detected in cancer tissues as potential prognostic biomarkers (Planque et al., 2010). Of these isoforms, the regular isoform 1 and isoform 2 encode a functional protein. As shown in Figure 1.6, Isoform 1 and 2 transcripts differ only in their exon 3 (or coding exon 2) sequences. KLK8 mRNA isoform 2 transcript has extra 45 amino acids at the N-terminus of its coding exon 2 making this isoform encompass a larger signal peptide, yet its pro activation sequence and full protease domain is the same as the classical isoform (Lu et al., 2009). Although isoform 1 is homologous with the mouse neuropsin mRNA, isoform 2 is absent in the mouse. Human KLK8 isoform 2 is believed to have risen later in evolution. A human-specific T to A point mutation has led to this novel form in the human brain, but not in the brains of chimpanzees or other species (Lu et al., 2007).

Human KLK8 and mouse neuropsin have 72% cDNA and amino acid similarity (Yoshida et al., 1998). Work in this thesis focuses on canonical KLK8 protein encoded by isoform 1, which has a secretion signal pre-peptide of 28 amino acids, followed by the pro-zymogen activation peptide of 4 amino acids and the mature chain of 228 amino acids with 1 potential N-linked glycosylation site and 12 Cys residues that participate in forming 6 disulfide bonds. Its catalytic triad of His86, Asp120, Ser212 is conserved and is essential for proteolytic activity.

6 Exons

1 2 3 4 5

H S D bp AA BC040887 304 178 8 70 170 263 134 156 46 1377 260

1 160 1013 260 NM_ 007196 171 8 70 263 134 156 51

2 171 8 70 263 134 156 51 1148 305 NM_144505 295

3 171 156 51 590 119 NM_144506 70

4 456 32 NM_144507 171 8 70 178

28 AA 4 AA 228 AA

Figure 1.6. Splice isoforms of the KLK8 gene. The start site is indicated with * and the termination codon with an arrow. The first exon is non-coding, and hence the only coding exons are labeled 1 to 5 in the diagram. Coding exons 2, 3 and 5 are important for activity as they encode H, D, and S, labeled to represent the positions of the catalytic triad. Only isoforms 1 and 2 produce functionally active proteins. The 28aa pre-signal , 4 pro-sequence and 228aa active domain shown describes the 260 aa protein formed by isoform 1. KLK8 isoform 2 has a longer pre-pro sequence but same 228 aa sequence of the active domain. Figure is modified from (Kurlender et al., 2005).

1.2.4 Kallikerin-related peptidase-8 knock out mouse

Neuropsin is the mouse homologue of KLK8 which shares 72% similarity. The first studies of neuropsin-deficient mice reported abnormalities in brain synapses and neurons (Chen et al., 1995; Kitayoshi et al., 1999; Yoshida and Shiosaka, 1999). Currently, the KLK8/neuropsin knock out mouse is the only available in vivo mouse model with targeted kallikrein-related peptidase disruption. Abolishing the KLK8 gene is not embryonically lethal, as mice grow without any major defects. The neuropsin-deficient mouse revealed that KLK8/neuropsin has major roles in the brain and skin. Brain-related studies will be summarized below, while skin- related findings will be discussed later in the skin section.

Neuropsin is expressed in limited regions in mouse brain including the hippocampus, lateral nucleus of the amygdala and other areas involved in learning and memory (Hirata et al. 2001). Neuropsin role in hippocampus plasticity is linked to kindling formation and long term potentiation (LTP) (Kishi et al., 1999). Kindling is a model of epilepsy, whereby repeated electric stimulations cause the brain to form synaptic structures and lowers its threshold, so that weaker stimulations can cause convulsions. Neuropsin mRNA expression is increased in the hippocampus after cumulative stimulations, and interference with neuropsin-specific antibody delays kindling (Yoshida, 2010). Furthermore, neurpopsin was implicated in the LTP of the synaptic plasticity process involved in learning and memory (Komai et al., 2000). Neuropsin protease activity is induced by activation of the ion channel glutamate receptor, N-methyl-D- aspartate (NMDA) and is blocked by NMDA receptor inhibitors (Matsumoto-Miyai et al., 2003). Studies have indicated that the neuropsin KO mouse displays slower learning. It is assumed that neuropsin remains inactive until stimulated by synaptic activation. An elegant recent study in Nature showed that neuropsin is also critical for stress-related plasticity in the amygdala of mice, as it regulates EphB2-NMDA-receptor interaction, Fkbp5 gene expression and anxiety-like behavior (Attwood et al., 2011). In the central nervous system, neuropsin is not expressed in the white matter or nerve fiber tract of healthy mice. Its expression is induced only after physical or chemical injury to the spinal cord by oligodendrocytes near the neuronal lesion (Terayama et al., 2007; Terayama et al., 2004). Neuropsin was also implicated in an experimental model of multiple sclerosis, an autoimmune disease in which oligodendrocytes are dead and myelin is degraded possibly through neuropsin actions (Terayama et al., 2005).

1.3 Kallikrein-related peptidases in normal human Epidermis

1.3.1 Normal skin structure and function

Human skin’s outer layer, the epidermis, is the body’s first line of defense against harsh environment, water loss, chemical and physical damages, UV-radiation, allergen and pathogens entry. It protects the body with its flexibility and toughness, all while acting as a sensory organ. The skin is composed of 3 main layers: the epidermis, dermis and subcutaneous tissue. Mature epidermis consists of 4 layers: the stratum basale (SB), stratum spinosum (SS), stratum granulosum (SG) and stratum corneum (SC), in order of increased differentiation from the lowest layer to the outer skin surface. The stratum basale displays characteristic small downward folds into the dermis known as ‘rete ridges’. Keratinocytes form the overwhelming majority of cells in the epidermis, with a few melanocytes and langerhan cells. Keratinocytes secrete over 30 types of keratins during their differentiation into distinct epidermal layers or ‘strata’. The dermis layer provides structural support, houses blood vessels and nerves, and is a base for skin appendages such as hair follicles, sebaceous and sweat glands. The main cells in this layer are the fibroblasts embedded in a matrix of collagen, glucosaminoglycans and glycoproteins which they produce. Below the dermis lies a subcutaneous tissue containing fat cells, known as adipocytes, which serves as a temperature insulator and a cushioning layer.

The majority of skin barrier functions are attributed to the uppermost epidermal layer, the stratum corneum. The stratum corneum (SC) layer gets renewed every 2-4 weeks via an elegant differentiation program of keratinocyte cells (Candi et al., 2005), which are the major cell constituents of the epidermis (Simpson et al., 2011). The formation of the stratified epidermis begins by the commitment of a single layer of multipotent ectodermal progenitor cells to a keratinocyte cell fate in the lower stratum basale. Keratinocytes at the basal layer (SB) withdraw from the cell cycle, detach from the basement membrane, and proliferate upwards to differentiate into intermediate spinous (SS) and granular layers (SG), pushing already formed cells higher up. At each stage the keratinocytes express a different set of proteins, and thus proliferating keratinocytes and differentiating ones have a different set of markers. For instance the nuclear Ki67 antigen is often used a marker of keratinocytes proliferation at the stratum basale, and involucrin is a cytoplasmic marker of differentiation at the SG. As shown in Figure 1.7, basal keratinocyte proliferation and spinous keratinocyte differentiation culminates with the processes

16 of terminal keratinocyte differentiation and cornification at the stratum granulosum/stratum corneum (SG/SC) interface, where granular keratinocytes:

1. Transport and secrete cargos via their lamellar granules (LG) into the SG/SC extracellular space. These cargos include structural proteins, adhesion proteins, lipids, lipid-processing enzymes, antimicrobial peptides and a cocktail of proteases and protease inhibitors

2. Replace their plasma membrane with a tough insoluble protein and lipid envelope known as the cornified envelope (CE)

3. Aggregate their keratin intermediate filaments via filaggrin (FLG), causing collapse of their cytoskeleton into flattened squames

4. Lose their nuclei and sub-cellular organelles to get terminally-differentiated into non- viable anucleated cells, known as ‘corneocytes’

The last step of skin barrier formation, shown in Figure 1.7, is known as ‘desquamation’. Skin desquamation refers to corneocyte shedding off the skin surface as a result of regulated degradation of adhesion proteins linking uppermost corneocytes, known as corneodesmosomes, by endogenous proteases (Milstone, 2004). Inherent terminal keratinocyte differentiation and corneocyte desquamation ensue in parallel to maintain the SC barrier thickness relatively constant.

Inv

K10 Epidermis Ki67

Dermis

Figure 1.7. Stratified human epidermis. Human skin is composed of outer epidermis and inner dermis. Normal human epidermis is a stratified epithelium of keratinocytes in the stratum basale (SB), stratum spinosum (SS), stratum granulosum (SG), and corneocytes in the uppermost stratum corneum (SC), in order of increasing differentiation. Common markers of keratinocyte proliferation and differentiation are shown on the left of the H&E human skin image next to their corresponding layer of expression. Ki67 is a marker of cell proliferation in the SB, keratin 10 or K10 in an early differentiation marker in the SS, and involucrin is a late differentiation marker of SG and SC layers. Epidermal programming is characterized by proliferation, differentiation, cornification, and desquamation events.

Up until the last two decades, the SC was viewed as a dead layer of non-viable corneocyte cells embedded in a lamellar lipid sea, which was often represented by the skin barrier “brick and mortar” model of vertically-stacked corneocyte ‘bricks’ held together by an extracellular lipid ‘mortar’ (Elias, 1983). With further studies, it became apparent that the SC layer is full of exciting metabolic activity and is more dynamic than previously thought, as several terminal keratinocyte differentiation products and metabolic processes take place in its extracellular milieu to regulate barrier functions.

Although the co-localized SC barrier functions are highly inter-dependent, their molecular and biochemical basis tend to differ. For example, in the SC, filaggrin serves as a template for the assembly of corneocytes’ cornified envelope forming flat and tough corneocyte ‘bricks’ in the outer barrier. Filaggrin ultimately dissociates to free amino acids that form the skin’s natural moisturizing factor (NMF), which creates a hydrated and acidic skin surface (Hachem et al., 2003; Rippke et al., 2004). Moreover, lipid precursor processing by β-glucocerebrosidase, acidic sphingomyelinase and secretory phospholipase A2 enzymes into ceramides and free fatty acids generates mature lipid lamellar-membranes that hamper transepidermal water loss (TEWL) and form the SC permeability barrier (Hachem et al., 2010; Ohman and Vahlquist, 1994). In addition to its hydrophobic content and acidic pH, the skin surface contains antimicrobial peptides (AP), and certain keratinocyte, eccrine and sebaceous gland-derived proteases and protease inhibitors which comprise its outermost antimicrobial shield (Braff et al., 2005a; Braff et al., 2005b; Lee et al., 2008; Nizet et al., 2001). Additionally, specialized SC extracellular adhesion proteins, known as corneodesmosomes (Candi et al., 2005; Simpson et al., 2011), are incorporated into the corneocyte envelope to adhere corneocytes together and maintain the SC structural barrier. It is important to note that KLK proteases co-localize with many of these barrier molecules in the epidermis, as discussed below. Thus, epidermal KLK serine protease activity is implicated in regulating many physiological features of a healthy skin barrier including lipid content, proper antimicrobial shield formation and corneodesmosome degradation.

1.3.2 Kallikrein expression in the skin

Multiple Kallikrein-related peptidases are expressed in the skin epidermis and its associated appendages. Of the 15 KLK-related peptidases present in the human body, eight KLKs co- localize in human epidermis in addition to the parent tissue KLK1 (Komatsu et al., 2005b;

Komatsu et al., 2006b). These KLKs are expressed in the SC, upper SG, sebaceous glands, eccrine sweat glands, hair follicles, and nerves. Kallikrein-related peptidases are detected by immunohistochemistry in glandular epithelia secretions confirming their extracellular localization (Komatsu et al., 2005b).

Kallikrein transcripts and/or proteins have been detected by RT-PCR and immunostained in the SC, upper SG, sebaceous glands, eccrine sweat glands, hair follicles, and nerves (Komatsu et al., 2005b; Komatsu et al., 2003). KLK mRNA expression in the inner and/or outer root sheath of hair follicle epithelia (Komatsu et al., 2003), suggests KLK involvement in hair development. KLK mRNAs and proteins are also intensely expressed in the basal layer of undifferentiated sebocytes, indicating their potential participation in sebaceous gland differentitation and sebum formation. Moreover, KLKs such as KLK6, 8, and 13 are found in the inner lumen of sweat gland ducts and hence are expected to be secreted in sweat (Komatsu et al., 2005b). Indeed, the expression of these three Kallikrein-related peptidases in human sweat and skin surface was confirmed recently by ELISA quantification, verifying previous immunohistochemistry results. KLK3 and 9 proteins have not been shown to be expressed in the epidermis (Komatsu et al., 2005a), conferring the epidermal chymotrypsin-like serine protease activity to KLK7 solely. In addition to the chymotrypsin-like KLK7, seven trypsin-like Kallikrein-related peptidases (KLKs 5, 6, 8, 10, 11, 13, and 14) have been detected in human SC and sweat from different body regions (Komatsu et al., 2006b). Komatsu et al. detected a broad range of SC trypsin-like KLK levels (KLK5, 6, 8, 10, 11, 13 and 14) where the most abundant KLKs (KLK8 and 11) are about 200-fold greater than the least abundant ones (KLK14 and 13). The levels of KLK8 and 11 are similar in the SC, but KLK8 concentration is significantly higher in sweat, representing up to 60% of the total trypsin-like KLK levels, as shown in Table 1.1. In a different study, Komatsu and colleagues showed that the total trypsin-like KLK concentration in the SC is approximately double the total chymotrypsin-like concentration (Komatsu et al., 2005a); although this does not necessarily signify a higher trypsin-like activity.

Studies have also reported visualizing KLKs, such as KLK5, KLK7 and KLK8, as they are distinctly transported by lammelar granules (LG) of granular keratinocytes in the SG and secreted to co-localize at SG and SC interstices (Ishida-Yamamoto et al., 2005; Ishida- Yamamoto et al., 2004). To date, three KLKs, namely KLK5, 7, and 14 have been extracted in active forms from SC tissues (Brattsand et al., 2005; Ekholm and Egelrud, 1999). Kallikrein-

20 related peptidases expression in human dermis had been less studied; nonetheless immunohistochemical studies have indicated that KLKs can also be expressed by endothelial cells in blood vessels. Our understanding of the expression and potential role of KLKs in dermal endothelial and immune cells is rudimentary and needs to be studied.

Profiling of epidermal Kallikrein-related peptidases based on enzymatic activity instead of protein levels is difficult, because activity results vary depending on the assay type used (Brattsand et al., 2005). The majority of protease assays used to measure endogenous kallikrein activity are not KLK-specific. Casein zymography analysis of SC tissue extracts indicates that KLK5 and KLK7 are the major active KLKs in the SC, while chromogenic peptide substrate studies attribute up to 50% of trypsin-like activity to KLK5, and the majority of the remaining activity to KLK14 (Brattsand et al., 2005). A separate study indicated that KLK14 accounts for 50% of the overall SC trypsin-like KLK activity (Stefansson et al., 2006), although denoted a minor SC trypsin-like kallikrein by Komatsu eta al. In general, quantification of epidermal KLKs based on enzymatic activity is a difficult mission because KLK activity in the epidermis is regulated by different endogenous and environmental factors.

Table 1.1 Relative trypsin-like KLK levels measured by enzyme-linked immunosorbent assay (ELISA) of stratum corneum tissue extracts and sweat of normal human epidermis

1.3.3 Dermatological roles of epidermal Kallikrein-related peptidases

In general, KLKs have two major functions in human epidermis: regulation of normal skin barrier thickness through differentiation and/or desquamation and modulation of innate and adaptive immune responses. Their role in desquamation is the best studied thus far, however mechanisms governing their roles in keratinocyte differentiation and innate/adaptive immunity are emerging. a) Skin Desquamation

Skin desquamation is a pH and calcium-dependent process, suggested to occur via initial proteolysis of non-peripheral corneodesmosomes at the transition from inner stratum compactum to outer stratum disjunctum of the stratum corneum layer, resulting in retention of corneodesmosomes at the lateral edges of corneocytes. These corneodesmosomes are degraded in the superficial SC leading to corneocyte shedding (Ishida-Yamamoto et al., 2005). KLK5 and KLK7 degrade DSG1, DSG4, and DSC1 corneodesmosomal isoforms(Borgono et al., 2007b; Descargues et al., 2006), as well as their CDSN glycoprotein(Simpson et al., 2011). KLK 6, 13, and 14 are potential desquamatory enzymes since they digest DSG1 and/or are inhibited by the epidermal KLK inhibitor, LEKTI (Borgono et al., 2007b). b) Lipid Permeability Barrier

Skin barrier function depends on the formation of mature lamellar membranes in the SC subsequent to proper extracellular lipid processing of lipid precursors secreted by LGs. Lipid precursors such as glycosylceramides, sphingomyelin, and phospholipids are released into the SG/SC interface to get processed by β-glucocerebrosidase, acidic sphingomyelinase, and secretory phospholipase A2 into ceramides and free fatty acids. The resulting lipids, particularly ceramides, are essential as they form extended hydrophobic lamellar sheets in SC extracellular spaces limiting water and electrolyte loss and composing the skin’s lipid permeability barrier (Hachem et al., 2006; Hachem et al., 2005).

Lamellar membrane organization is pH and calcium-dependent, as studies have shown that lipid processing enzymes exhibit optimal activity at acidic pH (Hachem et al., 2003). Experimental elevations of pH induce lipid processing defects visualized by the formation of immature lamellar membranes in the SC. The pH-induced lipid barrier dysfunction is suggested to be

22 mediated by the actions of epidermal kallikreins, as KLKs may regulate lamellar membrane formation via proteolytic degradation of SC lipid-processing enzymes (Hachem et al., 2003) or downregulation of their secretion by LGs (Hachem et al., 2006). Earlier studies showed that incubations of skin extracts with active recombinant KLK7 at an elevated pH of 7.6 results in decreased immunoblotting of both β-glucocerebrosidase and acidic sphingomyelinase lipid processing enzymes. However, later studies by the same group have revealed that inhibition of serine protease activity leads to permeability barrier recovery by enhancing LG secretion of lipids. Hence, the current premise suggested by Hachem et al. is that LG secretion, not lipid processing, is down-regulated by pH-induced increases in kallikrein activity, leading to barrier disruption. The deregulation of LG lipid secretion is suggested to occur via kallikrein-mediated activation of proteinase activated receptors, PARs (Hachem et al., 2006). c) Proteolytic Processing of Antimicrobial Peptides

Epidermal keratinocytes synthesize and secrete antimicrobial peptides that harbor the skin’s innate immunity against bacterial, fungal, and viral infections. β-defensins and cathelicidins are two major antimicrobial peptide families expressed by keratinocytes and neutrophils, where β- defensins are constitutively expressed (Ong et al., 2002), and cathelicidins are induced and deposited at inflammation sites upon infection (Braff et al., 2005b; Ong et al., 2002). Tight control of cathelicidin peptides expression is required to ensure their activity when defense against microbial invasion is required. Cathelicidins comprise a conserved N-terminal cathelin pro-domain and a variable C-terminal antimicrobial domain of 30-40 amino acids that becomes active after cleavage (Niyonsaba et al., 2010). KLK5 and KLK7 regulate cathelicidins’ proinflammatory activity by processing either the nascent pro-cathelicidin (hCAP18) or the mature peptide form (LL-37), serving as activators and inactivators. hCAP18 is biologically inactive (Zaiou et al., 2003). KLK processing of hCAP18 to active antimicrobial peptide forms, such as LL-37 stimulates host cell inflammatory reactions in response to infection as LL37 is known to act as a chemoattractant of neutrophils, monocytes, mast and T-cells upon tissue insult (Yamasaki et al., 2006). Subsequent to resolving the microbial challenge, KLK5 and KLK7 process LL-37 to peptide forms that lack pro-inflammatory activity, bringing the SC back to its normal immuno-barrier setting. Furthermore, cathelicidin processing has been shown to be altered in vivo in the absence of the epidermal serine protease inhibitor LEKTI (Yamasaki et al.,

2006), suggesting LEKTI involvement in antimicrobial peptide processing via its inhibitory effect on kallikreins. d) PAR-mediated effects

Proteinase-activated receptors (PARs 1-4) are members of the seven-transmembrane G-protein- coupled receptor (GCPR) family, activated by proteases such as trypsin, mast cell tryptase, cathepsin G, and thrombin. PARs are cell surface receptors expressed on keratinocytes (PAR2), melanocytes (PAR1), fibroblasts (PAR2), neurons (PAR2), and dermal capillaries (PAR1) (Rattenholl and Steinhoff, 2008). PAR activation occurs intramolecularly by irreversible proteolytic cleavage of the extracellular N-terminal peptide exposing a tethered ligand that binds the second extracellular loop of the receptor, initiating signaling. PARs mediate multiple signaling pathways by coupling to G-proteins and stimulating a variety of downstream targets. Growing evidence now attests to the role of kallikreins as modulators of PAR signaling. Recent in vitro and in vivo work by Oikonomopoulou et al. has demonstrated that PAR activity may be targeted by active KLK 5, 6, and 14. KLK5 and KLK6 were shown to activate PAR2, while

KLK14 was reported to inactivate PAR1 and activate PAR2 and PAR4 (Oikonomopoulou et al.,

2006). Among the four PARs, PAR2 is of prime interest as it is activated by trypsin cleavage and co-localized with tissue kallikreins in the stratum granulosum and in keratinocytes of hair follicles and sebaceous glands.

1.3.4 Regulation of epidermal Kallikrein-related peptidases a) Proteolytic Activation Cascade

Pro-Kallikrein-related peptidases are believed to be activated in a step-wise manner forming an activation cascade, where the active form of one kallikrein catalyzes the activation of the next pro-KLK. The occurrence of such a cascade in the skin is supported by the co-expression of multiple KLKs in upper epidermal layers and sweat at varying concentrations, and by the ability of some of these KLKs to auto-activate and activate other pro-KLKs (Brattsand et al., 2005; Yoon et al., 2007). A kallikrein may take on the role of the initiator, propagator, and/or executor within the cascade, depending on its concentration, specificity, and activity level. However, minute amounts of the initiator are sufficient to trigger the cascade, due to its catalytic nature. In vitro kinetic studies demonstrated that pro-KLK5 is activated by KLK14 and KLK5 itself, and

24 active KLK5 activates pro-KLK7, which are the main players of the skin desquamation cascade, as shown in Figure 1.8. Recently, Yoon and colleagues characterized the first extensive “KLK activome” upon examining the hydrolysis of 15 pro-KLKs by mature recmobinant KLKs (Yoon et al., 2007). Their novel findings allow expansion of the epidermal KLK activation cascade to include additional KLK members and thrombostasis proteases (Yoon et al., 2009; Yoon et al., 2008). The contribution of this in vito KLK activome to the skin barrier cascade remains to be elucidated. Furthermore, the characterization of initiators, propagators, and executors in the KLK proteolytic activation cascade poses a challenge that remains to be solved, although KLK5 is believed to be the cascade initiator (Brattsand et al., 2005). b) Lamellar Granule Trafficking

Lamellar granules (LGs), or lamellar bodies, are epidermal secretory granules that deliver cargos synthesized in upper granulocytes to SC interstices, including kallikrein serine proteases. In addition to epidermal transport and release of contents into intercellular spaces, LGs function as cargo storage sites (Braff et al., 2005b). LGs fuse with the apical membranes of uppermost granulocytes releasing their contents into extracellular spaces (Ishida-Yamamoto et al., 2005; Ishida-Yamamoto et al., 2004). Secretion from the uppermost differentiated granular keratinocytes allows KLKs to be delivered at close proximity to target substrates, such as corneodesmosomes, regulating their degradation which results in skin desquamation. Cargos, such as KLK5, 7, their epidermal inhibitor LEKTI, and target substrate corneodesmosin (CDSN), are separately co-localized and transported in different LG vesicles before their release into the SG/SC interface (Ishida-Yamamoto et al., 2005; Ishida-Yamamoto et al., 2004). LG separate transport averts any possible premature enzymatic activties among cargos, such as the proteolysis of CDSN by KLK5 or KLK7 which is known to occur at a pH of 5.5, similar to the environmental pH of LGs (Ishida-Yamamoto et al., 2004). In addition to transport, LG’s regulate the time and location of cargo secretion. For example, LEKTI inhibitor has been shown to be secreted earlier than KLKs into the superficial SG layer, while KLK5 and 7 are secreted into SC interstices. The exact mechanism for LG sorting, transport, and secretion of cargos is not fully understood, but selective aggregation and condensation of cargos has been suggested (Ishida- Yamamoto et al., 2005; Ishida-Yamamoto et al., 2004). Nonetheless, the temporal and spatial transportation and secretion of KLKs by LGs are critical regulatory events controlling epidermal kallikrein expression. Negative feedback loops regulating LG secretion of KLKs may also occur

25 in the epidermis as hyperactive Kallikrein-related peptidases have been reported to decrease LG secretions (Hachem et al., 2006). c) Colocalization with Epidermal Substrates and Inhibitors

Numerous skin-specific kallikrein substrates and inhibitors co-express with Kallikrein-related peptidases in human SG and SC layers. Co-localization with myriad of substrates in the SC, including: hCAP18 and LL37 cathelicidin antimicrobial peptides, and DSG1, DSG4, DSC1 and CDSN corneodesmosomal cadherins, allows for their efficient proteolysis by Kallikrein-related peptidases. On the other hand, co-localization with epidermal serine protease inhibitors, such as lympho-epithelial Kazal type inhibitor (LEKTI), elafin, and secretory leukocyte protease inhibitor (SLPI) results in regulation of KLK activity. Akin to other serine proteases, Kallikrein- related peptidases can be inhibited or trapped by forming a stable covalent complex with endogenous members of the superfamily of serine-protease inhibitors, serpins, such as α1- antitrypsin, whereby the serpin acylates the protease active serine resulting in a conformational change of the serpin’s reactive center and a destruction of the proteases’s active site. Elafin and SLPI do not inhibit KLK5, 6, 13 and 14 (Borgono et al., 2007b), but these inhibitors result in chymotrypsin-like KLK7 inhibition and cornocyte shedding in vitro (Franzke et al., 1996). Alternatively, several KLKs including KLK5, 6, 7, 13, and 14 are inhibited by LEKTI inhibitory domains (Borgono et al., 2007b; Deraison et al., 2007). Similar to KLKs, LEKTI is expressed in normal stratum corneum, stratum granulosum and skin appendages (Bitoun et al., 2003; Raghunath et al., 2004). LEKTI is a reversible inhibitor of 1064 amino acids encoded by SPINK5 (serine protease inhibitor Kazal type 5) gene and organized into fifteen serine protease inhibitory domains (D1-D15) (Bitoun et al., 2003). The full length protein is an inactive inhibitor of KLKs. Its intracellular cleavage by furin generates single or multidomain inhibitory fragments that get secreted by keratinocytes to inhibit KLKs (Deraison et al., 2007).

LEKTI domains display distinct inhibitory profiles as they are selective towards KLK (Egelrud et al., 2005; Schechter et al., 2005). For instance, KLK5 inhibition can be achieved by all LEKTI domain fragments, excluding D1. LEKTI domains D8-11 exhibited the strongest inhibition towards trypsin-like KLK5 and 14 with low Ki values of 3.7 nM and 3.1 nM, respectively, and a much lower inhibition of the chymotrypsin-like KLK7 with Ki of 34.8 nM (Deraison et al., 2007). These results are consistent with Borgono et al. findings of D1-8 inhibition of multiple

KLKs, D12-15 selective inhibition of KLK5 only, and D9-12 highest inhibition specificity towards KLK5 (Borgono et al., 2007b). Interestingly, neither KLK8 nor KLK1 were inhibited by LEKTI. The strongest inhibitory capacity of multiple LEKTI fragments towards KLK5 supports the paradigm of KLK5 being the initiator of the epidermal KLK activation cascade. c) Epidermal pH and Calcium Gradients

It is important to note that SC integrity and the majority, if not all, of its barrier functions are governed by its inherent calcium and pH gradients. Human SC extracellular calcium levels increase and pH levels decrease from the lower SG/SC border to the uppermost skin surface. The innate increase in epidermal calcium concentrations regulates terminal keratinocyte differentiation, lamellar granule (LG) secretion and cornified envelope formation, while the innate decrease in SC pH levels, from pH 7.0 to pH 5.0 at the skin surface, regulates desquamation, lipid permeability and antimicrobial barrier integrity.

Epidermal pH regulation of Kallikrein-related peptidases is bidirectional, as it modulates KLK inhibition, as well as activity. Experimental pH increase elevates KLK activity and results in over-degradation of SC structural proteins, such as DSG1 corneodesmosome, and of SC lipid- processing enzymes, such as β-glucocerebrosidase, leading to destruction of SC cohesion and lipid barrier, respectively (Hachem et al., 2005). Epidermal pH also regulates the kinetics of the interaction between KLKs and the serine protease inhibitor LEKTI. A recent in vitro study by Deraison et al. has shown that LEKTI fragments D8-11 tightly bind KLK5 forming stable complexes at pH 7.5, which mimics the pH of the SC/SG border. Increased dissociation of KLK5 from LEKTI fragments occurs upon decreasing pH from 7.5 to 4.5, akin to the SC pH gradient (Deraison et al., 2007). Thus, the processes of KLK binding to LEKTI fragments in the deeper SC and release of free KLKs in the superficial SC are intrinsically governed by the decreasing pH gradient along the SC, as shown in Figure 1.8. The removal of KLK inhibition combined with the retention of KLK proteolytic activity at acidic SC pH of 5.5 to 4.5 leads to regulated corneodesmosomal degradation and proper skin desquamation from the superficial SC layer (Caubet et al., 2004; Deraison et al., 2007). The pH-dependent regulation of KLK activity implicates SC homeostatic barrier functions other than desquamation, such as antimicrobial processing and lipid barrier formation.

pH ~ 5 pH ~ 7.5 2+ High Ca2+ Low Ca

Figure 1.8. Kallikrein skin barrier proteolytic activation cascade

Known activation interactions between the eight kallikreins expressed in the stratum corneum (SC) forming a SC tissue-specific activation cascade are indicated with solid black arrows while unknown ones are denoted with question marks at the top. Upon activation, KLKs (KLK5 and KLK7) can target corneodesmosomes (DSG1, DSG4, DSC1, and CDSN) leading to skin desquamation or (KLK5, 6, and 14) can target PAR2 activation in keratinocytes. Epidermal serine protease inhibitors (LEKTI, elafin, and SLPI) regulate KLK activity in the epidermis.

1.4 Kallikrein-related peptidases in inflammatory skin diseases

Aberrations in kallikrein levels and/or activity have been detected in inflammatory skin diseases such as: Atopic Dermatisis (AD), Netherton syndrome (NS), Psoriasis, peeling skin syndrome and Acne Rosacea. Accumulation of scales with increased numbers of persisting corneodesmosomes in the upper SC has been detected in many xeroses and hyperkeratosis skin states. Overexpression of trypsin- and chymotrypsin-like KLK levels was detected spanning the SC, SG and the lower epidermis of AD skin lesions, with chymotrypsin-like elevations being more prominent (Komatsu et al., 2007a). However, KLK overexpression in AD does not translate into any significant increase in SC trypsin or chymotrypsin-like activities, presenting a puzzling observation that remains to be illuminated (Komatsu et al., 2007a). Alternatively, hyperactivity of KLK7 and elevated levels of many trypsin-like KLKs were detected in psoriasis. Some trypsin-like KLKs (KLK6, 10, and 13) were also elevated in non-lesional SC of psoriatic patients (Komatsu et al., 2007b). Simiarly, KLK overexpression was detected in the SC of patients with peeling skin syndrome (Komatsu et al., 2006a). PAR2 receptors are overexpressed in the epidermis of AD and NS skin lesions exhibiting similar co-localization as human tissue kallikreins (Rattenholl and Steinhoff, 2008), which suggests KLK-PAR co-regulation and involvement in the pathogenesis of inflammatory skin diseases. KLKs have been suggested to induce inflammation in these skin disorders via PAR2 activation, in addition to inducing sweat- mediated itch in AD (Stefansson et al., 2008; Steinhoff et al., 2003).

The paramount importance of maintaining a physiological regulatory balance between KLKs and their epidermal inhibitors is demonstrated in the devastating rare skin disease Netherton syndrome (NS). NS is characterized by severe barrier dysfunction, ‘bamboo hair’, and atopic allergy-like symptoms resulting from SPINK5 gene mutations leading to loss or truncation of the serine protease inhibitor LEKTI (Chavanas et al., 2000; Descargues et al., 2005). The skin barrier dysfunction symptoms are mediated by KLK hyperactivity in the LEKTI-free NS epidermis (Komatsu et al., 2002). By employing the Netherton Syndrome (NS) mouse model and confirming results in human NS skin, Briot et.al demonstrated that KLK5 indeed induces inflammation and atopic-like lesions in NS skin via a PAR2-NFκB mediated cytokine burst that creates a pro-Th2 inflammatory microenvironment in the underlying dermis (Briot et al.,2010). In LEKTI-deficient epidermis, hyperactive KLK5 activates PAR2 by proteolytic cleavage, and

29 induces NFκB-mediated ICAM, IL-8, TNF-α and TSLP cytokine overexpression. Overexpression of the proallergic cytokine TSLP implicates KLK5 hyperactivity in an innate allergy regulatory pathway, which may explain the susceptibility of the majority of NS patients to develop AD.

KLKs are also known to mediate inflammation via their inherent ability to regulate skin antimicrobial peptide processing, activity and function. Cathelicidins are important effectors of the innate immune system, known for their role as ‘alarmins’ which protect the body from bacterial and viral infections. Cathelicidin (hCAP18) is activated in human epidermis by KLK5 trypsin-like processing near the C-terminal to release a 37 amino acid long antimicrobial peptide, named LL-37 (Yamasaki et al., 2006). LL-37 is a broad spectrum active antimicrobial peptide against Escherichia coli, Staphylococcus aureus and group A Streptococcus and it has antiviral activity. Trypsin-like KLK5 and chymotrypsin-like KLK7 target LL-37 antimicrobial peptide processing in human epidermis to generate shorter antimicrobial peptides that are active against Staphylococcus aureus. Individuals with the inflammatory skin disease ‘acne rosacea’ show facial inflammation in response to stimuli and have an exacerbated response to irritants and allergens as a result of barrier dysfunction. The facical skin of rosacea patients displays increased serine protease activity, LL-37 antimicrobial peptide overexpression and increased inflammation, compared to non-lesional areas. Furthermore, rosacea skin has unique cathelicidin peptides and abundant KLK5 expression compared to normal skin, suggesting KLK5 activity in rosacea pathogenesis. Subcutaneous injection of active KLK5 in mice, in amounts mimicking those observed in rosacea, increases cathelicidin processing and induces leukocyte infiltration and inflammation, confirming KLK5 pathogenic involvement in this disease (Yamasaki et al., 2007).

In addition to inhibition, KLK serine protease activity is also regulated by a pro-KLK zymogen activation proteolytic cascade, as mentioned above. Recently, a seminal study highlighted a novel role for a granular keratinocyte membrane-bound serine protease, known as matriptase, in activating KLK5 and KLK7 in vitro and in vivo. The matriptase-KLK signaling pathway was examined in LEKTI-deficient epidermis (Sales et al., 2010). Matriptase autoactivation is more efficient than KLK5 autoactivation, which makes it a better activator of KLK5 and a more likely ‘initiator’ of KLK activation cascades in the lower epidermis of diseased skin. A summary of our current knowledge of KLK roles in inflammatory skin diseases is provided in Table 1.2 and the importance of KLK serine proteases/protease inhibitor balance in the skin is shown in figure 1.9.

Table 1.2. Summary of KLK involvement in inflammatory skin disease pathologies

Skin Disease KLK Levels Possible pathological Ref Disease Pathogenesis pathways involving KLK activity

Atopic A common Higher Dermatitis chronic trypsin and  Higher SC pH (due to (Briot et al., (AD) inflammatory, dry, chymotrypsin-like epidermal FLG mutations 2009; Cork itchy, allergic skin KLK levels causing a decrease in SC (OMIM et al., 2009; disease involving acidity or due to barrier Elias and 603165) immune, disruptions) leads to Schmuth, endocrine, No change in increased kallikrein serine 2009; metabolic, and total trypsin or protease activity?? Hansson et infectious factors chymotrypsin like  KLK hyperactivity leads to al., 2002; Komatsu et activities increased desquamation, lipid permeability barrier al., 2007a) dysfunction, pain, inflammation and allergy

Psoriasis A common Higher Vulgaris chronic trypsin and  Higher SC pH leads to (Ekholm (PV) inflammatory chymotrypsin-like increased kallikrein serine and dermatosis and KLKs and protease activity Egelrud, autoimmune skin expanded lower  KLK7 hyperactivity leads to 1999; (OMIM disease, in the epidermis increased desquamation, IL- Komatsu et 177900) characterized by 1β activation, inflammation al., 2007b; Increased trypsin- erythematous and itch Kuwae et like activity in plaques and al., 2002) lesional skin only keratinocyte hyperproliferation

 KLK hyperactivity (due to Netherton A rare autosomal Higher (Briot et al., SPINK5 mutations causing Syndrome recessive mutation KLK expression, 2009; Briot LEKTI inhibitor et al., 2010; (NS) in SPINK5 gene expand lower in dysfunction) results in Descargues on chromosome the epidermis overdesquamation, et al., 2005; 5q32 causing inflammation and allergy Sales et al., truncation and/or (OMIM onset 2010) loss of the 256500) Increased activity  Matriptase activation of pro- epidermal serine of KLK5, KLK7 KLK zymogens contributes protease inhibitor and KLK14, but to the overwhelming KLK LEKTI NOT KLK8 hyperactivity in this disease  Aberrant cathelicidin

expression as a result of KLK hyperactivity

Rosacea An inflammatory Higher skin disorder KLK expression,  Abnormal processing of (Yamasaki characterized by where they hCAP18 and LL-37 et al., 2007; facial lesions with expand lower in cathelicidin peptides by Yamasaki erythema. The the epidermis, as hyperactive KLKs, leading et al., 2006) etiology is well as aberrant to inflammation, barrier unknown, but expression of dysfunction, and itching symptoms are cathelicidin exacerbated by peptides, factors that trigger compared to innate immune normal skin responses

Activators Inhibitors

Figure 1.9. The innate balance of KLK activity is integral in maintaining a healthy skin barrier. Future inflammatory skin disease therapy development may include targeting of KLK activity in the epidermis. It is important to note than none of these inhibitors inhibit KLK8. Thus, KLK8 hyperactivity may offset the serine protease/protease inhibitor balance in skin diseases.

1.5 Psoriasis and Atopic Dermatitis

Psoriasis and Atopic Dermatitis are two of the most common inflammatory skin diseases. Psoriasis affects approximately 2% of the population, while Atopic Dermatitis affects about 10%. Both diseases involve complex genetics and environment interplay, along with clear aberrations in the immune system and skin barrier. Similarities and contrasting features of these two diseases in terms of clinical and pathological features (Guttman-Yassky et al., 2011a), and dominating immune cell subsets (Guttman-Yassky et al., 2011b), have been extensively studied.

Most scientific research, including the work described here, refers to psoriasis vulgaris or plaque-type psoriasis, which is the most common form of psoriasis affecting about 85% to 90% of psoriatic patients (Nestle et al., 2009). About one-third of psoriasis vulgaris patients have moderate-to-severe disease, clinically classified on the basis of the lesions’ surface area and significant impact on patients quality of life, indicated with a Psoriasis Area and Severity Index (PASI) score of greater than 12 (Feldman, 2004). Psoriasis is associated with a high degree of morbidity. It mostly begins at a young age and is a lifelong condition. Patients feel depressed and embarrassed by the appearance of their skin disease, have lower employment and income, and often suffer from side effects and recurrence. The cost for long term treatment of this disease is also a major economic burden. Among psoriasis patients, 30 to 40% have an inflammatory, disabling joint arthritis known as psoriatic arthritis. Skin disease precedes joint disease by an average of 10 years in 85% of patients with psoriatic arthritis. Thus, dermatologists have a key role in the early detection and treatment of psoriatic arthritis (Gottlieb, 2005).

Psoriasis is characterized by red, raised scaly plaques that cover the body surface and immune infiltrate into the dermis and epidermis. The scaly skin is a result of epidermal hyperproliferation, premature differentiation of keratinocytes and incomplete cornification with retention of nuclei in the stratum corneum (parakeratosis) (Nestle et al., 2009). Psoriasis is often considered an autoimmune disease, yet the autoantigen remains unknown.

Two opposing paradigms have been proposed to explain concurrent barrier defects and inflammatory symptoms in atopic dermatitis and psoriasis. The ‘inside-out’ theory postulates that skin barrier breakdown is a secondary response to the inflammation process that occurs due to

34 activation of immune cells by autoantigens, allergen and/or irritants. On the other hand, the ‘outside-in’ theory postulates that skin barrier defects drive the inflammatory response.

Amounting evidence supports both theories for psoriasis and atopic dermatitis. The “inside-out” theory of AD pathogenesis is supported by genetic defects leading to overproduction of T-helper cells, Th2 cells, causing allergy via IgE overproduction, inflammation via cytokine release and skin barrier defects via neutrophil proteases (Meyer-Hoffert et al., 2004). On the other hand, environmental challenges (i.e. mechanical trauma, chemical detergents, pathogens, allergens like Der P 1 protease produced by house dust mites, etc) and genetic abnormalities in skin barrier proteins, such as filaggrin, affect one or more components of the stratum corneum barrier causing its breakdown (Elias and Steinhoff, 2008). A defective barrier with abnormally SC permits increased water loss and entry of pathogens and allergens (Ziegler and Artis, 2010). As a result, stressed keratinocytes secrete cytokines that recruit leukocytes and activate inflammation in response to barrier defects, hence the term ‘outside-in’. In psoriasis, the ‘outside in’ theory is supported by the fact that skin wounding triggers formation of psoriatic plaques, known as the Kobner phenomenon. On the other hand, aberrations in the immune system result in domination of Th1 and Th17 cells in psoriatic epidermis, which induce dramatic changes on the skin epidermal barrier. Effective psoriasis therapies are largely based on blocking T-cell derived cytokines such as TNFα, which clear psoriatic plaques in support of the ‘inside oustide’ pathogenesis (Lowes et al., 2007).

These theories remain under intense debate. However, aberrant lymphocyte activation is still viewed as the main root cause of psoriasis, as it remains considered an autoimmune disease in line with the ‘inside-out’ theory. On the other hand, atopic dermatitis is viewed as an epidermal disease where a defective thin skin barrier, results in hyperactivated immune system. Although both psoriasis and AD have skin barrier and immune abnormalities, these two common diseases are characterized by distinct and opposing expression of barrier proteins and innate/adaptive immune players, as highlighted in Table 1.3 below. The mutual antagonism of T-helper (Th) cells in psoriasis and atopic dermatitis pathogenesis is extensively studied in terms of polarization of Th1 cells in psoriasis versus Th2 in Atopic Dermatitis (Eyerich et al., 2011). With the discovery of Th17 cells in 2007, recent research is unraveling key roles of this new T-helper cell subset in psoriasis (Martin et al., 2012).

Table 1.3. Differences between Atopic Dermatitis and Psoriasis

Atopic Dermatitis Psoriasis Vulgaris

Skin - Reduced keratinocyte - Lipid depletion, with increased Barrier differentiation, cornification, differentiation and cornification Characteristics moisture and lipid content

- No scaling - Scaling and hyperplasia

- No parakeratosis - Parakeratosis

Neutrophils in - No - Accumulation of neutrophils epidermis

Eosinophils - Increased eosinophils and mast cells - No eosinophils, but mast cells in dermis are present

T-helper cell - Th2 cells secreting IL4 and IL13 - Th1/Th17 polarization with polarization Th22 present, cytokines - Th22 present implicated include TNFα, IFNγ, - Attenuated Th17 pathway IL17A and IL22 - Increased Th17 pathway

Host defense & - Reduced antimicrobial peptides - Increased antimicrobial peptides infection (AMP), leading to high frequency of (AMP), leading to lower frequency bacterial infections frequency of infections

Vasodilation and - Evidnce of vasodilation but no - Both vasodilation and Angiogenesis angiogenesis angiogenesis are well documented

- Few blood vessels - Dilated and tortuous blood vessels near epidermis

1.6 Kallikrein-related peptidase-8 in normal and inflamed skin

KLK8/neurposin protein expression is mostly noted in mouse and human skin. Normal stratum basale does not express KLK8/neuropsin mRNA, but it is expressed in the stratum spinosum and granulosum of wild type mice. KLK8/neuropsin expression is highest in the skin during development or under pathological conditions (Yoshida et al., 2010). Upon applying the phorbol ester, TPA, to normal mouse skin, KLK8 protein expression was found to increase and to correlate with increased thickness of mice epidermis (Kishibe et al., 2012). Phorbol esters induce keratinocyte proliferation resulting in hyperkeratosis, and are often used to model psoriasis. Administering the phorbol ester, TPA, to KLK8-KO mouse skin resulted in suppression of KLK6, KLK7 and PAR2 expression indicating potential KLK8 participation in an epidermal protease cascade upstream of KLKs and proteinase-activated receptors (PARs) (Kishibe et al., 2012). The number of stratum corneum layers and proliferating cells was found to be higher in KLK8-KO mouse compared to the wild type mice. Thus, KLK8/neuropsin is involved in regulating normal keratinocyte proliferation, differentiation and desquamation. No degradation of the corneodesmosome DSG1 or CDSN is apparent in the KLK8-KO mouse, endorsing its involvement in desquamation (Kishibe et al., 2007). A recent study showed that KLK8 is also involved in wound healing, as its expression is induced in regions near incisonal wounds and its ability to heal the wound is associated upregulation of KLK6 and PAR2 (Kishibe et al., 2012).

With regards to understanding KLK8 involvement in inflamed skin, mouse studies seem to suggest that it is involved in inducing hyperkeratosis in inflamed skin. SLS-induced hyperkeratosis and acanthosis was largely inhibited in KLK8/neuropsin KO mouse (Shingaki et al., 2010). SLS is an irritant used to induce skin inflammation. Two mechanisms were recently identified to explain the KLK8-mediated hyperkeratosis: (1) via inhibition of the transcription factor, activator protein-2α or AP-2α, resulting in induction of cell proliferation. Consistently, studies have shown that AP-2α knockout mice have thick skin due to a hyperproliferative defect (Wang et al., 2006). Alternatively, KLK8-mediated hyperkeratosis could be induced (2) via stimulation by the nerve growth factor (NGF-p75) pathway which has also been shown to induce hyperkeratosis in inflamed skin (Shingaki et al., 2012).

Thus, KLK8/neuropsin mouse studies have implicated KLK8 in the regulation of normal epidermal stratification and induction of hyperkeratosis in inflamed skins. These findings place

KLK8 as a potential key player in normal skin barrier functions and inflammatory skin disease pathologies such as psoriasis.

1.7 Rationale, hypotheses and objectives

1.7.1 Rationale

Kallikrein-related peptidase-8 is the most abundant KLK serine protease in human skin barrier, yet it is one of the least studied epidermal KLKs. Studies have revealed significant roles for KLK5, KLK7 and KLK14 in normal and diseased human skin, but our understanding of KLK8’s role in normal and diseased skin is lagging behind. To date, KLK5, KLK7 and KLK14 are the only KLKs isolated from human skin tissue extracts in active forms. The roles of these proteases in the skin are well-studied because they are direct targets of the serine protease inhibitor LEKTI, which is mutated in the rare skin disease Netherton Syndrome. Netherton Syndome is a great model to study KLK5, KLK7 and KLK14 functions in the skin. However, KLK8 is not inhibited by LEKTI or any of the currently known extracellular epidermal inhibitors, and thus its activity, regulation and function in human epidermis is overlooked and understudied.

In recent years, studies in KLK8/neuropsin KO mice suggested KLK8 involvement in normal epidermal proliferation, desquamation and wound healing. Despite being a barrier repair protease, KLK8 could play a damaging role in inflamed skin, where it induces hyperkeratosis. Hyperkeratosis and acanthosis in sodium lauryl sulphate (SLS)-stimulated skin is inhibited in KLK8/neuropsin KO mice. These recent findings could have tremendous implications for inflammatory skin diseases in humans, such as psoriasis. Consistently, the KLK8 gene was recently listed as one of the 130 overexpressed core set of psoriasis disease-specific genes (Ainali et al., 2012).

As outlined in Table 1.3, psoriasis and atopic dermatitis are characterized by opposing epidermal and immune mechanisms, yet KLKs are reported to be generally overexpressed in both diseases. Since KLK8 induces hyperkeratosis in inflamed mouse skin, then it should play a major role in psoriasis, but not atopic dermatitis. The previously reported KLK overexpression in atopic dermatitis (AD) lesions could simply be due to barrier disruption as a result of tape-stripping, since no increase in total trypsin activity was noted in lesional AD skin compared to normal (Komatsu et al., 2007a). In contrast, total trypsin-like activity was reported to be significantly

38 higher in psoriatic lesions compared to non-lesional and normal skin (Komatsu et al., 2007b). Thus, it is very likely that KLK8 is an active protease in normal human skin, which is overexpressed and hyperactive in lesional psoriatic skin, but not atopic dermatitis lesions.

In order to understand KLK8 role in normal skin and common inflammatory skin diseases such as psoriasis and atopic dermatitis better, the following points need to be addressed: (1) KLK8-specific activity in normal human skin surface, (2) KLK8 regulation by normal epidermal factors, (3) KLK8 substrate specificity and epidermal targets, (4) KLK8 regulation by immune cell subsets implicated in psoriasis and atopic dermatitis (5) the effect of KLK8 hyperactivity on normal skin and (6) KLK8 expression in psoriasis and atopic dermatitis skin lesions. If KLK8 proves to be active in normal human skin surface and if its overexpression is induced by immune factors governing psoriasis to play a pathogenic role in the disease, then it is likely to be an attractive target for topical therapeutic development to hamper its skin surface activity in psoriatic lesions.

1.7.2 Hypotheses

We hypothesized that Kallikrein-related peptidase-8 (KLK8) can be isolated from normal human stratum corneum and sweat in its active form. Since psoriasis is characterized by altered keratinocyte proliferation and differentiation, and by infiltration of T-helper Th1 and Th17 cells into the epidermis, we hypothesized that KLK8 is overexpressed in psoriasis due to keratinocytes’ cross talks with Th1 and Th17 immune cells, independent of barrier injury. KLK8 overexpression may induce epidermal hyperplasia and enhanced innate immune gene expression in psoriatic lesions, giving further support to the ‘inside-outside’ dogma of psoriasis. Consequently, KLK8 pathogenic role in psoriasis should be reduced by current systemic Th1 and Th17 blocking treatments. Novel KLK8-specific small molecule inhibitors can be identified by high throughput screening to be developed into topical psoriasis therapeutic agents.

1.7.3 Objectives

Objective 1: Characterization of previously unidentified KLK8 activity in normal skin (Chapter 2) a) To identify cellular sources of KLK8 protease in human epidermis by testing its expression by epidermal keratinocytes, epidermal melanocytes and dermal fibroblasts

39 b) To produce recombinant human KLK8 proteases in both latent pro-KLK8 and active mature KLK8 form as important reagents for in vitro assays c) To examine regulation of KLK8 activity by normal epidermal pH and ion content, and identify potential KLK8 activators and targets that augment its role in normal skin barrier function d) To develop an immunocapture pull-down assay and elucidate KLK8-specific serine protease activity in normal human epidermis and sweat ex vivo

Objective 2: Characterization of KLK8 in psoriasis (Chapter 3 and 4) a) To profile KLK8 secretion by cultured epidermal keratinocytes in response to treatment with T-helper cell-derived Th1, Th17 and Th2 cytokines, alone or in combination b) To examine KLK8 effect on cultured epidermal keratinocytes and 3D full thickness human skin model in terms of keratinocyte proliferation, differentiation and expression of innate defense genes c) To investigate in vivo KLK8 expression in psoriasis and atopic dermatitis patients and correlate data with in vitro findings of KLK8 epidermal expression in response to different cytokine subsets d) To test KLK8 levels in skin and sera of psoriasis patients before and after psoriasis treatment with common biologic TNFα and IL17A-blockers, and correlate levels with clinical measures of skin improvement and psoriasis clearance e) To examine the role of KLK8 as serum biomarker of psoriatic arthritis in psoriasis patients

Objective 3: Characterization of KLK8 signaling and inhibition patterns in comparison to other trypsin-like epidermal KLKs (Chapter 5) a) To demonstrate KLK8 differential signaling through proteinase-activated receptor-2 (PAR2) b) To identify KLK8-specific small molecule inhibitors by high throughput screening

Chapter 2 Kallikrein-related peptidase-8 is an active serine protease in human epidermis and sweat and is involved in a skin barrier proteolytic cascade

Sections of this chapter were reproduced from the following published manuscripts:

Eissa, A., Amodeo, V., Smith, C.R., and Diamandis, E.P. Kallikrein-related Peptidase-8 (KLK8) (2011). Is an Active Serine Protease in Human Epidermis and Sweat and Is Involved in a Skin Barrier Proteolytic Cascade. The Journal of biological chemistry 286, 687-706.

2 Kallikrein-related peptidase-8 (KLK8) is an active serine protease protease in human epidermis and sweat and is involved in a skin barrier proteolytic cascade

2.1 Introduction

Multiple trypsin-like KLK peptidases are co-expressed in human epidermis and associated appendages, such as hair follicles, sebaceous and sweat glands (Komatsu et al., 2005b; Komatsu et al., 2006b). Interestingly, KLK8 was found to be among the most abundant trypsin-like KLKs in normal human stratum corneum and sweat (Komatsu et al., 2006b).Work done in Klk8/neuropsin-null mice suggested that Klk8/neuropsin plays an important role in neural plasticity and skin barrier homeostasis. Healing of chemically-wounded or UV-irradiated mouse skin is largely impaired in the absence of Klk8/neuropsin (Kirihara et al., 2003; Kitayoshi et al., 1999). Additionally, the dramatic increase of KLK8 mRNA in hyperkeratotic skin of psoriasis vulgaris, seborrheic keratosis, lichen planus, and squamous cell carcinoma patients, compared to normal and basal cell carcinoma skin, suggested that human KLK8 is involved in keratinocyte differentiation and skin barrier formation (Kuwae et al., 2002). KLK8 protein was also detected expression in psoriasis, atopic dermatitis and peeling skin syndrome skin tissues (Komatsu et al., 2007a; Komatsu et al., 2007b; Komatsu et al., 2006a). Although KLK8 involvement in normal skin barrier formation and inflammatory skin disease pathology has recently become apparent, our basic understanding of KLK8 enzymatic regulation and activity in normal skin remains lacking. It is essential to probe KLK8 protease activity in normal human skin, in addition to continuing the ongoing investigation of KLK8 function and regulation in Klk8/neuropsin knock out (KO) mouse skin. This must be done while keeping in mind the anatomical and physiological differences between mouse and human skin, as well as differences at the molecular level. Also, the activation motif of human KLK8 (QEDK-VLGGH) differs from that of the mouse Klk8/neuropsin (QGSK-ILEGR), suggesting that endogenous activators of KLK8 may differ between mouse and human species, even though these proteases may play similar roles.

To investigate human KLK8 enzymatic properties and delineate its potential activators and downstream targets in normal skin, we produced recombinant human KLK8 in its latent zymogen (pro-KLK8) and active mature form (mat-KLK8) in yeast Pichia Pastoris for in vitro

42 activation and degradation assays. Recombinant KLK8 regulation by relevant epidermal pH and cations, potential epidermal activators and inhibitors was investigated in a series of enzymatic assays. We also examined recombinant mat-KLK8 substrate specificity via kinetic analysis of its cleavage of a panel of fluorogenic AMC substrates and a small positional-scanning library of internally-quenched FRET peptides. KLK8 ability to activate potential co-localized epidermal pro-KLKs and LL-37 antimicrobial peptide was also examined. We performed these in vitro biochemical characterization assays under the hypotheses that this protease is induced during terminal keratinocyte differentiation and is activated in the SC extracellular space to participate in barrier functions. Thus, we suspected that this serine protease is active in normal upper epidermis and sweat. Herein, we investigated KLK8 expression during terminal keratinocyte differentiation in culture and developed a sensitive and specific immunocapture assay to probe its activity in human epidermal extracts and sweat ex vivo. Our findings shed light on the orphan epidermal protease KLK8 and provide evidence that this KLK is indeed an active serine protease in human stratum corneum and sweat and is an intriguing member of a proteolytic cascade regulating skin barrier integrity.

2.2 Materials and Methods

Materials – The rapid endoprotease profiling library of fluorescence resonance energy transfer (FRET) quenched peptides (PepSets™REPLi) was purchased from Mimotopes Pty Ltd (Australia). The human antimicrobial LL-37 peptide (Leu-Leu-Gly-Asp-Phe-Phe-Arg-Lys-Ser- Lys-Glu-Lys-Ile-Gly-Lys-Glu-Phe-Lys-Arg-Ile-Val-Gln-Arg-Ile-Lys-Asp-Phe-Leu-Arg-Asn- Leu-Val-Pro-Arg-Thr-Glu-Ser) was purchased from Genemide Synthesis, Inc (San Antonio, USA). The majority of synthetic fluorogenic AMC substrates were purchased from Bachem Bioscience (King of Prussia, PA). AAPF-AMC and AAPV-AMC were obtained from Calbiochem. All AMC substrates were diluted in DMSO at a final concentration of 80 mM and stored at –20 °C. Recombinant KLK5 and KLK14 were produced in Pichia Pastoris as described previously (Borgono et al., 2007c; Michael et al., 2005). Recombinant pro-KLK11 was produced in Chinese Hamster Ovary cells and recombinant pro-KLK1 was produced in human embryonic kidney cell line, HEK293, as described previously (Emami and Diamandis, 2008; Luo et al., 2006).

2.2.1 Cloning, expression, and purification of recombinant human KLK8 proteins

Recombinant mature KLK8 protease was produced in the Original Pichia Pastoris expression system (Invitrogen). Briefly, PCR-amplified DNA fragment encoding mature KLK8 isoform-1 (amino acids 33-260 of NCBI gene bank accession no. NP_009127) flanked by XhoI and EcoRI restriction enzyme sites was cloned into pPIC9 expression vector, in-frame with its α-secretion signal and the alcohol oxidase AOX1 gene. Purified mat-KLK8-pPIC9 DNA construct was confirmed by sequencing using 5’-AOX1, 3’-AOX1, and α-secretion signal vector-specific primers and NCBI BLAST Align program. The mat-KLK8-pPIC9 construct was linearized with SacI and transformed into KM71 P.pastoris strain by electroporation. A stable KM71 transformant was grown in 1L BMGY media. After two days, yeast culture was centrifuged and the cell pellet was suspended in 300mL BMMY media (OD600= 10). Recombinant KLK8 expression was induced with 1% methanol for 5 days at 30ºC in a shaking incubator (250 rpm). Recombinant mat-KLK8 was purified from culture supernatant by ultra-concentration, serial dialysis and centrifugation procedures, followed by cation-exchange chromatography. One liter of culture containing secreted mat-KLK8 was centrifuged and supernatant was concentrated 10- fold by positive pressure ultracentrifugation in an Amicon TM stirring chamber (Millipore Corporation, Bedford, MA) with a 10 kDa cut-off regenerated cellulose membrane (Millipore). A series of bench-top purification experiments using an aliquot of KLK8-containing supernatant and SP Sepharose Fast Flow beads packed in Econo-Pac open column (Bio-Rad) were performed. We determined the optimal binding buffer for cation exchange purification of mat- KLK8 to be 0.01M acetic acid containing 50 mM NaCl (pH 4.76) and the optimal salt concentration for elution to be 250 mM NaCl. These findings were translated into an automated method where mat-KLK8 protein was purified using automated ÄKTA FPLC system on a pre- equilibrated 5 ml cation exchange HiTrap high performance sepharose HP-SP column (GE Healthcare), after serial dialysis (3X) against 0.01 M Acetic Acid, 50 mM NaCl (pH 4.76), running buffer A. Mat-KLK8 was eluted in 4 ml fractions via a step-wise salt gradient using 1M NaCl in 0.01M Acetic Acid (pH 4.76), Buffer B, at a flow rate of 1 ml/min, as follows: (a) 5% B for 25 min, (b) 10% B for 25 mins, (c) 15% for 25 mins, (d) 25% for 15 mins, (d) followed by a continuous gradient from 25%-100% B for 15 mins. Recombinant mat-KLK8 was further purified using 10 ml cation exchange Source15S Tricorn™ Column (GE Healthcare), which

44 resulted in elution of a very pure protein in 3 fractions that were pooled, concentrated, and stored at -80 ˚C.

Pro-KLK8 isoform 1 cDNA (amino acids 29 to 260) was cloned into pPIC9 Pichia Pastoris yeast vector and transformed into a stable GS115 yeast strain as described above for mat-KLK8. The recombinant colony was grown in 1L BMGY medium for 1 day and suspended in 2L

BMMY (OD600 =1.0). Pro-KLK8 expression of was induced with 1% methanol for 6 days at 30ºC in a shaking incubator (250 rpm). After concentrating the culture supernatant 20-fold, recombinant pro-KLK8 was purified after serial dialysis by cation-exchange chromatography using a HiTrap high performance sepharose HP-SP column connected to automated ÄKTA FPLC system. Pro-KLK8 was eluted in 4 ml fractions using the same step-gradient described above for mat-KLK8 purification. The protein eluted as a single peak and the protein-containing fractions were pooled, concentrated, and stored at -80 ˚C in 0.01 M acetic acid containing 250 mM NaCl (pH 4.76).

2.2.2 Detection of active mat-KLK8 and latent pro-KLK8 recombinant protein expression

The purity of recombinant proteins produced was assessed on silver-stained SDS-PAGE. Concentration was determined by the BCA method (Pierce), and protein identity was confirmed by mass spectrometry and N-terminal sequencing. SDS-PAGE was performed using the NuPAGE BisTris electroporesis system and precise 4-12% gradient polyacrylamide gels at 200 V for 45 min (Invitrogen). KLK8 Proteins were visualized with a Coomassie G-250 staining solution, SimplyBlueTM SafeStain (Invitrogen), and/or by silver staining with the Silver XpressTM Kit (Invitrogen), according to manufacturer’s instructions. For immunblotting of KLK8, proteins resolved by SDS-PAGE were transferred onto a Hybond-C Extra nitrocellulose membrane (GE Healthcare) at 30 V for 1 h. The membrane was blocked with Tris-buffered saline/Tween (0.1 mol/liter Tris-HCl buffer (pH 7.5) containing 0.15 mol/liter NaCl and 0.1% Tween 20) supplemented with 5% nonfat dry milk overnight at 4 °C and probed with a KLK8 polyclonal rabbit antibody (produced in-house; diluted 1:2000 in Tris-buffered saline/Tween) for 1 h at room temperature. The membrane was washed three times for 15 min with Tris-buffered saline/Tween and treated with alkaline phosphatase-conjugated goat anti-rabbit antibody (1:5,000 in Tris-buffered saline/Tween; Jackson ImmunoResearch) for 1 h at room temperature. Finally,

45 the membranes were washed again as above, and the signal was detected on x-ray film using chemiluminescent substrate (Diagnostic Products Corp., Los Angeles).

Mass spectrometry analysis for positive identification of both recombinant pro- and mat-KLK8 proteins was performed. N-terminal sequencing was performed by the Edman degradation. Briefly, proteins were transferred by electroblotting to polyvinylidene difluoride membrane and visualized with Coomassie Blue Stain. The bands were excised and applied to the sequencer.

2.2.3 Gelatin Zymography

Mat-KLK8 proteolytic activity was visualized by gelatin zymography (Novex® 10% Zymogram, Gelatin, Invitrogen), according to manufacturer’s instructions. Briefly, mat-KLK8 was diluted 1:1 in Tris-glycine SDS sample buffer and electrophoresed for 2 hrs at 125V at 4 ºC. After electrophoresis, the gels were incubated in renaturing buffer for two- 30 min intervals at room temperature, followed by incubation in developing buffer for 4 hrs at 37 ºC. Gels were stained with SimplyBlueTM SafeStain and destained until the white lytic bands corresponding to areas of protease activity were visible against a dark blue background.

2.2.4 AMC substrate profiling and kinetics constant determination

Mat-KLK8 hydrolysis of 16 fluorogenic AMC-peptides was investigated using the same KLK8 concentration (12nM) and increasing concentrations of AMC peptides (0.03, 0.06, 0.12, 0.25, 0.50, 0.75, 1.0 mM) in KLK8 activity buffer (100mM phosphate, 0.01% Tween 20, pH 8.5). The trypsin-like substrate peptides tested were VPR-AMC, GGR-AMC, FSR-AMC, PFR-AMC, LKR-AMC, LRR-AMC, QRR-AMC, QAR-AMC, QGR-AMC, GPR-AMC, GPK-AMC, EKK- AMC, VLK-AMC. AAPF-AMC and LLVY-AMC were the chymotrypsin-like substrate peptides tested. The known neutrophil elastase substrate AAPV-AMC was used as a negative control. KLK8-free reactions, for each peptide concentration, were used as negative controls and background counts were subtracted from each value. Free AMC fluorescence was measured on the Wallac 1420 Victor2TM fluorometer (PerkinElmer LifeSciences) with excitation and emission filters set at 380 and 480 nm, respectively, at 1-min intervals for 20 min at 37 °C. A standard curve was constructed using known concentrations of AMC in order to calculate the rate of free AMC emission. The slope of the resultant AMC standard curve was 19.18 AMC fluorescence counts/nM free AMC. The steady-state (Michaelis-Menten) kinetic constants (kcat/Km) were

46 then calculated by non-linear regression analysis using Enzyme Kinetics Module 1.1 (Sigma Plot, SSPS, Chicago, IL). All experiments were performed in triplicate and repeated at least twice.

2.2.5 Cleavage of a positional-scanning rapid endopeptidase library (RepLi) of FRET-quenched peptides

The RepLi library contains FRET-quenched peptide pools lyophilized into 512 wells in a total of six 96-well plate format. Each well contains 8 peptides with the same amino acid combinations in their variable tri-peptide core, which allows screening of 512 peptide pools with 3375 potential cleavage sites. The soluble peptide library pools (i.e. 512 wells) in six 96-well plates were diluted with mat-KLK8 activity buffer, 100 mM sodium phosphate buffer without Tween- 20 (pH 8.50) to a final concentration of 50 µM. Tween-20 was not included because it is not compatible with mass spectrometry analysis. After agitating the plate for 1 min, 20 µl aliquots of each well were collected as background controls. Background readings were measured using Envision 2103 Multilabel Reader (excitation λ=320 nm, emission λ=400 nm). After measuring background readings, 10 µl of mat-KLK8 was added (10 nM final) to each well of the six 96- well RepLi plates, prior to incubating plates at 37˚C for 1 hr. This library was incubated with minimal amount of active enzyme (10 nM) for 1 hr to avoid selection of peptides containing non- optimal cleavage sites. Fluorescence data were analyzed before and after protease addition. Cleavage was determined by assigning “strong, moderate, weak, and no cleavage” identifiers to wells generating a signal to background ratio (S:B) of “ ≥ 2, between 1.50-2.0, between 1.25- 1.50, and ≤1.25”, respectively. The cleavage sites of selected wells that showed the highest fluorescence readings were determined by LC-MS analysis, comparing the sample before and after mat-KLK8 addition.

2.2.6 pH, divalent cations, and glycolsylation effect on mat-KLK8 activity

Four buffer systems were assessed to determine the optimal pH for mat-KLK8 activity; 1 M potassium phosphate buffer (pH 5.0-6.5), PBS ( pH 7.0-7.5), 50 mM Tris-HCl (pH 8.0-9.0), and

100 mM sodium phosphate (pH 7.0-9.0). Solutions prepared from salts of ZnCl2, MgCl2, CaCl2, NaCl, and KCl were added to optimal activity buffer containing 0.25 mM VPR-AMC at a final concentration of (0, 10-2, 10-3, 10-4, 10-5, 10-6 and 10-7 nM) in a final volume of 100 µl. At this point, KLK8 (12 nM) was applied to each reaction mixture, and the plate was agitated for 1 min.

Residual KLK8 activity against VPR-AMC after incubation in each buffer pH or with each individual cation was calculated. Alternatively, mat-KLK8 was treated with PNGase F to remove N-glycans without denaturation. The same amount of PNGase-treated mat-KLK8 and mock treated mat-KLK8 (12nM) were added to activity buffer containing VPR-AMC (0.25 mM) in a final volume of 100µl, and AMC fluorescence was measured to test the deglycosylation effect on KLK8 activity.

2.2.7 Mat-KLK8 autodegradation

Aliquots of intact mat-KLK8 enzyme (100 ng) were incubated in KLK8 activity buffer at 4˚C, 25˚C, and 37 ˚C 0, 0.5, 1, 2, 4, 6, 12, 24 and 34 hrs. Autodegradation fragments were detected by reduced silver-stained SDS-PAGE and their activity was tested against VPR-AMC substrate, as described above.

2.2.8 Pro-KLK8 zymogen activation by KLK5, KLK1, and lysyl- endopeptidase

Activation studies of pro-KLK8 by potential recombinant activators were done in two consecutive steps, an “activation step” followed by a “detection step”. In the activation step by mat-KLK5, pro-KLK8 (200 nM) was added to 20 nM active KLK5 at increasing incubation times at 37 °C (1, 3, 18, 24, 48 hr) and at 25 °C (day 1, 2 and 4) in KLK5-optimized activity assay buffer (100 mM phosphate buffer, 0.01% Tween 20, pH 8.0) in a total volume of 35 µl. In the detection step, pro-KLK8 activation was monitored as an increase in the fluorescence of cleaved AMC, off VPR-AMC, after adding 10 µl of the activation mix to 90µl KLK8-optimized assay buffer (100 mM phosphate buffer, 0.01% Tween 20, pH 8.5) containing 0.1 mM VPR- AMC. To avoid confounding results due to KLK5 similar activity towards VPR-AMC, we included a duplicate activation mix where α1-antitrypsin inhibitor (AT) is added to quench KLK5 activity prior to detecting pro-KLK8 activation. α1-antitrypsin (AT) was added at a 5-fold molar excess and incubated for an additional hour at 37 °C. Activity towards VPR-AMC was measured for both reaction mixtures, with or without AT in triplicates. To test activation of pro- KLK8 by KLK1, 200 nM pro-KLK8 was incubated with 20 nM active mat-KLK1 for 1 hr and 3 hr at 37 °C in 35 µl activity assay buffer (100 mM phosphate buffer, 0.01% Tween 20, pH 8.0). Pro-KLK8 activation by lysyl endopeptidase was performed by incubating 200 nM pro-KLK8 with lysyl endopeptidase in its optimal activity buffer (50mM Tris, 10mM CaCl2,150 mM NaCl ,

48 pH 9.0) at an activator to pro-KLK8 molar ratio of 1:1000 for 1 hr at 37 oC in 35 µl total volume. “Detection” of activation was done in triplicates where 10 µl of each activation mix was added to 90 µl KLK8-optimized assay buffer (100 mM phosphate buffer, 0.01% Tween 20, pH 8.5) containing 0.1 mM VPR-AMC. The fluorescence values obtained for the activation mix, pro- KLK8 alone and activator alone reaction controls were subtracted from raw values of the no enzyme background control. The reaction rate was then calculated by measuring the slope in FU/min and converting it to free AMC (nM)/min.

2.2.9 Inhibition of KLK8 by epidermal inhibitors and general serpins

Mat-KLK8 (12 nM and 3 nM) was incubated with 30 nM of each of the four LEKTI domains (D1-6, D6-9, D9-12, D12-15) for 1 hr at 37˚C in optimal KLK8 activity buffer. To detect the potential inhibitory effect of each LEKTI fragment, 10 µl of each mix was added to 90 µl KLK8- optimized assay buffer containing 0.25 mM VPR-AMC. 12 nM of KLK5 was also incubated with each inhibitory LEKTI fragment as a positive control. SLPI or elafin (60 nM and 600 nM) were incubated separately with mat-KLK8 (6nM) in a final volume of 20 µl for 1hr at 37 °C. Control reactions, i.e. elastase, elafin, and SLPI incubated alone, were also performed. 6 nM neutrophil elastase (NE) was tested as a positive control for SLPI and elafin inhibition. KLK8 activity was also tested upon incubating with 0.1 mg/ml or 0.01 mg/ml soybean trypsin inhibitor (STI) or aprotinin, 1 mM PMSF, 1 mg/mL α1-antitrypsin inhibitor and 1 mg/mL chymostatin for 1 hr at 37˚C in optimal KLK8 activity buffer. 10 µl aliquots of each inhibitor-treated and non- treated reaction mix were added to 90 µl KLK8-optimized assay buffer containing 0.25 mM VPR-AMC in triplicates in a 96-well plate, so that the final KLK8 concentration in each well is 6 nM. Enzyme free reactions were included to be used as background controls. KLK5 was also treated with the same inhibitor concentration and incubation time as a control and for comparison purposes.

2.2.10 Activation of Pro-KLK1, pro-KLK11, and pro-KLK5 by KLK8

Pro-KLK1 and active KLK8 were incubated at a 1:1 molar ratio in a total volume of 50 µl at 37 °C for 10 min and 30 min time points. Reactions were done in triplicates. Given the issues of pro-KLK1 auto-activation and similar trypsin-like activity of mat-KLK8, KLK1-specific activity was measured by detecting fluorescence release of pulled-down KLK1 before and after incubation with mat-KLK8, as previously described (Emami and Diamandis, 2008).

Pro-KLK11 (1 µM) and active KLK8 (10 nM) were incubated at a 1:100 molar ratio in a total volume of 50 µl at 37 °C for 0.5, 1.5 and 3 hr time points in optimal KLK8 activity buffer. Detection of activation was done in triplicates where 10 µl of the activation mix was added to 90 µl KLK11-optimized assay buffer (50 mM Tris, 1.0 M NaCl, 0.01% Tween-20, pH 8.5) containing 0.25 mM PFR-AMC.

KLK5 (20nM) and active KLK8 (20 nM) were incubated in a total volume of 50 µl at 37 °C for 1 hr and 3 hr time points in optimal KLK8 activity buffer. Detection of activation was tested in triplicates where 10 µl of the activation mix was added to 90 µl KLK5-optimized assay buffer (100 mM sodium phosphate, 0.01% Tween-20, pH 8.0) containing 0.1 mM FSR-AMC. The increase in fluorescence signal was measured on a Wallac Victor fluorometer, as described above.

2.2.11 Proteolytic processing of LL-37cathelicidin antimicrobial peptide

For analysis of LL-37 processing by mat-KLK8, l40 µM of LL-37 synthetic peptide was incubated with 20 nM KLK8 for 0, 2, and 6 h at 37˚C in total volume of 400µl of 50mM Tris- HCl buffer containing 2 M NaCl (pH 8.50). After incubation, peptides were separated by two independent methods: 1) peptide separation by reverse-phase HPLC (Agilent Eclipse XDB-C18, 5µm) followed by peptide identification by LC/MS, and 2) direct peptide separation and identification of the LL-37/KLK mixture by LC/MS/MS.

In the first method, the C18-column was equilibrated in 10% acetonitrile with 0.1% trifluoroacetic acid at a flow rate of 0.8 ml/min for 10 min, and cleaved peptides were eluted using a 30 min gradient of 10-100% acetonitrile. Column effluent was monitored at 214 nm and 280 nm, and one fraction was collected per minute. All collected 0.8 ml fractions were lyophilized prior to pre-concentration using the OMIX C18MB (Varian) tips and eluted with 5 µL of buffer A (0.1% formic acid and 0.02% trifluoroacetic acid in 65% acetonitrile). In the second method, the incubation mix was purified and pre-concentrated using the OMIX C18MB

(Varian) tips and immediately applied to a mass-spectrometer set-up with C18 trap column using the EASY-nLC system (Proxeon Biosystems, Odense, Denmark). Recombinant KLK14 ability to process LL-37 was tested given that this possibility was not previously investigated. KLK5 cleavage of LL-37 was included as a positive control.

2.2.12 Calcium induction of terminal keratinocyte differentiation and KLK8 secretion

HaCat cells were cultured in low-serum EpiLife medium (Invitrogen) containing 0.06 mM calcium. One million HaCat cells were seeded in two T-175 flasks from the same passage (passage 3). In the calcium-treated flasks, HaCat cells in low calcium (0.06 mM) basal condition were switched to high calcium (2.0 mM) at 20% confluency. Calcium treated and non-treated cells were allowed to reach 70% confluency prior to switching to Epilife serum-free medium (SFM) containing no added supplements. One mL aliquots of the SFM were collected each day for three days to measure total protein and KLK8 levels.

2.2.13 Collection and preparation of sweat and stratum corneum extracts

Sweat samples were collected from the face, arms, legs, stomach and abdomen of 7 healthy donors during a dry sauna session. Volunteers showered the night before, and had not applied any topical agents to their skin. Sweat samples were collected using 1mL pipettes into 15 mL tubes and snap frozen on dry ice prior to storage at -20ºC. On the other hand, stratum corneum flakes were collected from 8 volunteers using previously described ‘tape-stripping’ and ‘scraping’ methods (Bernard et al., 2003). The ‘scraping method’ generated a higher number of total protein extracts during method optimization and hence this procedure was used for the actual study. Briefly, scraping buffer (50mM sodium phosphate buffer (pH 7.2) containing 5mM EDTA, 150mM NaCl, 0.1% Tween-20) was applied to each volunteer’s forearm and spread evenly to moisten. A microscope slide was then used to scrape the skin surface until corneocyte cells were visible on the slide. The corneocytes were washed off the slide into a 50mL tube with 10 ml buffer and soaked for 10 mins prior to storage at -20ºC. For processing, sweat and SC samples were thawed, vortexed for 10 min, and centrifuged at 4000rpm, 4ºC for 15 min. Sweat samples were pooled and dialyzed using a 3 kDa membrane to remove salts. Dialyzed pooled sweat and pooled SC samples were next passed through a Millipore 0.22 µm filter (Nalgene Syringe Filters, 0.2um; 25mm) and finally concentrated 20X using Amicon Ultra Centrifugal Filters with a 3 kDa molecular mass cut-off. Concentrated and pooled sweat and SC tissue extracts were stored at -20ºC until analysis. Sweat and SC solubilized total protein amounts were quantified using the BCA assay (Pierce).

2.2.14 KLK8 expression in human sweat, stratum corneum extracts, and skin cell cultures

KLK8 concentration in sweat and SC tissue extracts, as well as in the culture media of HaCat keratinocytes, primary human epidermal keratinocytes, epidermal melanocytes, and dermal fibroblasts was determined by a monoclonal-monoclonal KLK8 sandwich-type ELISA. In the case of culture media, primary epidermal keratinocytes (pHEK), primary epidermal melanocytes (pHEM), and primary dermal fibroblasts (pHDF) from neonatal human foreskin were purchased from Cascade Biologics (Invitrogen) and cultured according to the manufacturer’s instructions. The HaCat keratinocyte cell line was grown in DMEM medium containing 10% FBS. HaCat cells were plated at a seeding density of 3.0x104 cells/well while primary pHEK, pHEM and pHDF cells were seeded at a density of 2.0x105 cells/well in 6-well plates. The medium was changed after 48 hours, when cells were 60-80% confluent, to a fresh medium (5ml) and this day was marked as day 0. Aliquots of the medium from each well of the 6-well plates were collected daily for 12 days. Secretion of epidermal KLKs such as KLK5, 6, 7, 11, 13, and 14 was also investigated by KLK-specific ELISAs. To control for the viability of cells, the levels of the intracellular enzyme lactate dehydrogenase (LDH) were measured as an internal control in the culture media to indicate cell membrane rupture and cell death over time.

2.2.15 Immunocapture of KLK8 activity in sweat and SC epidermal protein extracts

Physiologically-relevant KLK8-specific activity was measured by detecting AMC fluorescence emission increase after adding VPR-AMC substrate to wells containing immunocaptured or pulled-down KLK8 compared to background. Briefly, 500 ng of KLK8-specific monoclonal antibody (Mono Ab 19-10) were immobilized overnight on a 96-well plate in coating buffer (50 mmol/liter Tris, 0.05% Tween 20, pH 7.8). The plate was washed three times with washing buffer (50 mmol/liter Tris, 150 mmol/liter NaCl, 0.05% Tween 20, pH 7.8). About 30 µL of sweat (~40 ng KLK8), 40 µL of SC extracts (~4 ng KLK8), and 30 µL of recombinant mat- KLK8 (~250 ng), as well as 10X diluted samples in a total volume of 100 µL 100mM sodium phosphate buffer (pH 7.5) were loaded per KLK8-antibody-coated well in triplicates. The plate was incubated at room temperature with gentle shaking for 3 hrs, and then washed six times with the washing buffer, above, to remove contaminants as well as non-bound KLK8. Subsequently, 200 µL of 0.50 mM VPR-AMC substrate in KLK8-activity buffer at optimal pH 8.5 or 5.0 was

52 added to each well. The substrate was incubated with the immunocaptured KLK8 for a total of 24 hrs at 37 ºC. The increase in fluorescence release was measured in real time at 20 minute- intervals on a Wallac Victor fluorometer, set at 355 nm for excitation and 460 nm for emission. For controls, recombinant active mat-KLK8, mat-KLK5, and lysyl endopeptidase were loaded into wells coated with KLK8-antibody in the same plate. To test if the sweat and SC contain latent pro-KLK8 that can be activated by potential activators, sweat and SC samples were spiked with active KLK5 or lysyl endopeptidase at 1:100 and 1:1000 molar ratios, respectively, overnight at 37ºC prior to loading into wells coated with KLK8-antibody.

2.3 Results

2.3.1 Recombinant mat-KLK8 and pro-KLK8 enzyme production and characterization

Mature KLK8 and pro-KLK8 proteins were produced in their native forms, without any fusion tags, in yeast (P. pastoris). Both proteins were secreted in the yeast culture supernatant after 1 day of 1% methanol induction, with the highest levels produced on day 6. Recombinant proteins were obtained with > 95% purity, as verified by silver-stained reduced SDS-PAGE and confirmed by mass-spectrometry. The yield of purified mat-KLK8 and pro-KLK8 from 1L culture supernatants was in the range of 0.8-1.5 mg, as determined by ELISA and BCA total protein assays. The apparent mass of both purified pro-KLK8 and mat-KLK8 recombinant proteins on a reduced SDS-PAGE was higher (~31 kDa) than their predicted molecular mass (~28 kDa). KLK8 has one predicted glycosylation site at N100SS. We detected recombinant KLK8 glycosylation upon treating both mat-KLK8 and pro-KLK8 enzymes with PNGase F. The non-glycosylated reduced form of KLK8 shifted lower from an apparent molecular mass of 31 kDa to 28 kDa.

The purified pro-KLK8 was visualized as a single glycosylated band of 31 kDa, with an N- terminal sequence of QEDKV as expected. Although purified mat-KLK8 appeared as an intact 31 kDa band at the time of purification, we detected 3 lower bands at 21, 11, and 8 kDa after keeping the enzyme at 4˚C for a week in PBS buffer (pH of 7.4) (Figure 2.1A lanes 1-3). Western blotting and N-terminal sequence analysis identified the low molecular mass (<28 kDa) bands as internal fragments of mat-KLK8, likely arising from auto-proteolytic cleavage, (Figure 2.1B and C). The N-terminal sequence of the top two bands, band I and band II, was determined

53 to be VLGGHE by Edman degradation, which corresponds to the N-terminal sequence of active mature KLK8. The lower two bands were identified as autodegradation products of mat-KLK8 having an internal N-terminal sequence of ENFPDT, indicating auto-cleavage after Arg164 (Figure 2.1C). By homology modeling using the pymol software, we found that Arg164 resides in an exposed, solvent-accessible surface loop, which is consistent with being susceptible to autolysis, Figure 2.1D. It is highly unlikely that the N-glycan attached to N100 of KLK8 participates in its substrate binding as it is directed away from the catalytic triad and substrate binding pocket (Figure 2.1D). Our results confirmed that deglycosylation had no effect on mat- KLK8 ability to cleave VPR-AMC.

A) B)

kDa 1 2 3 4 kDa 31 38 I

28 II 21 17

14 III

6 IV

Gelatin Silver WB Zymogram

C) 28 32 1 MGRPRPRAAK TWMFLLLLGG AWAGHSRA QE DK VLGGHECQ PHSQPWQAAL FQGQQLLCG 86 * 110 120 * 61 VLVGGNWVLT AAHCKKPKYT VRLGD(H ) SLQN KDGPEQEIPV VQSIPHPCY N SSDVEDHNH (D ) 164 121 LMLLQLRDQA SLGSKVKPIS LADHCTQPGQ KCTVSGWGTV TSPR ENFPDTLNCAEVKIFP 206 212 * 181 QKKCEDAYPG QITDGMVCAG SSKGA D TCQG D (S ) GGPLVCDG ALQGITSWGS DPCGRSDKPG 241 VYTNICRYLD WIKKIIGSK

E) F)

1200 G) Yeast mat-KLK8 1000

800 600

400 (nM AMC/min) 200

Rate of VPR-AMC hydrolysis 0

Mat-KLK8 Mat-KLK8 * 1 mM EDTA 1 mM PMSF 1 mg/mL AT Cocktail Inhibitor

1 mg/mL chymostatin

Figure 2.1. Activity and autodegradation of recombinant mat-KLK8. A. Silver-stained reduced SDS-PAGE of purified mat-KLK8 (lanes 1-4). Lane 4 represents intact purified recombinant mat-KLK8 protein at 31 kDa. Lanes 1-3 represent the purified mat-KLK8 protease after storage in PBS buffer (pH 7.4) at 4˚C for 7 days. B. Detection of active mat-KLK8 and degraded fragments in replicate silver-stained SDS-PAGE, western Blotting, and gelatin-

56 zymography. Intact KLK8 corresponded to the 31 kDa band. Lower molecular weight autodegradation fragments of KLK8 are labeled II-IV having an apparent molecular mass of 21, 11, and 8 kDa. Only bands I and II were active as revealed by the two white bands in the gelatin zymogram. C. Location of the N-terminal sequences obtained by Edman degradation of each KLK8 autodegradation fragment within the primary KLK8 protein sequence. Pre-pro-KLK8 is formed of a signal peptide, followed by a short 4 amino acid pro-peptide (bolded and underlined) and the mature KLK8 N-terminal sequence (bolded and boxed). The N-terminal sequence of KLK8 degradation fragments II, III, IV is boxed, with the corresponding label above. The R164 amino acid where autolytic cleavage occurs is bolded. The catalytic triad (H86, D120, S212) is indicated with an asterisk (*). The site of putative processing by a signal peptidase is C terminal to A28, K32 marks the activation site of the pro-KLK8 protease, N110 glycosylation site, and D206, which confers trypsin-like specificity of the mat-KLK8 protease are bolded and labelled with their position in the KLK8 primary sequence. KLK8 sequence is numbered from the N- terminus of pre-pro-KLK8 based on NCBI gene bank accession no. NP_009127. D. Location of key residues and the autolytic cleavage site within the theoretical tertiary structure of mature KLK8, as predicted by Pymol homology modeling. The ribbon plot of mature KLK8 is shown in the traditional serine protease standard orientation (i.e. looking into the active site cleft). Secondary structure elements are displayed as arrows (β-strands) and ribbons (α-helices). N- and C-terminal residues are shown in black. The side chains of the catalytic triad residues are shown in green. D206 is shown in red at the base of the active-site pocket. The glycosylation site is coloured orange. KLK8 auto-cleavage site after R164 at P1 is shown in magenta. E. Silver- stained SDS-PAGE displaying mat-KLK8 autodegradation in a time-course study. Cleaved fragments represented the majority of KLK8 detected after 12 hr incubations at 37˚C. F. Mat- KLK8 autolysis resulted in enzymatic inactivation detected by the drastic decrease in residual KLK8 activity towards VPR-AMC at time points 12, 24, and 34 hrs, corresponding to residual activity of 78%, 18% and 9%, respectively. G. Mat-KLK8 activity in the presence of inhibitors of different protease classes. The mat-KLK8 labeled with an asterisk was not incubated at 37˚C, while the remaining samples were incubated for 12 hr at 37˚C.

2.3.2 Pro-KLK8 zymogen activation in an epidermal cascade

To avoid confounding activity results due to overlapping KLK5 and KLK8 trypsin-like activity, α1-antitrypsin (AT) inhibitor was used in this study to quench KLK5 activity prior to detecting pro-KLK8 activation. As a control, we showed that AT decreased mat-KLK5 activity to less that 3% of its original activity, and had no inhibitory effect on mat-KLK8 activity at all (Figure 2.2). The significant increase in activity in the pro-KLK8/mat-KLK5 mix detected after 18 hrs at 37˚C, post quenching KLK5 activity with AT, indicated activation of pro-KLK8 (Figure 2.2). Although KLK5 is indeed an in vitro activator of pro-KLK8, this activation process was slow as it occurred after 18 hr incubation at 37˚C (Figure 2.2) or 1 day at 25˚C. Nonetheless, KLK5 activation of pro-KLK8 may be important physiologically as normal human epidermal cell turnover occurs in the span of 2-4 weeks. Furthermore, expression data suggest that immunoreactive KLK8 concentration in normal human SC tissue extracts is about 4-fold higher than KLK5 (Komatsu et al., 2005a). Hence, this activation process may occur quicker had we used a pro-KLK8: KLK5 molar ratio of 1:4 or 1:1 instead of 1:10.

We also tested if KLK8 is activated by KLK1 given that KLK1 is active in normal human sweat (Hibino et al., 1994) and co-localizes with pro-KLK8 in the SC. We used a pro-KLK1 preparation known to autoactivate and found that the pro-KLK8 was not activated by KLK1. Autoactivation of pro-KLK8 was not reported previously, however, we detected a time and concentration dependent increase in AMC fluorescence emission after incubating pro-KLK8 for 48 hr at 37˚C. This very slow autoactivation could be facilitated by host proteases. We thus carried out a stability time-course study using two recombinant pro-KLK8 enzymes produced in baculovirus and yeast expression systems. Pro-KLK8 proteases were incubated in the presence of inhibitors of different protease classes, similar to the experiment done above to investigate mat-KLK8 autodegradation. We detected increase in activity upon incubating both proteases alone at 37˚C for 48 hours. But unlike mat-KLK8 autodegradation, pro-KLK8 activation was facilitated by host serine proteases that were inhibited by AT treatment. For in vitro activation of pro-KLK8, lysyl endopeptidase was the best pro-KLK8 activator, due to its specific cleavage after lysine residues, where it resulted in rapid activation of pro-KLK8 (16.5-fold increase) within 1 hr incubation at 37˚C with 1:1000 (activator:pro-KLK8) ratio (data not shown). Thus, our data show that pro-KK8 does not autoactivate or get activated by KLK1, but it is activated by KLK5 and lysyl-endopeptidase in vitro.

37 C

500 - AT + AT 400

300

200 (nM AMC/min) 100

Rate of VPR-AMC hydrolysis 0

KLK5 (1hr)KLK5 (3hr) KLK5 (18 hr) pro-KLK8pro-KLK8 (1hr) (3hr) pro-KLK8 (18 h)

KLK5 + pro-KLK8KLK5 + pro-KLK8 (1hr) (3hr) KLK5 + pro-KLK8 (18hr)

Figure 2.2. Pro-KLK8 activation by active KLK5. Activation or pro-KLK8 by mat-KLK5 was carried at a molar ratio of (10:1), where a duplicate mix was included with α1-antitrypsin inhibitor (AT) as a KLK5 activity quencher. Each activation mix was incubated for varying time points (1, 3, 18, at 37 °C) with or without an additional 1 hr incubation with AT. Activity towards VPR-AMC substrate was measured for the same reaction mixures with or without AT in triplicates.

2.3.3 Effect of cations on KLK8 activity

KLK8 activity ability to hydrolyze VPR-AMC in the presence of the relevant epidermal cations Zn2+, Ca2+, Mg2+, Na+, and K+ was examined, as shown in Table 2.1. KLK8 was activated to a significant extent by Ca2+ ions at all concentrations examined. Mg2+ ions activated KLK8 but to a lower extent compared to Ca2+ and at higher concentrations. On the other hand, Zn2+ attenuated mat-KLK8 activity as expected given its inhibitory effect on numerous metal binding enzymes including metalloproteases and other KLKs. Zn2+ inhibition of 10 nM mat-KLK8 was pronounced in the µM and mM range but not in the nM range. Na+ and K+ cations had no significant effect on KLK8 activity at the concentrations tested. These results support the involvement of metal ions, particularly Ca2+ and Zn2+, at the active site of mat-KLK8. To date, the crystal structure of human KLK8 remains to be resolved, thus the exact mechanisms by which zinc and calcium bind human KLK8 active site remain to be elucidated.

2.3.4 Differential inhibition by skin specific inhibitors and general serpins

We investigated the inhibitory effects of three serine protease inhibitors known to be present in human SC: the lympho-epithelial kazal type inhibitor (LEKTI), SLPI, and elafin. SLPI and elafin did not inhibit mat-KLK8 activity. These two inhibitors inhibit chymotrypsin-like KLK7 activity, but do not exert any inhibitory effect on epidermal trypsin-like KLKs (Borgono et al., 2007b). Alternatively, inhibitory LEKTI domains (D1-15) inhibit distinct trypsin-like KLKs and chymotrypsin-like KLK7 with different potencies (Deraison et al., 2007; Descargues et al., 2005). Unlike other epidermal KLKs such as KLK5, KLK6, KLK7, KLK13, and KLK14, and similar only to KLK1 (Borgono et al., 2007b), all LEKTI domains had no inhibitory effect on mat-KLK8 activity, even at 10-fold higher molarity. All LEKTI domains tested inhibited KLK5 as a positive control.

We further tested mat-KLK8 inhibition by general serine protease inhibitors (serpins). We confirmed KLK8 inhibition by α2-antiplasmin, protein C inhibitor, and aprotinin. Unlike KLK5 and KLK14, mat-KLK8 was inhibited by the chymotrypsin-like inhibitor chymostatin, but not inhibited by the trypsin-like inhibitor α1-antitrypsin. Taken together, our results show that KLK8 regulation by endogenous inhibitors and general serpins is different from the two other epidermal trypsin-like KLK5 and KLK14.

Table 2.1. Divalent Ion effect on mat-KLK8 activity

Ion 1 Concentration 2 Molar ratio Residual Activity (mM) (KLK8:cation) (100%)

Ca 2+ 0.0001 (1:10) 114.9 0.001 (1:100) 116.5 0.10 (1:10,000) 121.0 1.00 (1:100,000) 145.0 10.0 (1:1000,000) 150.2 Mg 2+ 0.10 (1:10,000) 106.1 1.00 (1:100,000) 112.8 10.0 (1:1000,000) 142.0 Zn 2+ 0.0001 (1:10) 105.4 0.001 (1:1,00) 101.2

0.01 (1:1,000) 107.3

0.10 (1:10,000) 76.6

1.00 (1:100,000) 39.6

10.0 (1:1000,000) 28.6 Na + 10.0 (1:1000,000) 100 K + 10.0 (1:1000,000) 100

2+ + 1. Ca and Na data corresponds to incubation of KLK8 with CaCl or NaCl, respectively. 2

2. KLK8 final concentration was 0.00001 mM (or 10 nM) in all experiments

2.3.5 KLK8 AMC substrate profiling and steady-state kinetic constants

The substrate specificity of KLK8 was assessed by profiling its kinetic parameters (i.e. Km and kcat) against a panel of 16 tripeptide synthetic substrates containing an AMC fluorogenic leaving group. Among these AMC-peptides, thirteen were candidate trypsin-like enzyme substrates (10 with Arg and 3 with Lys basic residues at P1 position according to the Schechter and Berger notation), and two for chymotrypsin-like enzymes (with bulky, hydrophobic amino acids Tyr and Phe), and one substrate for neutrophil elastase (with small aliphatic Val at P1 position). As predicted by the presence of Asp206, close to Ser212 of the catalytic triad, KLK8 was confirmed to have trypsin-like, but not chymotrypsin-like activity, since no reaction was observed for the two chymotrypsin-like enzyme substrates (AAPF-AMC, LLVY-AMC). Mat-KLK8 displayed trypsin-like specificity with a greater catalytic efficiency for Arg vs. Lys at the P1 position, as substrates with the highest kcat/Km values contained P1-Arg. Although KLK8 did not cleave GPK, it cleaved VLK, indicating that is it capable of cleaving after lysine, depending on adjacent residues. The P2 specificity of KLK8 was examined by comparing the kcat/Km values among substrates with invariable P1 and P3 residues as follows: 1) QAR-AMC, QGR-AMC, and QRR- AMC; 2) LKR-AMC and LRR-AMC; and 3) GPR-AMC and GRR-AMC. KLK8 preferred A>R>G, R>K, G>P at P2. This suggested that P2 position may not influence mat-KLK8 specificity significantly, but this observation was based on comparing a small number of fluorogenic tripeptides. The P3 preference of KLK8 was assessed by examining kcat/Km values in substrates bearing the same P1 and P2 amino acids as follows: 1) VPR-AMC and GPR-AMC; 2) QGR-AMC, and GGR-AMC; and 3) QRR-AMC and LRR-AMC. Glycine was a highly unfavored residue at this position. The best substrates for mat-KLK8 were VPR-AMC, also a substrate for α-thrombin, and QAR-AMC, also a substrate for trypsin.

2.3.6 Rapid endopeptidase library screening of mat-KLK8 P2-P2’ substrate specificity

A rapid endopeptidase profiling library of quenched FRET peptides was utilized to reveal information about non-prime and prime side substrate specificity of mat-KLK8. The principle of this assay and how it differed from non-prime AMC substrate profiling is illustrated in Figure 4. Our data suggest that mat-KLK8 is a specific trypsin-like enzyme, where all top 29 peptide pools displaying strong and moderate cleavage contained R/K. Of the 57 peptide pools cleaved, 47 contained R/K in at least one variable core region (Figure 2.3A), consistent with the trypsin-like

62 specificity of KLK8 on the C-terminal side of basic residues. Interestingly, 10 of the peptide pools that showed weak cleavage (S:B = 1.25-1.50) contained no R/K, where 9 pools contained F/Y and one peptide pool contained I/L, suggesting restricted weak chymotrypsin-like activity of mat-KLK8. Mat-KLK8 enzyme is likely to function as a regulatory trypsin-like protease in upper epidermis rather than a degrading enzyme with broad specificity, as 122 out of the total 169 peptide pools containing at least one R/K (~ 72% of trypsin-like peptides) remained uncleaved after 1 hr incubation (S:B< 1.25). KLK8 selectivity can be seen by its in vitro ability to activate the pro-form of merpin-β, but not merpin-α, even though activation of both epidermal metalloproteases requires cleavage after arginine (Ohler et al., 2010). The top peptide hit for KLK8 with highest fluorescence increase contained the core motif (RK)-(S/T)-(A/V) and 6 out of the top 14 peptide hits contained (R/K)-(S/T) bond, indicating a strong preference for Ser/Thr at P1'. Using this peptide as a reference for positional-scanning profiling of mat-KLK8 specificity we found that mat-KLK8 displayed strong preference for hydrophobic I/L and F/Y amino acids at P2, specific preference for R/K at P1, restricted preference for S/T and F/Y at P1', and A/V, R/K, and F/Y at P2', (Figure 2.3B). All peptides containing negatively charged amino acids (D or E) at P'1 or P'2 in this library were not hydrolyzed by mat-KLK8. Furthermore, we detected potential subsite cooperativity in the active site pocket, where the presence of two (R/K) residues close to each other enhanced cleavage. These results were consistent with M. Debela, E.L. Schinder, C.S. Craik unpublished findings regarding mat-KLK8 non-prime substrate specificity where preference for R over K at P1, hydrophobic residues at P2, and R/K residues at P3 was observed (Ohler et al., 2010). Herein, we also show that the prime side of the scissile bond is important, where the highest fluorescence signal was obtained for the (R/K)-(S/T)-(A/V) FRET-quenched peptides. We confirmed KLK8-cleavage of this eight peptide pool after arginine by mass-spectrometry MS1 and identified the fluorescent tag and the cleaved N-terminal side by MS2 analysis.

Figure 2.3. Kallikrein-related peptidase-8 displays restricted substrate specificity based on cleavage of FRET-tripeptides. (A) Pie chart of mat-KLK8 cleavage of peptides after 1 hr incubations with 10 nM mat-KLK8 at 37˚C. (B) Mat-KLK8 specificity based on positional scanning of the top peptide hit result from the Rapid Endoprotease Library (RepLi) screenin

2.3.7 KLK8 activation of co-localized epidermal pro-KLKs

Pro-KLK5, pro-KLK11, and pro-KLK1 are potential downstream targets of mat-KLK8 given their co-localization with KLK8 in normal human sweat and SC and the requirement for cleavage after Arg for their activation. Pro-KLK10 is an unlikely substrate of mat-KLK8 because its activation sequence NDTRLDP contains two acidic D residues, P3-D and P2’-D, which are highly unfavored residue for mat-KLK8 based on our RepLi screening. Unfortunately, we were unable to draw any conclusions regarding KLK5 activation by KLK8, due to the confounding effect of KLK5 rapid autoactivation. In the case of pro-KLK1, which autoactivates, we utilized a sandwitch-type pull-down assay to capture KLK1 and completely eliminate mat-KLK8 activity. Although KLK8 exhibits activity towards PFR-AMC, no enzymatic activity was observed for the pulled-down KLK8 on KLK1 antibody-coated wells (Figure 2.4A). KLK8 ability to activate KLK1 was detected in a time-dependent manner, Figure 2.4A. In the case of pro-KLK11, time- dependent pro-KLK8 activation was seen within 30 min incubation period at 37˚C at 1:100 (KLK8: pro-KLK11) molar ratio Figure 2.4B. Hence, we demonstrated KLK8 ability to target in vitro activation of two potential substrates, pro-KLK11 and pro-KLK1, in human SC and sweat.

Figure 2.4. KLK8 activation of pro-KLK1 and pro-KLK11. (A) Pro-KLK1 activation by mat-KLK8 was performed using a sandwitch-type pull-down activity assay of pro-KLK1. Enzymatic activity of pulled down KLK1 was measured, using 0.25 mM PFR-AMC. (B) Pro- KLK11 activation by mat-KLK8. Time-dependent activation of pro-KLK11 was detected at a 1:100 molar ratio at 37 °C in optimal KLK8 activity buffer. Pro-KLK11 activation is measured by detecting fluorescence release off 0.25 mM PFR-AMC over time for the pro-KLK11 alone, mat-KLK8 alone, and the pro-KLK11/mat-KLK8 activation mixture as shown above.

2.3.8 KLK8 processing of LL-37 antimicrobial peptide

Cathelicidin antimicrobial peptides (APs) are effector molecules of the innate immune system detected in human SC, sweat, and wound secretions with microbicidal and proinflammatory activities (43 Gallo -45 Murakami). The 37-amino acid-long C-terminus of cathelicidin is referred to as LL-37 and represents the active peptide which displays direct antimicrobial activity by interaction with the cell membranes of microorganisms. We investigated KLK8 ability to process LL-37 synthetic peptide into shorter active antimicrobial peptides. We detected a single peak by RP-HPLC on a C18 column eluting at 55% B, Acetonitrile, corresponding to the LL-37 peptide alone which remained present after 2 and 6 hrs incubations at 37˚C. Processing of the LL37 peptide was observed upon incubation with 20 nM mat-KLK8, where the LL-37 peak seen at 25 minute decreased and broadened, and two earlier sharp peaks were detected indicating cleavage of LL-37. The two peaks corresponded to small peptides (charge state z = 2) with m/z ratio of 434.2390 and 458.2481, which were identified by LC/MS to represent the LL-7 and NL- 8 peptides, respectively. However, the abundance of the remaining cleavage products in the lyophilized RP-HPLC fractions was insufficient to be detected by LC/MS. Thus, we used an alternative method where the LL-37 control peptide and LL-37/KLK incubation mixtures were purified by C18 OMIX tips prior to separating on nano-LC C18 column attached directly to LTQ/Orbitrap mass-spectrometer. This sensitive method allowed us to identify all peptide fragments present in each sample. We identified the LL-7 and NL-8 fragments as well as IK14, LL-23, and LL-37 in the KLK8-treated sample. LL-37 peptide was treated with active KLK5 as a positive control and LL-7, NL-8, and IK-6 were identified as cleavage products, in addition to their other halves, KS-30*, LL-29*, KS-22*, which represents the active antimicrobial peptides marked with asterisks, respectively (Lundstrom et al., 1994). Our results show that active KLK8 and KLK14 process LL-37 AP after Arg residues generating KS-30*/LL-7, LL-29*/NL-8, and IK-14/LL-23* peptides. Figure 6 displays identified fragments in each LL-37/KLK incubation mix versus LL-37 peptide incubated alone for 6 hours. IK-14/LL-23* was not identified in the KLK5-treated sample because the IK-14 peptide was likely further proteolysed to IK-6 and NL- 8.

c:\chris\...\ll-37-klk5-6hrdigest_01c:\chris\...\ll-37-klk5-6hrdigest_01 11/17/200911/17/2009 3:06:37 PM 3:06:37 PM c:\chris\...\ll-37-klk5-6hrdigest_01 c:\chris\...\ll-37-klk5-6hrdigest_01LL-37 11/17/2009 3:06:37 PMLL-7 11/17/2009 3:06:37 PMNL-8 IK-14 m/z = 899.1226 m/z = 432.2390 m/z =458.2481 m/z = 563.3206 RT: 0.00 - 23.03 RT:SM0.00 : 7B - 23.03 zSM = :57B RT: 0.00 - 23.03RT:zSM =0.00 :27B - 23.03 SM : 7B RT:z =0.00 2 - 23.03RT:SM0.00 : 7B - 23.03 SM : 7B z = 3 RT: 0.00 - 23.03RT:SM0.00 : 7B - 23.03 SM : 7B RT: 0.00 - 23.03 SM : 7B RT: 0.00 - 23.03 SMRT: : 0.007B - 23.03 SM : 7B RT: 0.00 - 23.03 SMRT: : 0.007B - 23.03 SM : 7B RT: 0.00 - 23.03 SMRT: : 0.007B - 23.03 SM : 7B RT: 0.00 - 23.03 SM : 7B RT: NL:15.16 9.27E6 RT: 15.16NL: 9.27E6 NL: 9.27E6 NL:RT: 7.80E4 17.55 NL:RT: 7.80E4 17.55 NL: 7.80E4 RT: 12.85NL: 3.45E4 RT: 12.85NL: 3.45E4 NL: 3.45E4 RT: 8.67 NL: 3.21E4RT: 8.67 NL: 3.21E4 NL: 3.21E4 RT: 15.16 RT: 15.16 NL:RT: 9.27E6 17.55 RT: 17.55 RT: 12.85NL: 7.80E4 RT: 12.85 RT: 8.67 NL: 3.45E4 RT: 8.67 NL: 3.21E4 AA: m/150613447 z= AA: m/150613447 z= m/ z= m/AA: z= 292366 m/AA: z= 292366 m/ z= AA: 474056m/ z= AA: 474056m/ z= m/ z= AA: 390314m/ z=AA: 390314 m/ z= m/ z= AA: 150613447NO Enzyme m/ z=AA: 292366 AA: 474056m/ z= AA: 390314 m/ z= m/ z= 100 100 100 899.5137-899.5317 899.5137-899.5317100 AA: 150613447 899.5137-899.5317100 100 434.2352- 434.2352-100 434.2352-AA: 292366100 100 458.2430- 458.2430-100AA: 474056458.2430- 100 100 457.6086-457.6178 457.6086-457.6178AA: 390314 457.6086-457.6178 100 899.5137-899.5317 100 434.2352- 100 458.2430- 100 457.6086-457.6178 MS Genesis MS Genesis MS Genesis 434.2438 MS 434.2438 MS 434.2438 MS 458.2522 MS 458.2522 MS 458.2522 MS MS Genesis MS Genesis MS Genesis MS Genesis RT: 9.92 434.2438 MS 458.2522 MS MS Genesis ll-37-control- ll-37-control- ll-37-control-RT: 9.92 RT: 9.92 Genesis Genesis Genesis Genesis Genesis Genesis ll-37-control- ll-37-control- ll-37-control- ll-37-control- RT: 9.92 Genesis RT: 12.51 RT: 12.51 Genesis ll-37-control- 6hdigat37c_01 6hdigat37c_01 6hdigat37c_01AA: 602461 AA: 602461ll-37-control-AA: 602461 ll-37-control- ll-37-control-RT: 12.51 ll-37-control- ll-37-control-RT: 12.51 ll-37-control- 6hdigat37c_01 6hdigat37c_01 6hdigat37c_01 6hdigat37c_01 6hdigat37c_01AA: 6024616hdigat37c_01 ll-37-control-AA: 12562 AA: 12562 6hdigat37c_01 6hdigat37c_01 ll-37-control- 6hdigat37c_01 50 50 50 50 50 50 6hdigat37c_01 50 AA: 5012562 50 6hdigat37c_01 AA: 5012562 50 50 50 RT: 14.11 50 RT: 14.11 6hdigat37c_01RT: 4.89 RT: 4.89 50 6hdigat37c_01 50 RT: 14.11 RT: RT:14.11 4.89 RT: 4.89 AA: 24893 AA: 24893 AA: 85652 AA: 85652 AA: 24893 AA:AA: 24893 85652 AA: 85652 0 0 0 0 0 0 0 0 0 0 0 0 RT:NL: 15.49 6.54E60 RT: NL:15.49 6.54E6 NL: 6.54E6 RT: 9.86 NL: 1.98E7RT: 09.86 NL: 1.98E7 NL: 1.98E7 RT: 4.90 RT: 4.90NL: 4.79E60 NL: 4.79E6 NL: 4.79E6 RT: 8.76 NL: 1.44E4RT: 8.760 NL: 1.44E4 NL: 1.44E4 RT: 15.49 RT: 15.49RT: 9.86 NL: 6.54E6 RT: 9.86 RT: 4.90 NL: 1.98E7 RT: 4.90 RT: 8.76 NL: 4.79E6 RT: 8.76 NL: 1.44E4 AA:m/ 93940079 z= AA:m/ 93940079 z= m/ z= AA: 177596707m/ z=AA: 177596707m/ z= m/ z= AA: 61020732 AA: m/61020732 z= m/ z= m/ z= AA: 146250m/ z=AA: 146250 m/ z= m/ z= AA: 93940079KLK5 AA: 177596707m/ z= AA: 61020732 m/ z= AA: 146250 m/ z= m/ z= 100 100 100 899.5137-899.5317 899.5137-899.5317100 AA: 899.5137-899.531793940079100 100 434.2352- 434.2352-AA: 100177596707434.2352-100 100 458.2430- AA: 61020732458.2430-100 458.2430- 100 100 457.6086-457.6178RT: 12.62 RT: 457.6086-457.6178 AA:12.62 146250 457.6086-457.6178 100 MS Genesis 899.5137-899.5317 100 434.2438 MS 434.2352- 100 458.2522 MS 458.2430-RT: 12.62 100 MS Genesis 457.6086-457.6178 MS Genesis MS Genesis 434.2438 MS 434.2438 MS 458.2522 MS 458.2522 MS MS Genesis AA: MS 40697 Genesis RT: 12.62 ll-37-klk5- ll-37-klk5- MS Genesis Genesis Genesis 434.2438 MS Genesis Genesis 458.2522AA: 40697 MS AA: 40697 ll-37-klk5- ll-37-klk5- MS Genesis ll-37-klk5- ll-37-klk5- Genesis Genesis Genesis RT: 7.66 GenesisRT: 7.66 RT:ll-37-klk5- 7.66 AA: 40697 ll-37-klk5- 6hrdigest_01 6hrdigest_01 6hrdigest_01 ll-37-klk5- ll-37-klk5- ll-37-klk5- ll-37-klk5- ll-37-klk5- ll-37-klk5- 6hrdigest_01RT: 15.28 RT:RT: 7.666hrdigest_01 15.28 6hrdigest_01 6hrdigest_01 6hrdigest_01 ll-37-klk5- AA:6hrdigest_01 63449 ll-37-klk5-AA:RT: 6344915.28 AA: 63449 6hrdigest_01 50 50 50 50 6hrdigest_01 6hrdigest_01 50 50 6hrdigest_01 6hrdigest_01 50 50 AA:AA: 63449 11718 RT: 15.28 50 50 50 50 50 6hrdigest_01 50 50 6hrdigest_01AA: 11718 AA: 1171850 RT: 7.08 RT: 7.08 AA: 11718 RT: 7.08 RT: 7.08 AA: 3311 AA: 3311

AA: 3311 AA: 3311

Relative Abundance Relative Abundance Relative Abundance Relative Abundance Relative

Relative Abundance Relative Abundance Relative Abundance Relative

Relative Abundance Relative 0 0 0 0

Relative Abundance Relative Abundance Relative Abundance Relative 0 0 Abundance Relative 0 0 0 0 0 0 RT: 15.68 0 RT:NL: 15.68 3.41E6 NL: 3.41E6 RT: 9.84 RT:0 9.84 NL: 7.30E5 NL: 7.30E5 RT: 4.90 RT: 4.90 0 NL: 3.83E5 NL: 3.83E5 RT: 8.44 RT: 8.440 NL: 8.66E4 NL: 8.66E4 RT: 15.68 NL: 3.41E6 RT: 15.68RT: 9.84 NL: 3.41E6 NL: 7.30E5 RT: 9.84 RT: 4.90 NL: 7.30E5 NL: 3.83E5 RT: 4.90 RT: 8.44 NL: 3.83E5 NL: 8.66E4 RT: 8.44 NL: 8.66E4 AA:m/ 48301780 z= m/ z= AA: 10242151m/ z= m/ z= AA: 4492169 m/ z= m/ z= AA: 1387272 m/ z= m/ z= AA: 48301780 AA:m/ 48301780 z= AA: 10242151m/ z= AA: 10242151m/ z= AA: 4492169 AA:m/ z= 4492169 m/ z= AA: 1387272 m/ z= AA: 1387272m/ z= m/ z= 100 100 899.5137-899.5317AA: 899.5137-899.5317 48301780100 100 434.2352-AA: 10242151434.2352-100 100 AA: 4492169458.2430- 458.2430- 100 100 AA:457.6086-457.6178 1387272 457.6086-457.6178 100 KLK8899.5137-899.5317100 100 899.5137-899.5317 434.2352-100 100 434.2352- 458.2430-100 100 458.2430- 457.6086-457.6178100 457.6086-457.6178 MS Genesis MS Genesis 434.2438 MS 434.2438 MS 458.2522 MS 458.2522 MS MS Genesis MS Genesis MS Genesis MS Genesis 434.2438 MS 434.2438 MS 458.2522 MS 458.2522 MS MS Genesis MS Genesis LL-37-KLK8- LL-37-KLK8- Genesis Genesis Genesis Genesis LL-37-KLK8- LL-37-KLK8- LL-37-KLK8- LL-37-KLK8- Genesis Genesis Genesis Genesis LL-37-KLK8- LL-37-KLK8- 6hrdigest_01 6hrdigest_01 6hrdigest_01 LL-37-KLK8- LL-37-KLK8- LL-37-KLK8- LL-37-KLK8- LL-37-KLK8- LL-37-KLK8- 6hrdigest_01 6hrdigest_01 6hrdigest_01 6hrdigest_01 6hrdigest_01 LL-37-KLK8- 6hrdigest_01 LL-37-KLK8- 6hrdigest_01 50 50 50 50 50 50 6hrdigest_01 6hrdigest_0150 50 50 6hrdigest_01 6hrdigest_0150 50 50 50 50 RT: 17.75 6hrdigest_01 50 6hrdigest_01 50 RT: 17.75 RT: 17.75 RT: 17.75 AA: 1665381 AA: 1665381 AA: 1665381 AA: 1665381 0 0 0 0 0 0 0 0 0 0 0 0 RT:NL: 15.64 4.04E60 RT:NL: 15.64 4.04E6 NL: 4.04E6 NL:RT: 6.40E7 8.980 NL: 6.40E7 NL: 6.40E7 RT: 4.80 RT: 4.80NL: 6.56E60 NL: 6.56E6 NL: 6.56E6 NL:RT: 2.03E6 7.930 NL: 2.03E6 NL: 2.03E6 RT: 15.64 RT: 15.64RT: 8.98 NL: 4.04E6RT: 8.98 RT: 8.98 RT: 4.80 NL: 6.40E7 RT: 4.80 RT: 7.93 NL: 6.56E6RT: 7.93 RT: 7.93 NL: 2.03E6 m/ z= AA:m/ 18225518 z= m/ z= m/AA: z= 2080941934m/ z= m/ z= AA: 64525892m/ z= m/ z= m/ z= m/AA: z= 17746784 m/ z= m/ z= AA: 18225518 AA: 18225518 AA: 18225518AA: 2080941934m/ z=AA: 2080941934 AA: 64525892 AA:m/ 64525892 z= AA: 64525892 AA: 17746784 m/ z= AA: 17746784 m/ z= 100 100 100KLK14899.5137-899.5317 899.5137-899.5317100 899.5137-899.5317100 100 434.2352- AA:434.2352- 2080941934100 434.2352-100 100 458.2430- 458.2430-100 458.2430- 100 100 457.6086-457.6178 AA:457.6086-457.6178 17746784 457.6086-457.6178 100 MS Genesis 899.5137-899.5317 100 434.2438 MS 434.2352- 100 458.2522 MS 458.2430- 100 MS Genesis 457.6086-457.6178 MS Genesis MS Genesis MS Genesis 434.2438 MS 434.2438 MS 434.2438 MS 458.2522 MS 458.2522 MS 458.2522 MS MS Genesis MS Genesis MS Genesis ll-37-klk14- ll-37-klk14- ll-37-klk14- Genesis Genesis Genesis Genesis Genesis Genesis ll-37-klk14- ll-37-klk14- ll-37-klk14- 6hrdigest_01 ll-37-klk14- ll-37-klk14- Genesis ll-37-klk14- Genesis 6hrdigest_01 ll-37-klk14- 6hrdigest_01 6hrdigest_01 6hrdigest_01 ll-37-klk14- ll-37-klk14- ll-37-klk14- ll-37-klk14- ll-37-klk14- ll-37-klk14- 6hrdigest_01 6hrdigest_01 6hrdigest_01 50 50 6hrdigest_01 6hrdigest_01 50 6hrdigest_01 6hrdigest_01 50 50 50 50 50 50 6hrdigest_0150 50 50 6hrdigest_01 6hrdigest_0150 50 50 6hrdigest_01 50

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 5 10 15 20 0 5 10 15 20 0 5 10 15 20 0 5 10 15 20 0 5 100 515 2010 15 020 5 10 0 155 20100 155 2010 15 020 5 10 0 155 2010 0 155 2010 15 0 20 5 10 0 155 2010 0 15 5 2010 15 0 20 5 10 15 20 Time (min) Time (min) Time (min) Time (min) Time (min) Time (min) Time (min) Time (min) Time (min) Time (min) Time (min) Time (min) Time (min) Time (min) Time (min) Time (min)

ll-37-klk5-6hrdigest_01 # 1801-2421 RT: 12.39-15.63 AV: 90 ll-37-klk14-6hrdigest_01 # 1212-1890 RT: 8.15-11.43 AV: 98 NL: ll-37-klk14-6hrdigest_01 # 237-1068 RT: 2.53-7.51 AV: 124 NL: ll-37-klk14-6hrdigest_01 # 806-1752 RT: 6.29-10.80 AV: 136 ll-37-klk5-6hrdigest_01ll-37-klk5-6hrdigest_01 # 1801-2421 RT: #12.39-15.631801-2421ll-37-klk5-6hrdigest_01RT:AV: 12.39-15.6390 ll-37-klk14-6hrdigest_01AV: # 1801-242190 ll-37-klk14-6hrdigest_01RT: 12.39-15.63 # 1212-1890AV:RT:90 8.15-11.43# 1212-1890ll-37-klk14-6hrdigest_01AV:RT:988.15-11.43NL: ll-37-klk14-6hrdigest_01AV: #981212-1890NL: ll-37-klk14-6hrdigest_01RT: 8.15-11.43 # 237-1068AV:RT:982.53-7.51 #NL:237-1068ll-37-klk14-6hrdigest_01AV:RT:124 2.53-7.51NL: ll-37-klk14-6hrdigest_01AV: #124237-1068NL: RT:ll-37-klk14-6hrdigest_012.53-7.51 # 806-1752AV:RT:1246.29-10.80NL: # 806-1752ll-37-klk14-6hrdigest_01AV:RT:1366.29-10.80 AV: # 806-1752136 RT: 6.29-10.80 AV: 136 T: FTM S + p NSI Full ms [350.00-1455.22] T: FTM S + p NSI Full ms [350.00-1455.22] T: FTM S + p NSI Full ms [350.00-1455.22] T: FTM S + p NSI Full ms [350.00-1455.22] T: FTM S + p NSI T:FullFTM ms [350.00-1455.22] S + p NSI Full ms [350.00-1455.22] T: FTM S + p NSI FullT: msFTM [350.00-1455.22] S + p NSI FullT: FTM ms [350.00-1455.22] S + p NSI Full ms [350.00-1455.22]T: FTM S + p NSI FullT: msFTM [350.00-1455.22] S + p NSI FullT: msFTM [350.00-1455.22] S + p NSI Full ms [350.00-1455.22]T: FTM S + p NSI FullT: msFTM [350.00-1455.22] S + p NSI FullT: msFTM [350.00-1455.22] S + p NSI Full ms [350.00-1455.22]T: FTM S + p NSI Full ms [350.00-1455.22] 899.5231 434.2396 458.2480 563.6496 899.5231 899.5231 899.5231 434.2396 434.2396 434.2396 458.2480 458.2480 458.2480 563.6496 563.6496 563.6496 z=5 z=2 z=2 z=3 z=5 100 z=5 z=5 100 z=2 z=2 z=2 z=2100 z=2 z=2 100 z=3 z=3 z=3 100 100 100 100 100 100 100 100 100 100 100 100 95 Figure 2.5. Proteolytic processing95 of the LL-37 antimicrobial95 peptide by kallikreins.95 95 95 95 899.722995 95 95 95 95 95 95 95 95 899.7229 899.7229 899.7229 90 z=5 90 90 90 90 90 z=5 Identificationz=590 90 of cleavedz=5 90 fragments 90off LL-3790 peptide90 after 6 hr incubation90 at90 37°C alone90 or with 90 85 85 899.3228 85 85 85 85 85 85 899.322885 899.3228 active85 KLK5,899.3228 KLK8,85 or KLK14. The85 MS1 elution profile85 with integrated85 peaks is shown85 for the 85 z=5 80 z=5 80 80 80 80 80 z=5 80 z=580 80 80 80 80 80 80 80 80 75 75 75 parent75 LL-3775 peptide,75 and the75 LL-7,75 NL-8, 75and IK-1475 cleaved75 fragments75 (labelled75 at75 the top75 of 75 70 899.9231 70 70 70 70 70 899.9231 the899.9231 70Figure with70 their899.923170 corresponding70 m/z ratio70 and z 70charge state) for70 each of70 the LL70-37 peptide 70 z=5 z=5 z=5 65 z=5 65 65 65 65 65 65 65 65 65 65 65 65 65 65 65 60 incubated with no enzyme, KLK5,60 KLK8, and KLK14 as indicated60 on the left side of the Figure60 . 60 60 60 60 60 60 60 60 60 60 60 60 55 55 55 55 55 55 The55 retention55 times 55(RT) and integrated55 area55 under 55curve (AA) are55 displayed55 as well.55 Each of 55 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 these peptides was confirmed by MS2 in the enzyme-treated samples (data not shown).563.3203 563.3203 563.3203 45 45 45 45 563.3203 45 45 45 900.123445 45 45 45 45 45 45 45 45 z=3

900.1234 900.1234 900.1234 z=3 z=3 z=3

Relative Abundance Relative Abundance Relative Abundance Relative

Relative Abundance Relative z=5

Relative Abundance Relative Abundance Relative Abundance Relative Abundance Relative

Relative Abundance Relative Abundance Relative Abundance Relative

Relative Abundance Relative 40 40 40 40

Relative Abundance Relative Abundance Relative Abundance Relative Relative Abundance Relative 40 40 z=5 40z=5 40 z=540 40 40 40 40 40 40 40 35 35 35 35 35 35 35 35 35 35 35 35 35 35 35 35 899.1226 30 899.1226 899.122630 899.122630 30 30 30 30 30 30 z=530 30 30 30 30 30 30 z=5 z=5 z=5 25 25 25 25 25 25 25 25 25 25 25 25 25 900.3238 25 25 25 900.3238 900.3238 900.3238 20 z=5 20 20 20 20 20 z=5 20 z=5 20 20 z=5 20 20 20 20 20 20 20 900.5287 15 15 900.5287 15 900.5287 15 900.5287 15 15 15 15 15 15 15 15 15 z=5 15 15 15 z=5 z=5 z=5 458.2162 458.2162 458.2162 563.2133 10 10 10 458.2162 563.2133 10563.2133 563.2133 10 10 10 10 10 10 10 10 z=? 458.2790 10 z=? 458.279010 z=? 458.279010 10z=? 900.7253 900.7253 900.7253 457.9447 457.9447z=? 458.2790457.9447 z=? z=? 898.5205 900.7253 457.9447 z=3 z=? 563.5550 563.8162 5 898.5205 5 5 898.5205 5 433.5831z=5 433.7677 434.08195 433.5831434.3971433.7677434.67015 433.5831434.0819433.76775 434.3971434.0819458.0771434.6701434.3971 434.67015 z=3 z=3458.4187458.07715 z=3 458.07715 563.0768458.4187z=3 458.4187563.55505 563.0768563.81625 563.0768563.8162 563.5550 563.8162 5 898.5205 z=5 z=5 5 433.5831z=5 433.7677 434.0819 434.3971 434.6701z=3 5 z=3 458.0771 z=3 458.4187 5 563.0768 563.5550 z=? z=? z=? z=? z=2 z=? z=51 z=6 z=? z=? z=?z=? z=? z=? z=? z=? z=? z=? z=? z=? 0 z=2 z=? z=2 z=51 0z=? z=6 z=51 z=? z=2z=6 z=?z=? z=51 z=? z=6 0z=?z=? z=? z=?z=? z=?z=? 0 z=? z=? 0 0 0 0 0 0 0 0 0 0 0 0 899 900 901 433.5 434.0 434.5 458.0 458.2 458.4 563.0 563.2 563.4 563.6 563.8 899 900899 901900 901899 433.5900 433.5434.0901 434.0434.5 433.5 434.5 434.0458.0 458.2458.0434.5 458.4458.2 458.0458.4 563.0458.2 563.2 563.0458.4563.4 563.2563.6 563.4563.8 563.0563.6 563.2563.8 563.4 563.6 563.8 m/z m/z m/z m/z m/z m/z m/z m/z m/z m/z m/z m/z m/z m/z m/z m/z 68

2.3.9 Human KLK8 is a keratinocyte-specific protease induced during terminal keratinocyte differentiation

In order to support KLK8 involvement in normal skin barrier functions, we investigated KLK8 secretion by skin cells in culture. Given that KLKs are constitutively expressed in the granular layer of epidermis, we cultured HaCat keratinocytes in low versus high calcium medium, a manipulation thought to mimic the calcium gradient of upper epidermis (Hennings and Holbrook, 1983). We found that increasing exogenous Ca2+ in the medium changed keratinocyte cell morphology, induced corneocyte formation as expected, Figure 2.6B (Menon and Elias, 1991; Menon et al., 1994) and significantly increased KLK8 protein secretion into the medium in a time-dependent manner. We detected over 14-fold increase in KLK8 levels in high versus low calcium serum-free medium in day 3, (Figure 2.6A). We also investigated the ability of primary epidermal keratinocytes to co-secrete KLK proteins, including KLK8, as well as primary melanocytes and fibroblasts. We found that KLK8 was exclusively secreted by keratinocytes, whereby primary epidermal melanocytes (pHEM) and dermal fibroblasts (pHDF) secreted no detectable KLK8 even after day 12 in culture. KLK8 levels in the HaCat immortalized keratinocyte and primary neonatal keratinocyte culture media continued to increase with increased keratinocyte proliferation, while LDH levels remained constant. This confirmed that the elevation in KLK8 levels was due to increased secretion rather that cell rupture or death over time. Hence, our data suggest that KLK8 is a keratinocyte-specific protease secreted during epidermal keratinocyte proliferation and abundantly secreted upon their terminal differentiation in culture, Figure 2.6A. However, not all epidermal KLKs evaluated were keratinocyte-specific. KLK7 was secreted by all epidermal cells investigated, while KLK5 and KLK8 were by far the most abundant secreted keratinocyte-specific KLKs.

2.3.10 KLK8 is expressed in a free form in human sweat and non- palmoplantar stratum corneum

We pooled processed normal human sweat and non-palmo-plantar stratum corneum (SC) extracts to study KLK8 expression and activity ex vivo. The sweat and SC samples contained 8.0 mg and 3.5 mg total protein, respectively. KLK8 was detected in both biological samples at a molecular mass similar to recombinant mat-KLK8 (~31 kDa) in a reduced SDS-PAGE, and processed froms of KLK8 in the sweat (~31 and 21 kDa) were also detected, (Figure 2.6C).

A) B) C)

Figure 2.6. KLK8 is a skin barrier protease. (A) KLK8 secretion is induced by calcium- mediated stimulation of terminal keratinocyte differentiation and cornification in culture. The black bars represent undifferentiated keratinocytes and the grey bars represent differentiated keratinocytes grown in high calcium medium over 3 days period. (B) Terminal keratinocyte differentiation and cornification in culture (high calcium), compared proliferating keratinocytes (low calcium) in day 3. (C) KLK8 is expressed in a free non-complexed form in normal human sweat and non-palmoplantar stratum corneum skin extracts.

2.3.11 KLK8 is catalytically active in human sweat and non-palmoplantar stratum corneum

We sought to investigate KLK8-specific activity using an ex vivo skin model and recombinant mat-KLK8 as a positive control. We first confirmed the co-expression of multiple KLKs in sweat and SC epidermal extracts. According to our KLK-specific ELISA results, the pooled sweat sample contained 172 ng KLK8/mg total protein, 35 ng KLK11/mg total protein and 2.2 ng KLK5/mg total protein. On the other hand, the pooled SC epidermal extract contained 28 ng KLK8/mg total protein, 63 ng KLK11/mg total protein, and 210 ng KLK5/mg total protein. Hence, we detected 78-fold higher concentration of KLK8 compared to KLK5 in sweat, and 7.5- fold lower concentration of KLK8 compared to KLK5 in the stratum corneum extracts. KLK11 was in the middle range between KLK5 and KLK8, as indicated above.

To detect KLK8 activity specifically, and no other KLK or serine protease activity, we immunocaptured KLK8 and tested its ability to cleave fluorogenic VPR-AMC substrate at different pH levels. The principle of the KLK8 immuocapture-activity assay we developed for this purpose is shown in Figure 2.7A. We validated first that the monoclonal KLK8 antibody we used to coat the plate was a non-neutralizing antibody. Then, we performed a series of optimization steps using active recombinant mat-KLK8 protein. The sensitivity of the KLK8 immunocapture assay was optimized to detect a concentration-dependent increase in activity when increased amount of active recombinant mat-KLK8 was loaded into KLK8-Ab coated wells. Assay specificity was also confirmed upon detecting no activity as a result of loading active KLK5 into wells coated with KLK8 antibody.

Approximately 40 ng of sweat KLK8, 4 ng of SC KLK8, and 250 ng of mat-KLK8 as well as 10X diluted samples of each were loaded in triplicate wells in the same 96-well plate coated with KLK8-antibody. We demonstrated a concentration and time dependent increase in AMC- fluorescence emission in the non-diluted (1X) and (10X) diluted sweat and SC samples, confirming KLK8 activity in normal human skin surface (Figure 2.7B and 2.7C). The immunocaptured recombinant mat-KLK8 positive control reached saturation quicker than the immunocaptured sweat and SC KLK8, as expected, due to loading a significantly higher amount into the wells (5-10 fold higher than sweat-KLK8 and SC-KLK8). Immunocaptured SC and sweat KLK8 displayed optimal activity at pH 8.5, Figure 2.7B and 2.7C, and retained lower, yet

71 significant, activity at pH 5.0. Hence, we elucidated KLK8 activity in upper skin surface within the normal physiological pH gradient of human stratum corneum.

We also investigated if sweat and SC KLK8 contains some inactive pro-KLK8 proportion that can be activated by exogenous KLK5 or lysyl-endopeptidase. Our results indicate that the majority of sweat and SC KLK8 was catalytically active (Figure 2.8A and 2.8B). These results, combined with our immunodetection of KLK5 in the same sweat and SC samples, suggest that KLK5 may have already activated a large proportion of sweat and SC KLK8. When recombinant pro-KLK8 was incubated with active KLK5 (1:100 molar ratio) or lysyl endopeptidase (1:1000 molar ratio), and immunocaptured as a positive control in the same KLK8 antibody-coated plate, we detected pro-KLK8 activation by both proteases (Figure 2.8C).

B) C)

Figure 2.7. Immunocapture of KLK8 activity in normal human sweat and non- palmoplantar stratum corneum ex vivo. (A) A schematic of the principle of the assay. KLK8 is immunocaptured using a specific non-neutralizing monoclonal antibody. Potential non-specific binding of other protease contaminants is eliminated through a series of stringent washes. The activity of the immobilized sweat or SC KLK8 is measured by monitoring fluorescence emission of VPR-AMC substrate compared to no-enzyme background control. (B) Time and concentration-dependent activity of immunocaptured sweat and SC KLK8 monitored in real-time for a total of 24 hours at 37ºC and pH 8.5. (C) Time and concentration-dependent activity of immunocaptured recombinant mat-KLK8 activity as a positive control monitored in the same plate in real-time for a total of 24 hours at 37ºC and pH 8.5.

Figure 2.8. The majority of sweat and SC KLK8 is catalytically active. No significant increase in sweat and SC KLK8 activity was detected upon adding active KLK5 (A) or active lysyl-endopeptidase (B) to the samples prior to the KLK8 immunocapture-activity assays, compared to the immunocaptured recombinant pro-KLK8 positive control (C). KLK5 and lysyl endopeptidase alone reaction rates were zero ensuring assay specificity

2.4 Discussion

We sought in this study to investigate KLK8 activity, regulation, and downstream targets in order to support its potential functional involvement in a proteolytic activation cascade regulating skin desquamation and antimicrobial defence. We previously described that multiple KLKs are co-localized in human epidermis and sweat and noted particularly an abundant expression of KLK8 (Komatsu et al., 2006a). KLK8 is transported and exocytosed by lamellar granules into the stratum granulosum/stratum corneum (SG/SC) interface (Ishida-Yamamoto et al., 2004), and is thus likely activated in the SC extracellular space to play a role in SC barrier functions. Once activated, KLK8 activity is possibly regulated by many of the factors that control SC barrier integrity, such as epidermal pH and calcium ion gradients as well as endogenous serine protease/serine protease inhibitors. Herein, we produced recombinant KLK8 in its precursor (pro-KL8) and active (mat-KLK8) forms and investigated a potential SC cascade-mediated role of KLK8 by examining in vitro its potential to: 1) be regulated by pH and ions, 2) be activated by co-localized serine proteases, 3) be inhibited by co-localized serine protease inhibitors, 3) activate co-localized pro-KLKs, and to 4) target co-localized LL-37 antimicrobial peptide activation.

Our results showed that recombinant KLK8 activity is pH-dependent, since it displayed maximal activity at alkali pH of 8.5 and retained lower activity at pH 5. This suggests a role for KLK8 in human SC barrier where the pH decreases from 7.5 to 5 at the uppermost surface. Although the alkaline pH (where KLK8 activity is optimal) occurs in lower SC, it is possible that less active enzyme is present there where the majority is in a latent pro-KLK8 form or is bound to endogenous inhibitors. More active enzyme may be present in the upper SC as a result of pro- KLK8 activation, being freed of a potential inhibitor, and/or the calcium ion gradient shift. Our studies indicate that KLK8 activity is enhanced by calcium and to a lesser degree by magnesium, two ions known to have higher concentrations in uppermost SC compared to lower layers. Interestingly, topical application of 10 mM MgCl2 and CaCl2 was shown to accelerate mouse skin barrier recovery after acute chemical disruptions (Denda et al., 1999). It is possible that magnesium and calcium ions influence barrier recovery via their activation of serine proteases such as KLK8. This possibility requires further investigation.

Since Zn2+ levels in human sweat (Crew et al., 2008) and in the skin of mammals may reach the millimolar range (Nitzan et al., 2004), this ion could be an important regulator of KLK8 activity in the skin. We showed that zinc ions, within physiological range, attenuate KLK8 activity. Zinc had a significantly lower inhibitory effect on KLK8 activity compared to other active epidermal KLKs, as 1:10 molar ratio of KLK to zinc ions resulted in 97.5% and 95% inhibition of KLK5 and KLK14 activity (Michael et al., 2005; Borgono et al., 2007c), respectively, compared to 0% inhibition of KLK8. This suggests a possibly different binding mode of zinc to KLK8 structure. However, the crystal structure of human KLK8 has not been resolved to date and the mechanisms of zinc or calcium binding remain unknown.

Another important mode of regulating a protease irreversible cleavage of substrates is its pro- zymogen activation to generate the mature active form. We examined the ability of recombinant pro-KLK8 to be activated by epidermal tryspin-like serine proteases. Akin to pro-KLK8, the proenzymes of KLK7 and 14 require cleavage after lysine for activation. Pro-KLK7 and pro-KLK14 are activated by KLK5 in human epidermis (Brattsand et al., 2005). Given that KLK5 is active in human SC as a result of autoactivation, we suspected that it activates pro-KLK8 in the deepest layers of the SC at the close-to-neutral pH. Our results show slow activation of pro-KLK8 by KLK5 within a day at room temperature at 10:1 molar ratio. This slow activation is similar to previous reports of KLK5 activation of pro-KLK7 (Brattsand et al., 2005), which may be important in normal skin physiology where SC layers get renewed every 2-4 weeks (Milstone, 2004). Our in vitro results suggest that pro-KLK8 does not undergo autoactivation or activation by KLK1, but that it is activated slowly by KLK5 and rapidly by lysl-endopeptidase. Lysyl- endopeptidase was a better activator of pro-KLK8 due to it specific cleavage after lysine residues. Hence, we put forward the lysine-specific enteropeptidase, discovered recently in human skin granular layer (Nakanishi et al., 2010), as a potent potential endogenous activator of pro-KLK8.

KLK8 activity was not inhibited by any of the currently known endogenous epidermal inhibitors (LEKTI domains, SLPI, or elafin) in our study. Mat-KLK8 in vitro activity is inhibited by general serine protease inhibitors such as α2-antiplasmin, aprotinin, protein C inhibitor, chymostatin and to a low extent by soybean trypsin inhibitor. Unlike KLK5 and KLK14, α1- antitrypsin does not inhibit mat-KLK8 activity at all (Luo and Jiang, 2006). The P1-Arg preference of KLK8 and the presence of P1-Arg in α2-antiplasmin and P1-Met in α1-antitrypsin

76 in these serpins may explain their different inhibitory potencies towards KLK8. Perhaps more interesting from a dermatological perspective, is the finding that unlike epidermal KLK5, 6, 7, 13, and 14, and similar only to KLK1, KLK8 was not inhibited by any of the LEKTI domains implicated in the devastating skin disease, Netherton Syndrome (Deraison et al., 2007), (Descargues et al., 2006). KLK8 activity was also recently shown to be completely unaffected by the new epidermal LEKTI inhibitors encoded by SPINK6 and SPINK9, even though they inhibit KLK5, 7, and 14 (Brattsand et al., 2009; Meyer-Hoffert et al. 2010). We demonstrated here that KLK8 activity could be inhibited by auto-cleavage after a solvent-exposed Arg164 in its computed tertiary structure, which was not previously reported. It is plausible that active co- localized trypsin-like KLKs, such as KLK14, may degrade KLK8 after Arg164 and reduce its activity. However, this possibility requires further assessment.

A recent study described KLK8 as a desquamatory enzyme which regulates corneodesmosome’s degradation, through the actions of other kallikreins, in normal and barrier-disrupted mouse epidermis (Kishibe et al., 2007). KLK5 has not been detected in mouse epidermis to date, although a major active enzyme in human SC and a potential activator of KLK8 as we showed here. We thus investigated the possibility that active KLK8 may target activation of pro-KLK11, which is also not yet detected in mouse epidermis, but has been detected in human epidermis and sweat with unknown activators and undemonstrated activity/functionality to date. We elucidated KLK8 ability to activate pro-KLK11 in vitro using full recombinant KLK proteins which retain their native tertiary structure. KLK8 was also able to activate pro-KLK1 in vitro. Tissue kallikrein (KLK1) is active in human sweat (Hibino et al., 1994) and our study is the first to identify KLK8 as a possible endogenous protease activator of KLK1 in sweat. Our activation results are in accord with Yoon et al findings with pro-KLK peptide-fusion proteins (Yoon et al., 2007), except for our finding that KLK1 does not activate pro-KLK8. Overall, our data implicate KLK5 as an activation initiator and KLK8 as an upstream activator of KLK1 and KLK11 in a SC and sweat activation cascade.

SC serine proteases can also target LL-37 antimicrobial peptide processing in human skin to generate shorter antimicrobial peptides that are active against Staphylococcus aureus (Yamasaki et al., 2006). We demonstrated KLK8 ability to process LL-37 synthetic peptide in vitro, leading to the formation of active KS-30, LL-29, and LL-23 antimicrobial peptides by trypsin-like cleavage. Yamasaki et al. previously showed that trypsin-like KLK5 and chymotrypsin-like

KLK7 are responsible for LL-37 processing in human skin surface (Yamasaki et al., 2006), although other yet unknown serine proteases may be equally important in this process. Our in vitro data suggest KLK8 as a new potential regulator of LL-37 antimicrobial activity in human skin and sweat.

We showed here that chymostatin inhibits KLK8 activity and previously demonstrated that this chymotrypsin-like inhibitor is a better inhibitor of KLK8 than the trypsin-like inhibitor, leupeptin, with IC50 of 8 µM compared to 66 µM, respectively (Kishi et al., 2006). Epidermal KLK5 and KLK14 trypsin-like activity is efficiently inhibited by leupeptin, but not by chymostatin (Brattsand et al., 2005). Interestingly, Yamasaki et al detected 80% reduction in skin surface serine protease activity by chymostatin and a minor reduction by leupeptin, and suggested accordingly that the chymotrypsin-like KLK7 is a more potent enzyme than trypsin- like KLKs in human skin surface. Our data lead us to infer that trypsin-like KLK8 was present in the skin samples tested by Yamasaki et al., contributing to the major serine protease activity inhibited by chymostatin and leading to LL-37 antimbicrobial peptide trypsin-like processing. Furthermore, our detection of enhanced KLK8 in vitro expression and activity as a result of calcium induction of terminal keratinocyte differentiation, and of pro-KLK8 activation by KLK5 and lysyl-endopeptidase, also suggested that KLK8 is active in uppermost epidermis.

We sought after elucidating KLK8 activity in non-palmoplantar stratum corneum and sweat ex vivo by a designing a KLK8-specific immunocapture-activity assay using fluoreogenic VPR- AMC substrate. This method allowed us to pull down KLK8 and identify its serine protease activity in human epidermis and sweat for the first time, supporting our in vitro findings. The majority of SC and sweat KLK8 was present in a catalytically-active form, displaying optimal activity at pH 8.5 and retaining activity at pH 5, similar to our recombinant human KLK8. Given that soap-treated, inflamed, atopic-dermatitis and scaly psoriatic skins show alkali to neutral pH near KLK8 activity optimum (Hachem et al., 2010), KLK8 activity may contribute to their overdesquamation and inflammation symptoms, which remains to be studied.

To date, few active serine proteases were demonstrated in human sweat including tissue kallikrein (KLK1) and kininase II (Hibino et al., 1994). In the stratum corneum (SC), kallikrein- related peptidase 5, 7, and 14 are the only described active serine proteases (Brattsand et al., 2005). Herein, we elucidated the presence of KLK8 as an active serine protease in human sweat

78 and non-palmoplantar stratum corneum, raising its potential functional involvement in skin desquamation and antimicrobial proteolytic cascades. We identified the substrate-specificity as well as potential endogenous activators and targets of this new active epidermal protease. As mentioned above, none of the currently known endogenous serine protease inhibitors exert an effect on KLK8 activity. Thus, KLK8 activity may affect the serine protease/serine protease inhibitor balance in normal human epidermis and skin diseases. Further understanding of KLK8’s ‘unique’ inhibition mechanism is needed for the development KLK8 activity-based probing tools to study its function in vivo and for the development of KLK8-specific inhibitors as potential skin care and disease drug agents.

Chapter 3 Kallikrein-related peptidase-8 is induced by TNFα and IL17A resulting in epidermal hyperplasia and elevation of psoriasis- related innate immunity gene expression

This chapter contains original unpublished data for a first author manuscript in preparation.

3 Kallikrein-related peptidase-8 (KLK8) is induced by TNFα and IL17A resulting in epidermal hyperplasia and elevation of psoriasis-related innate immunity gene expression

3.1 Introduction

Psoriasis is a complex multifactorial autoimmune skin disease with a devastating negative impact on patients’ quality of life. This chronic disease primarily affects keratinocytes in the epidermis. Psoriatic keratinocytes proliferate about ten times higher than normal skin keratinocytes (Crow, 2012) and fail to undergo proper differentiation and cornification. Recent genomic studies associated psoriasis pathogenesis to epidermal innate immunity and listed the Kallikrein-related peptidase-8 (KLK8) gene as one of the core set of 130 overexpressed psoriasis disease-specific genes (Ainali et al., 2012). Further, KLK8 mRNA and protein overexpression was reported in multiple skin diseases, including psoriasis and atopic dermatitis (Bowcock et al., 2001; Komatsu et al., 2007b; Komatsu et al., 2005b). However, the molecular mechanisms behind KLK8 dysregulation in psoriasis and other skin diseases remain unknown.

We investigated herein KLK8 expression in psoriasis and atopic dermatitis skin under the hypothesis that this protease is differentially regulated, by the distinct T-helper (Th) cell immune milieu in these common inflammatory skin diseases, independent of barrier injury or infections. Unlike AD lesions, psoriatic lesions are characterized by absence of a functional stratum granulosum (SG), hyperplasia and retention of nuclei in the stratum corneum (parakeratosis) (Guttman-Yassky et al., 2011a). Since granular keratinocytes of the SG are the major producers of KLK8 in normal human skin barrier, we asked the question: how is KLK8 overexpressed in psoriatic lesions then? Is it possible that KLK8 overexpression in psoriasis is an epidermal response to ‘inside’ T-cell mediated-mechanisms rather than ‘outside’ epidermal mechanisms such as skin injury? The expression of KLK8 is controlled by epidermal variables such as differentiation, calcium, vitamin D, retinoic acids, and wounds, all of which are known to influence human skin innate immunity (Morizane et al., 2010). Yet, KLK8 epidermal regulation by major cytokine subsets implicated in psoriasis and atopic dermatitis was never examined and the role of KLK8 protease activity in modulating psoriatic lesions architecture and innate immunity remains unclear.

Since psoriasis is characterized by altered keratinocyte proliferation and differentiation, and by infiltration of T-helper Th1 and Th17 cells into the epidermis (Lowes et al., 2007), we postulated that KLK8 is overexpressed in psoriasis due to keratinocytes’ cross talks with Th1 and Th17 immune cells, independent of barrier injury. In turn, KLK8 overexpression may induce epidermal hyperplasia and enhanced innate immune gene expression in psoriatic lesions, giving further support to the ‘inside-outside’ dogma of psoriasis (Elias et al., 2008). On the other hand, prototypic atopic dermatitis-associated Th2 cytokines likely reduce KLK8 expression by keratinocytes, given that AD is characterized by a reduction in barrier function and terminal keratinocyte differentiation.

Using HaCat keratinocytes as a differentiation model, we profiled keratinocyte secretion of KLK8 post-treatment with Th1, Th17 and Th2 cytokines alone or in combination, and investigated the effect of KLK8 overexpression on terminal keratinocyte differentiation and innate immunity gene expression in cultured keratinocytes and 3D full-thickness human skin equivalents. KLK8 expression was also examined in lesional and non-lesional skin of psoriasis and atopic dermatitis patients. Moreover, psoriasis treatment with common biologic drugs that target specific aberrant immune pathways in psoriasis should decrease KLK8 levels in the skin and serum of psoriatic patients. This study addresses the missing gaps in our understanding of KLK8 regulation and role in diseased human skin by focusing on keratinocytes’ cross-talks with immune cells. We provide new insights into the regulation and function of KLK8 in normal and psoriatic skin, which expand beyond KLK8 role in barrier repair responses, to support its pathogenic involvement in the ‘inside-outside’ dogma of psoriasis pathogenesis.

3.2 Materials and Methods

3.2.1 HaCat keratinocyte cell differentiation model and cytokine stimulations

Immortalized HaCat keratinocyte cells were grown in 6-well plates in DMEM media with 10%FBS, until they reach full confluence. Cells were then washed with PBS three times before adding serum-free chemically-defined CD-CHO (Life Technologies, Cat# 10743-029) medium. Calcium levels and cell confluence were constant to allow cells to undergo adhesion-mediated

82 differentiation post-confluency, as previously described (Alameda et al., 2011). All cytokines were purchased from R&D and stored in aliquots at -20ºC according to manufactures instructions. The only variable was the cytokine(s) included in the serum-free CD-CHO medium. After 3X washes with PBS, confluent cells were treated with 10ng/mL of a panel of cytokines, alone or in combination with other cytokines, in 4mL CD-CHO medium for 6 days. 500 µL of medium were collected on day 3 and day 6 for ELISA measurements and cells were collected 24 and 48 hrs later and frozen at – 80 ºC for mRNA analysis. To confirm that the effects of Th1, Th17 or Th2 cytokines occur in proliferating keratinocytes, instead of differentiating ones, we performed similar cytokine stimulation experiments in serum-containing media, with dose- response cytokine stimulations. All experiments were done in triplicates and repeated at least twice.

3.2.2 Enzyme-linked immunosorbent assays (ELISAs) and LDH assays

KLKs were measured via ELISAs, as described previously (Komatsu et al., 2005a), in skin washes, serum samples and media of cytokine-treated keratinocytes. To control for the viability of cultured keratinocytes during cytokine stimulation experiments, the levels of the intercellular enzyme lactate dehydrogenase (LDH) were measured in the culture media as an internal control to indicate cell membrane rupture and cell death.

3.2.3 BrdU cell proliferation assay

BrdU Cell Proliferation Assay Kit (Cat #6813) was purchased from Cell Signaling Technologies and used in quadruplicate wells per condition, according to manufacturer’s instructions.

3.2.4 KLK8 treatment of human full thickness 3D epidermis equivalents

Active recombinant human KLK8 protease was produced and purified as previously done (Eissa et al., 2011). EpiDerm-FT™ full-thickness human skin equivalents (EFT-400) and serum-free EFT-400-MM medium were purchased from MatTek Corporation (Ashland, MA). Mattek Epiderm full thickness model is composed of neonatal human-derived dermal fibroblasts and epidermal keratinocytes co-cultured in a collagen matrix to form a multi-layered, highly differentiated model of the human dermis and epidermis at the air-liquid interface, with the epidermis differentiating up and the dermis remaining below at the liquid/medium level, separated by a functional skin basement membrane (Kubilus et al., 2004). The skin equivalents

83 were placed in 2 ml of EFT-400-MM in 6-well culture plates for overnight equilibration at 37° C in a humidified incubator. The next morning, medium was aspirated and the skin equivalents were removed from the plates and immediately placed in the same 6-well plates in fresh EFT- 400-MM. After 24 hr incubation, cells were washed in PBS and 5 ml of fresh medium or KLK8- containing medium (200 ng/mL or 2000 ng/mL KLK8) was added to wells containing tissues. The cultures were then incubated at 37°C for 5 days for IHC analysis or for 2 days for PCR analysis. Analysis of the effects of KLK8 on Epiderm-FT™ was performed in triplicates. For histological analysis, tissues were removed from the supports and placed in 3% paraformaldehyde in PBS and paraffin embedded. Tissue sections (4 μm) were stained with hematoxylin and eosin (H&E). In the 2 day-treatment experiments, the epidermis was placed in RNA later for mRNA analysis.

3.2.5 Immunohistochemistry and immunocytochemistry

4m formalin-fixed paraffin-embedded sections were dewaxed in 5 changes of xylene and brought down to water through graded alcohols. Endogenous peroxidase and biotin activities were blocked respectively using 3% hydrogen peroxide and avidin/ biotin blocking kit (Vector Labs Cat#SP2001) and then followed 10% normal serum (from the species where the secondary antibody is obtained) blocking for 10 min. Sections were incubated accordingly at room temperature with the appropriate primary antibodies using conditions previously optimized. This was followed with a biotinylated secondary (Vector labs) for 30 min and alkaline phosphatase streptavidin labeling reagent (Vector labs Cat#SA5100.) for 30 min. After washing well in TBS, color development was done with freshly prepared Vector Red solution (Vector labs Cat# SK5100) for involucrin or KLK8 while DAB (Vector labs Cat# SK4100) was used for Ki67. Finally, sections were counterstained lightly with Mayer’s Hematoxylin, dehydrated in alcohols, cleared in xylene and mounted in Permount (Fisher, cat# SP15-500). For the single and double immunocytochemistry staining of stimulated HaCat cells, we used the ImmPress universal antibody (anti-mouse Ig/anti-rabbit Ig, peroxidase) polymer detection kit (Vector Laboratories, Cat # MP-7500), following manfacturers’ instructions. Primary antibodies for Ki67 and involucrin (Abcam) and KLK8 (R&D) were applied at ratios of 1:100; 1:1380; 1:200, respectively. In double immunocytochemistry experiment, VIP stain (dark red) was used for cytoplasmic Involucrin and DAB for nuclear Ki67 (brown).

3.2.6 Reverse-transcription and quantitative PCR

Briefly, FT epiderm tissues were homogenized (Polytron PT3100, Capitol Scientific, USA) and RNA was isolated using QIAGEN RNeasy purification kit following the manufacturer's protocol. To obtain high quality samples, genomic DNA contaminants were removed by DNase I treatment followed by the RNA Cleanup kit. Total RNA was isolated from control and KLK8- treated Epiderm tissues or cells using an RNeasy Kit (Qiagen Hilden, Germany). cDNA was generated from 1 μg of total RNA using the Superscript II cDNA synthesis kit (Invitrogen). Quantitative PCR was conducted using 1X SYBR reagent (Applied Biosystems, Foster City, CA), and transcript levels of S100A7, hBD4, IL6, IL17, TSLP, FLG were measured on a 7500 ABI system. All quantitative PCR data were normalized to TATA-binding protein expression and analyzed via delta delta Ct method.

3.2.7 Clinical samples from patients

Skin washes from lesional and non-lesional skin of patients with psoriasis (n=12) and atopic dermatitis (n=4) were collected as previously described (Harder et al., 2010). Lesional and non- lsional skin tissues from age and localization-matched psoriatic (n=4) and atopic dermatitis patients (n=4) were provided by Dr. Ulf Meyer-Hoffert for immunohistochemical analysis. Our study also included frozen lesional skin tissues from psoriatic patients before and after treatment with etanercept (n=3) provided by Dr. Martin Steinhoff. Serum samples from a large cohort (n=60) of sex and age-matched psoriatic patients before and after psoriasis treatment with TNFα- blockers were collected from the Toronto Western Hospital with research ethics board approval. The demographics of the patients are listed in Appendix Table 3.1.

3.2.8 Statistical analysis

Statistical analysis was done using GraphPad Prism software version 4.03 (GraphPad Software Inc., La Jolla, CA). One-way ANOVA was used for comparison of different treatment regimens. If two groups were compared, Student's t test was applied. p values <0.05 were considered significant (*). In figures with bar graphs or tables, data are expressed as means ± S.E. of at least three independent experiments unless stated otherwise.

3.3 Results

3.3.1 Keratinocyte secretion of KLK8 is differentially-regulated by Th1, Th17 and Th2 cytokines

We previously showed that proliferating HaCat and primary keratinocyte cells secrete KLK8, with calcium induction of terminal keratinocyte differentiation causing a dramatic increase in KLK8 secretion. In this study, we profiled the secretion of KLK8 into the media of confluent HaCat cells treated with various cytokines in day 3 and day 6 post-treatment. All treatments were performed in serum-free media to confluent cells unless otherwise indicated. As shown in Figure 3.1A, individual treatments with Th1 and Th17/22 cytokines: TNF-α, IL-22 and IL17A cytokines enhanced KLK8 secretion in day 3, while IFNγ and the Th2 cytokines: IL4, IL13 and IL25 caused no significant changes in day 3. Combined treatments of TNFα+IL22 doubled KLK8 levels the media, while TNFα+IL17A treatments and TNFα +IL17A+IL22 treatments induced more than 3-fold increase in KLK8 secretion. Interestingly, more dramatic changes were observed for the combined TNFα and IL17A treatment by day 6. TNFα+IL17A induced synergistic and potent secretion of KLK8 compared to un-treated control cells and those treated with TNFα only and IL17A only. This synergistic effect was detected at two different doses of TNFα+IL17A, 10 ng/mL of TNFα with 10ng/mL IL17A and also when 100 ng/mL IL17A was combined with 10ng/mL TNFα. Combining TNFα and IL-22 increased KLK8 secretion significantly compared to control cells, but it did not induce KLK8 secretion synergistically when compared with TNFα and IL22 individual treatments (Figure 3.1A). TNFα+IFNγ treatment resulted in keratinocyte apoptosis characterized by clear morphological features, such as membrane blebbing, nuclear fragmentation and a dramatic elevation of LDH levels in the media compared to control cells (data not shown). On the other hand, the Th2 cytokines, IL4, IL13 and IL25 reduced KLK8 secretion significantly by day 6 compared to the untreated cells, Figure 3.1B. Thus, KLK8 secretion by keratinocytes is differentially regulated by Th1, Th17 and Th2 cytokines, with TNFα+IL17A inducing a significant overexpression of this epidermal protease.

It is important to note that KLK8 hypersecretion by TNFα+IL17A-stimulated keratinocytes was accompanied with unique and dramatic morphological changes, Figure 3.1B, C and D. We observed accumulation of cells in circular web-like structures of enucleated cells conntected to nodes that resembled the ‘stratification domes’ previously reported to be induced by adhesion- mediated terminal differentiation of HaCat keratincoytes in serum-free medium and cyclinD

86 overexpression in HaCat cells. Cell remodelling and formation of the web-like structures was visible in the TNFα+IL17A-treated cells in day 3 post-treatment and these structures became very pronounced with enhanced differentiation in culture (day 6 versus day 3). Unlike the TNFα+IFNγ-treated cells, the TNFα+IL17A-treated cells had lower levels of LDH levels compared to their respective control cells in day 3 and day 6, indicating that KLK8 hypersecretion by these cells was due to enhanced keratinocyte proliferation or differentiation, but not cell rupture and death.

Furthermore, we confirmed that this combined cytokine effect on KLK8 hyper-secretion was stratification and serum independent. We observed similar results of TNFα+IL17A stimulatory effect on KLK8 secretion by proliferating keratinocytes in serum-containing media (data not shown), although no effect on cell polarity or stratification domes formation was observed when TNFα+IL17A cytokines were added in serum-containing media. Significant KLK8 reduction in media of proliferating cells in serum-containing media, treated with combined IL4/IL13/IL25 was also detected (data not shown) confirming that keratinocytes respond to these cytokines regardless of growth factor signalling. To our knowledge, the synergistic effect of TNFα and IL17A cytokines on HaCat keratinocyte differentiation in serum-free medium, cell remodelling into web-like structures, stratification dome formation (Figure 3.1E and 3.1F), and KLK8 secretion (Figure 3.1A) were never reported before. Based on these in vitro results, we suspected that TNFα and IL17A-treated keratinocytes likely recapitulate the psoriatic phenotype of altered keratinocyte differentiation and that the web-like structures may resemble psoriatic rete ridges and epidermal scaling.

*** *** *** *** *** ***

** * * * * * * * * * ** ** ** **

B) II

IV V

Figure 3.1. Differential Kallikrein-8 secretion by differentiating keratinocytes in response to Th1 (TNFα and IFNγ), Th17 (IL17A, IL22) and Th2 (IL4, IL13, IL25) cytokines. (A) KLK8 secretion in HaCat conditioned medium on day 3 and day 6 after individual and cocktail

88 cytokine stimulation. All cytokines were added at 10ng/mL in SFM, except in the case of IL17A* where the asterisk indicates higher concentration of 100ng/mL (***p<0.001, **p<0.01, ***p<0.05). (B) Microscopic images (4X) of cells: I) HaCat control, day 6. II) 10 ng/mL TNFα- treated, day 6. III) 10 ng/mL IL17A-treated, day 6. IV) 10 ng/mL TNFα + 10 ng/mL IL17A, day 6. V) 10 ng/mL TNFα + 100 ng/mL IL17A*, day 6. Note the web-like structures and stratification domes indicated with arrows in TNFα + IL17 and TNFα + IL17A*-treated cells.

3.3.2 TNFα and IL17A-treated keratinocytes have an altered differentiation program and mimic lesional psoriatic skin

To confirm that the web-like structure seen after TNFα+IL17A treatment represent epidermal scales, we performed BrDU cell proliferation assays and double-immunocytochemistry with Ki67 proliferation and involucrin terminal differentiation marker. All treatments, except for TNFα which is a known anti-proliferative, increased BrdU incorporation compared to untreated control cells, Figure 3.2A. However, unlike the consistently increased proliferation induced by IL17 alone, the TNFα+IL17A treatment reduced BrDU incorporation over time, resulting in a negative slope in Figure 3.2A, indicating reduced proliferation and perhaps an accelerated differentiation program. Consistently, TNFα+IL17A–treated cells displayed intense staining for the terminal differentiated marker, involucrin, in the web-like structures and stratification domes (Figure 3.2B). Over 70% of the TNFα+IL17A-keratinocytes were differentiated by day 6 as indicated by involucrin staining in the enucleated stratification domes where it displays the highest intensity. Hence, our data suggests that KLK8 overexpression by TNFα+IL17A-treated keratinocytes is a likely outcome of altered keratinocyte differentiation rather than hyperproliferation. We immunolocalized KLK8 and LL-37 proteins to the epidermal scales of TNFα+IL17A-treated HaCat cells in Figure 3.2C. Finally, to prove that TNFα+IL17A-treated HaCat keratinocytes indeed mimics psoriatic skin, we measured gene expression of psoriasin (S100A7) and human β-defensin-4 (hBD4), IL6 and filaggrin. Synergistic upregulation of the psoriasis-related genes S100A7 and hBD4 to 114-fold and 218-fold, respectively, was detected in addition to elevation of IL6 by 6-fold and reduction of filaggrin to 0.25-fold compared to untreated controls, Figure 3.2D. The S100A7 and hBD4 gene expression was significantly reduced in IL4-treated keratinocytes to 0.01-fold and 0.05-fold, (date not shown) confirming their differential response to Th1/Th17 versus Th2 cytokine milieu and their psoriasis specificity.

A) B)

NT TNFα+IL17A

Anti- INV

4X 4X

C) D)

NT TNFα+IL17A

Anti- KLK8

Anti- LL37

Figure 3.2. TNFα+IL-17A treatment induces changes in HaCat keratinocytes that mimic psoriatic skin. (A) TNFα+IL-17A stimulation enhances keratinocyte proliferation and accelerates differentiation program over time. (B) Web-like structures and stratification domes display intense positive staining for involucrin as well as (C) KLK8 and LL-37 vs. NT controls. (D) TNFα+IL-17 treated keratinocyte-induced gene expression of psoriasis-related genes. (*** p<0.001)

3.3.3 Overexpression of KLK8 alters keratinocyte differentiation program, induces epidermal hyperplasia and up-regulates innate immunity gene expression

Upon examining the kinetics of KLK8 treatment on HaCat keratinocyte proliferation in culture, we observed that KLK8 induces keratinocyte proliferation compared to non-treated controls on day 3 and reduces proliferation significantly by day 6, similar to the kinetics of TNFα and IL17A effect on keratinocyte proliferation shown in Figure 3.2A. As shown in Figure 3.3, the KLK8- mediated reduction of BrdU incorporation and cell proliferation was concentration dependent and it was paralleled with enhanced involucrin staining on day 6, consistent with having a role in inducing keratinocyte differentiation. Thus our data show that KLK8 effect on keratinocyte proliferation and differentiation is dose-dependent, where higher KLK8 concentrations reduce proliferation and enhance differentiation.

Figure 3.3. KLK8 treatment reduces cell proliferation and enhances differentiation of HaCat keratinocytes in a concentration-dependent manner.

Given that HaCat keratinocytes are an immortalized cell line, we also studied the role of KLK8 in epidermal differentiation and remodelling in a 3D epidermis model, where full-thickness human epidermis equivalents of normal primary epidermal keratinocytes and fibroblasts from a single donor were grown in collagen matrix and treated with two doses of active KLK8 protease (200 ng/mL and 2000 ng/mL). The lower dose of KLK8 increased SC and overall epidermis thickness, as shown in Figure 3.4. KLK8 treatment enhanced gene expression of S100A7 by 3- fold, hBD4 by 1.8-fold and IL6 by 1.5-fold in HaCat keratinocytes within 24 hrs (data not shown), and a similar trend was noted in the human FT-epiderm model, where KLK8 treatment resulted in 3.2-fold, 7.8-fold and 2-fold increase in S100A7, hBD4 and IL6 genes after 48 hrs, respectively. Interestingly, we also detected a 2.5-fold increase in TSLP gene.

Further, the keratinocytes of KLK8-treated epidermis displayed increased and more intense staining of involucrin in multiple layers and reduced Ki67 staining in the stratum basale on day 5, whereby basal keratinocytes appeared more differentiated and columnar in shape compared to control (Figure 3.6B versus Figure 3.6A). Interestingly, unlike the non-treated epidermis, we observed co-localization of Ki67 and involucrin in cytoplasmic regions of upper layers of the KLK8-treated epidermis, suggesting nuclear rupture and accelerated differentiation program. These results were consistent with our BrdU cell proliferation assay results of KLK8-treated keratinocytes (Figure 3.3A). Hence, the KLK8-treated epidermis exhibited altered differentiation and expression of innate immunity genes.

Treating the epidermis model with a higher KLK8 dose, on the other hand, resulted in a dramatic destruction of the barrier visualized by absence of proper differentiation of the basal layer, SC detachment, epidermal thickening and retention of nuclei in upper layers (Figure 3.5B and 3.5C compared to 3.5A). Interestingly, we also detected changes in dermal fibroblasts in epidermis treated with the higher KLK8-dose. Although the dermal fibroblasts were similar in numbers between KLK8-treated and non-treated epidermis, they were elongated in shape and their focal length was significantly higher than normal human epidermis fibroblasts as shown in the H&E stained epidermis in Figure 3.6.

A) B)

SG SS

C) D)

250 p<0.01 10 Control **

m) 200 KLK8-treated  8

150 p<0.01 6

100 4 ** ** *

50 2 Mean thickness ( thickness Mean 0 0 change fold gene Normalized IL6 hBD4 TSLP S100A7 Epidermis

Stratum corneum Figure 3.4. KLK8 treatment enhances differentiation of normal full thickness human epidermis model and alters innate immunity gene expression. (A) Normal human full- thickness epidermis (FT-Epiderm) equivalent after 5 days of culture at the air-liquid interface. Alterations in basal keratinocytes following KLK8 treatment are indicated with an arrow. (B) KLK8-treated epidermis equivalent after 5 days of culture at the air-liquid interface. (C) Mean stratum corneum and epidermis thickness of control and KLK8-treated epidermis equivalents. (D) Changes in innate immune gene expression 48 hrs post-KLK8-treatment of FT-Epiderm normalized to GAPDH and non-treated controls via the ∆∆Ct method.

A) B)

C) C) D)

Figure 3.5. Alterations in proliferation and differentiation markers in KLK8-treated full thickness epidermis model and psoriatic skin. Double immunohitochemical staining of cytoplasmic involucrin (pink red) differentiation marker and nuclear Ki67 (brown) proliferation markers in: (A) Normal human Epidermis equivalent after 5 days of culture at the air-liquid interface. (B) KLK8-treated epidermis equivalent after 5 days of culture at the air-liquid interface. Intense involucrin staining in all layers and arrow indicates brown staining in cytoplasmic regions in the KLK8-treated epidermis equivalent, further suggesting enhanced differentiation and nuclear rupture to form a thicker stratum corneum compared to untreated control where Ki67 is restricted to the SB. (C) Non-lesional psoriatic skin and (D) matched lesional psoriatic skin from the same psoriatic patient display enhanced involucrin and Ki67 staining.

A) B)

C) D)

500 p<0.01 Control

400 KLK8 300

200 p>0.05 100

Fibroblasts Count Figure 3.6. KLK8 overexpression induces drastic changes Focal in interface full length thickness epidermis model. KLK8 induces abnormal differentiation, SC detachment, retention of nuclei in upper layers (parakeratosis), and elongation of fibroblasts in normal full-thickness epidermis equivalents. H&E stained 20X images of (A) Normal epidermis. (B and C) Epidermis tissues treated with high KLK8 dose. (D) Mean fibroblast count and focal length as a measure of morphological change upon KLK8-treatment.

3.3.4 KLK8 is significantly elevated in lesional psoriatic skin and reduced in lesional acute atopic dermatitis skin

Our observed TNFα and IL17A synergistic effects on keratinocytes may explain KLK8 overexpression in psoriatic lesions. Further, the reduction of KLK8 secretion by Th2 cytokines (IL4, IL13 and combined IL4/IL13/IL25 treatments) suggests that KLK8 is reduced in atopic dermatitis lesions. To validate that our above in vitro organotypic tissue culture findings are relevant in vivo, we analyzed KLK8 expression in skin washes and tissue biopsies from non- lesional and lesional skins of psoriatic and atopic dermatitis patients. Our results show that KLK8 is significantly elevated in skin washes of lesional psoriatic compared to non-lesional skin of the same patient. Conversely, KLK8 levels seemed to be reduced or comparable in lesional versus non-lesional skins of atopic dermatitis patients Figure 3.7. Since we had a smaller number of AD (n=4) compared to psoriatic patient volunteers (n=12) for skin wash experiments, we also investigated the expression pattern of KLK8 in lesional and non-lesional skins of psoriasis and AD skins by immunohistochemistry. KLK8 overexpression in lesional psoriatic skin and reduction in lesional AD skin was also observed compared to non-lesional skin from the same patients, as shown in Figure 3.8. Intriguingly, unlike acute AD lesions, the dermis of psoriatic lesions displayed intense staining of KLK8, in the immune cell infiltrate near the epidermis as shown in Figure 3.9.

P<0.05 B)

C) P<0.05 P<0.05

Figure 3.7. KLK8 is significantly overexpressed in lesional psoriatic skin washes only, unlike other KLKs. ELISA levels of (A) KLK5, (B) KLK7 and (C) KLK8 in lesional (L) and non lesional (NL) skin washes from psoriasis (n=12) and atopic dermatitis patients (n=4). In Figure s, Ps refers to psoriasis and AD to atopic dermatitis.

LESIONAL NON-LESIONAL

PSORIASIS

ATOPIC

DERMATITIS

Figure 3.8. KLK8 epidermal expression is elevated in lesional psoriasis and reduced in lesional atopic dermatitis skin, compare to respective non-lesional counterparts. Microscopic IHC images (20X) of KLK8 staining in skin biopsies from (A) a psoriatic patient and (B) an atopic dermatitis patient indicated with arrows.

100

A) Epidermis

Dermis PSORIASIS

ATOPIC DERMATITIS

Figure 3.9. KLK8 overexpression in lesional psoriatic skin, is not restricted to the epidermis, but is also seen in dermis immune infiltrate near the epidermis, unlike atopic dermatitis skin. Representative microscopic images (20X) of KLK8 IHC staining in lesional (A) psoriasis and (B) atopic dermatitis skin.

101

3.3.5 KLK8 elevation in psoriatic patients’ lesional skin and sera is significantly reduced after effective treatment with the TNFα – blockers

Since our data strongly suggest that KLK8 is linked to major immune players in psoriasis, TNFα and IL17A, we hypothesized that common biologic psoriasis therapy, such as etanercept, will reduce KLK8 expression. Since KLK8 overexpression is restricted to lesional psoriatic lesions only, its reduction by psoriatic therapy targeting TNFα and IL17A should correlate positively with psoriasis clearance. Indeed, we found that KLK8 gene expression was reduced in the skins of psoriatic post-etanercept treatment (p<0.05), and confirmed dramatic reduction of psoriasis- related and KLK8-induced genes such as hBD4 (p<0.001) post-treatment. SA1007 was also reduced post-treatment, but with p=0.07, as shown in Figure 3.10.

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p<0.001 20000 15000 Pre-treatment 10000 Post-treatment 5000 5000 4000 3000 p<0.05 2000 1000 200 150 100 50 0

Gene fold normalized to normal skin to normal normalized fold Gene IL17 FLG KLK8 hBD4 S100A7

Figure 3.10. Expression of KLK8 and other innate immunity genes in lesional psoriatic skin pre and post-treatment with the TNFα-blocker, etanercept. KLK8 and other innate immunity gene (such as S100A7 and hBD4) expression are reduced in pooled psoriatic patients (n=3) skin lesions post-treatment with the TNF-blocker etanercept. Changes in gene expression were normalized to GAPDH and normal skin control via the ∆∆Ct method.

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Furthermore, consistent with our clinical findings (discussed in Chapter 4 of this thesis) of significant KLK8 elevation in the sera of psoriatic patients and its positive correlation with clinical variables of psoriasis severity measured by the Psoriasis Area and Severity Index (PASI) score, our data here show that indeed KLK8 levels in the sera of 60 psoriatic patients are significantly reduced after treatment with TNFα-blockers (p=0.006), unlike other KLKs, as shown in Appendix Table 3.2. High sensitivity C-reactive protein (hs-CRP) was also reduced in serum samples post-treatment and was included in the analysis as an internal control of treatment effect and correlation with PASI. KLK8 reduction in the sera of patients correlated positively with psoriasis clearance and PASI reduction with a correlation coefficient of 0.544 and a p- value<0.0001 (data not shown). Our results show that KLK8 decreases with treatment (p=0.0001), and is a better indicator than hsCRP for patients’ response to treatment as shown in Table 3.1.

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Table 3.1. KLK8 serum levels predict positive response to TNFα-blockers

Univaraite Multivariate

Covariate Estimate SE P-value Estimate SE P-value

Age (1 year 0.088 0.100 0.383 0.029 0.099 0.769 increase)

Sex (Males vs. -3.500 2.499 0.167 0.077 2.349 0.674 Females)

Psoriasis -0.036 0.094 0.701 -0.034 0.090 0.704 Duration (1 year increase)

KLK8 reduction - 1.004 0.213 <0.000 -0.931 0.220 0.0001 (1 unit increase) 1

hsCRP reduction - 0.179 0.077 0.024 -0.140 0.072 0.057 (1 unit increase)

. POPULATION: patients who take anti-TNF and have pre and post-treatment medication sample values (n=60); 55 patients had pre and post treatment information on PASI scores . RESPONSE: PASI score . COVARIATES: hsCRP and KLK8 reduction adjusting for age, sex and duration of Ps; . MODEL: Multiple Linear regression models with reduced model obtained by stepwise elimination and the R-squared method.

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3.4 Discussion

Psoriatic epidermal response shares many similar features with epidermal regeneration during wound healing. Not only do keratinocytes get activated, but also angiogenesis and inflammatory responses are stimulated and antimicrobial peptides are induced to prevent infections around the wound (Nickoloff et al., 2006). Psoriasis is often triggered with skin injury that develops later into plaques, known as the Kobner phenomenon. Thus, wound repair pathways are implicated in psoriasis pathogenesis.

The role of KLK8 in epidermal regeneration and wound healing was recently reported (Kishibe et al., 2012). The mouse homologue of KLK8 is able to induce hyperkeratosis in SLS-induced mouse skin inflammation (Shingaki et al., 2012). Here, we hypothesized that human KLK8 is overexpressed in psoriasis to induce hyperkeratosis (or hyperplasia) and host defense gene expression, which are characteristics of psoriatic lesions. We postulated that KLK8 overexpression in psoriatic lesions is an ‘outside’ exacerbated epidermal response to ‘inside’ immune aberrations known to dominate this devastating autoimmune disease. The T helper 17 cells (Th17) are active new players in psoriasis. Th17 are developmentally distinct from Th1 and Th2 cells, which have been shown to drive psoriasis and atopic dermatitis pathogenesis, respectively (Guttman-Yassky et al., 2011). Our results demonstrated that KLK8 epidermal expression in psoriasis and atopic dermatitis is a differential response to Th1/Th17 and Th2 cytokines, independent of barrier injury, and identified ways in which KLK8 may contribute to psoriasis skin architecture and innate immunity.

HaCat keratinocytes were used as a differentiation model as they recapitulate the process of terminal keratinocyte differentiation in culture through the formation of visible stratification domes of enucleated corneocytes. Overexpressing the cell cycle regulator, cyclin D, implicated in psoriasis hyperproliferation (Belso et al., 2008), in HaCat cells induced visible stratification domes, underscoring the role of cyclin D as an epidermal differentiation regulator in a recent study (Alameda et al., 2011). Here, we show original data demonstrating that combined TNFα and IL17A treatment induces dramatic changes in keratinocytes cell polarity and differentiation

106 through formation of unique ‘web-like structures’ and stratification domes. To our knowledge, this observation has never been shown. Yet, it provides strong evidence for the immune bases of psoriasis. Our cell proliferation assays and immunohistochemical expression of the terminal differentiation marker involucrin indicate a key role for TNFα and IL17A cytokines in accelerating keratinocyte differentiation and inducing scaling of cultured keratinocytes. Th1 and Th17 immune cells, which secrete TNFα and IL17A, respectively, are usually absent in normal human epidermis, but they are abundant in psoriatic epidermis. Certainly, our TNFα+IL17A- treated keratinocytes mimicked psoriatic kerationcytes as we detected synergistic elevation of the psoriasis-related host defense genes: psoriasin (S100A7) and human beta-defensin-4 (hBD4) and upregulation of IL6, but not filaggrin, consistent with previous findings (Chiricozzi et al., 2011).

TNFα and IL17A acted synergistically to induce the most potent upregulation of KLK8 protein by keratinocytes, compared to non-treated cells and all other cytokine treatments, including TNFα+IL22. Combining TNFα+IL17A or TNFα+IL17A+IL22 resulted in comparable levels of KLK8 secretion suggesting a key role for TNFα+IL17A in modulating KLK8 expression. Overexpression of KLK8, LL37 also immunolocalized to the web-like structures and stratification domes of TNFα and IL17A-stimulated cells. Interestingly, KLK8 hypersecretion by keratinocytes post TNFα and IL17A treatment was independent of keratinocytes’ state of differentiation. Thus, our data suggest that TNFα+IL17A induce proliferating and differentiating keratinocytes to secrete KLK8 and provide a conceivable explanation for KLK8 overexpression in lesional psoriatic skin characterized by absence of a functional stratum granulosum. Our analysis of patients’ skin biopsies and skin surface washes confirmed that our in vitro findings are relevant in vivo, as we detected significant KLK8 overexpression in lesional psoriatic skin only, compared to matched non-lesional control and lesional and non-lesional atopic dermatitis (AD) skin. We noted significant reduction in KLK8 secretion by keratinocytes post-treatment with Th2 cytokines (IL4, IL13 and IL25) implicated in AD, which was mirrored by reduced KLK8 immunohistochemical expression in lesional AD skin compared to its matched control. Our data is consistent with a previous study indicating enhanced trypsin-like activity in psoriatic lesions, but not AD lesions. Although KLK8 was reported to be elevated in AD stratum corneum, the study was based on analysis of tape-stripped skin, which could have activated the wound healing response. Our current study investigated KLK8 expression in cultured keratinocytes and skin biopsies independent of barrier disruption.

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We hypothesized that KLK8 overexpression in lesional psoriatic skin contributes to the development and/or maintenance of psoriatic lesions. We confirmed this notion upon examining the effect of KLK8 on the differentiation, proliferation and innate immune gene expression in cultured normal keratinocytes and full-thickness 3D skin equivalents of epidermal keratinocytes and dermal fibroblasts. Keratinocytes within these skin equivalents differentiate, forming distinct basal, spinous and granular layers, as well as a stratum corneum, when placed at an air-liquid interface. Our data show that KLK8 activity induces keratinocyte differentiation leading to increased thickness of the stratum corneum and full epidermis, and enhanced involucrin terminal differentiation marker expression in all differentiating layers. The Ki67-positive proliferating cells, which were at the basal layer moved to suprabasal layers and their nuclei ruptured to release their contents, confirming enhanced differentiation. However, adding ten-fold higher concentration of KLK8 to the skin model induced dramatic changes that mimicked psoriatic skin, such as stratum corneum detachment, epidermal thickening and retention of keratinocytes’ nuclei in the stratum corneum (parakeratosis).

KLK8 expression must be relevant to host defense as it enhances differentiation and thickening of the stratum corneum, thereby providing increased protection from injury or infection. We show that KLK8 induces expression of the psoriasis-related antimicrobial S100A7 and hBD4 genes in cultured keratinocytes and 3D full thickness equivalents, confirming its role in regulating cutaneous innate immunity. Thus, given KLK8 ability to induce a psoriatic phenotype in vitro, it is very likely to induce a similar effect in response to TNFα and IL17A in vivo.

Our findings support the recently elucidated role of KLK8 in inducing hyperkeratosis in inflamed mouse skin. We provide a missing regulatory link and a possible upstream mechanism for KLK8 overexpression and induction of hyperkeratosis in psoriatic lesions. Our work demonstrates that KLK8 is a key epidermal protease regulated by immune and epidermal barrier crosstalks in psoriatic lesions, to induce epidermal hyperplasia and enhanced host defense gene expression. The mechanisms by which KLK8 can cause a thickened epidermis may involve inhibition of AP-2α expression leading to hyperproliferation (Shingaki et al., 2010). Consistently, AP-2α knockout mice have thick skin due to a hyperproliferative defect (Wang et al., 2006). Another possible mechanism may include IL6, which is known to act as an autocrine regulator of keratinocytes differentiation and mediator of hyperplasia in active psoriatic lesions (Gottlieb, 1990; Lindroos et al., 2011). IL6 is overexpressed in psoriatic lesions compared to

108 non-lesional and normal skin as well as in the sera of psoriatic patients (Lo et al., 2010; Suttle et al., 2012). Almost all classical psoriatic therapies normalize IL6 expression in psoriasis. Further, IL6 activity and its receptor are involved in the Koebner phenomenon and wound healing response (Suttle et al., 2012). TNFα and IL1 activate epidermal keratinocytes to produce IL6 (Fujisawa et al., 1997) and our data here suggest KLK8 as a new inducer of IL6 expression by keratinocytes. A third possible mechanism is that the altered skin epidermis phenotype following KLK8 treatment reflects increased proteolytic degradation of structures responsible for basal cell cohesion in the basement membrane such as collagen IV, which is expressed in full thickness epidermis and is a known substrate for KLK8. Keratinocyte differentiation and proliferation were recently shown to be regulated by adhesion to the 3D meshwork of type IV collagen in reconstructed skin equivalents (Fujisaki et al., 2008) and KLK8 proteolytic activity may affect this regulation. Further studies are required to confirm this possibility.

We also detected elevation of the Thymic Stromal Lymphopoietin (TSLP) gene, which serves as a link between innate and adaptive immunity. TSLP elevation has been reported in lesional psoriatic skin although not associated with inducing allergy in psoriatic patients. Furthermore, KLK8 overexpression induced elongation of dermal fibroblasts in full thickness epidermis equivalents. We previously showed that KLK8 is not secreted by primary dermal fibroblasts (Eissa et al., 2011), but it is possible that KLK8 is involved in paracrine signalling in the dermis where it remodels dermal fibroblasts and/or endothelial cells (which are not present in the full- thickness epidermis model) but are dilated in psoriatic lesions. Further studies are required to investigate KLK8 role in dermal psoriatic lesions and its effect on dermal fibroblasts and endothelial cells, as well as its expression by these cells post TNFα+IL17A stimulation. Our data pointed to a dramatic KLK8 overexpression by dermal cells in psoriatic lesions compared to matched non-lesional skin. Potential candidates may include dendritic cells, endothelial cells, mast cells or neutrophils known to secrete serine proteases. KLK8 was previously localized in mast cells of mice (Wong et al., 2003). IL17A-positive mast cells and neutrophils are found in high levels at sites of skin and joint disease in humans (Kirkham et al., 2013). Thus, the possibility of KLK8 overexpression by any of these cells in psoriatic lesions is not farfetched.

Our data show that effective biologic psoriasis treatment diminished KLK8 gene expression in lesional psoriatic skin post-treatment, along with the hBD4 gene. Unlike other KLKs, KLK8 protein levels in the serum were also significantly reduced in psoriatic patients post treatment

109 with common TNFα-blockers in the clinic, such as etanercept. The reduction in KLK8 correlated significantly with psoriasis clearance. Etanercept is an immunoglobulin fusion protein that blocks tumor necrosis factorα (TNFα) receptor. Blockade of TNFα is considered to be its primary action, but recent clinical trials showed that effective treatment of psoriasis with etanercept is a result of its early inhibitory effects on the newly discovered Th17 cells (Zaba et al., 2007). Newly developed IL17A antagonists in clinical trials will also likely reduce KLK8 epidermal and serum levels in psoriasis, given that it is one of the synergistically-induced epidermal proteins by these two cytokines in lesional psoriatic skin. Together, our data provide new insights into KLK8 distinct regulation and pathogenic involvement in psoriatic skin. Inhibiting KLK8-specific activity in lesional psoriatic skin will reduce psoriatic plaques, and opens a new future avenue for topical psoriasis drug development.

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Chapter 4 Serum Kallikrein-related peptidase-8 levels correlate with skin activity, but not psoriatic arthritis, in patients with psoriatic disease

Sections of this chapter were reproduced from the following published manuscripts:

Eissa, A. Cretu, D., Soosaipillai, A., Thavaneswaran, A., Pellett, F., Diamandis, E.P., Cevikbas, F., Steinhoff, M., Gladman, D., Chandran, Vinod. Serum kallikrein-8 correlates with skin activity, but not psoriatic arthritis, in patients with psoriatic disease. 2012. Clinical Chemistry Laboratory Medicine: 1434-6621

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4 Serum Kallikrein-related peptidase-8 levels correlate with skin activity, but not psoriatic arthritis, in patients with psoriatic disease

4.1 Introduction

As mentioned in previous chapters, psoriasis is a very common inflammatory skin disease affecting about 2% of the North American population (Kurd and Gelfand, 2009). This multifactorial chronic disease is characterized by epidermal hyper-proliferation and dermal inflammation that vary in severity from minor, localized patches to involvement of the entire skin surface (Lowes et al., 2007). About one-third of patients with psoriasis suffer from moderate-to-severe disease and report that the disease has a substantial negative impact on their quality of life. The concept of ‘psoriatic disease’ encompasses additional manifestations often associated with the occurrence of psoriatic skin lesions, including musculoskeletal and cardiovascular systems (Nograles et al., 2009; Scarpa et al., 2010). Approximately 30% of psoriasis patients develop arthritis which contributes additional morbidity to psoriasis patients (Langley et al., 2005). Psoriatic arthritis (PsA) is an inflammatory joint disease associated with cutaneous psoriasis and seronegative for rheumatoid factor. There is a high prevalence of undiagnosed PsA among psoriasis patients seen in dermatology clinics (Reich et al., 2009). The diagnosis of PsA is usually made by a rheumatologist after a clinical evaluation; no diagnostic test is available. Soluble biomarkers of PsA are of particular interest to dermatologists and rheumatologists, as they may aid in screening, early detection and treatment, leading to amelioration of progressive joint damage and disability, and improvement in the quality of life and function.

Given that the majority of PsA patients initially present with cutaneous psoriasis, we hypothesized that epidermal proteins implicated in skin barrier function and innate immunity, such as KLKs, may act as serum biomarkers of psoriasis severity and may aid in screening and early detection of PsA.

Currently, there is no validated single or panel of blood/serum biomarkers for PsA in the clinic. The diagnosis of PsA is considered when inflammatory musculoskeletal disease is recognised in the presence of psoriasis. PsA is classified using the CASPAR (ClASsification criteria for

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Psoriatic ARthritis) criteria (Taylor et al., 2006). PsA disease activity is primarily assessed by counting the number of tender or swollen joints. Since early diagnosis of PsA is associated with less joint damage progression (Gladman et al., 2011), it is imperative that psoriasis patients are screened regularly for the presence of PsA. Screening tools for PsA including questionnaires, imaging, genetic and cellular biomarkers are being investigated (Chandran and Gladman, 2012). Preliminary studies have suggested soluble PsA markers, including acute phase reactants (such as hsCRP), markers of extracellular matrix-destruction (MMPs) and cytokines such as tumour necrosis factor (TNF) may discriminate patients with PsA from those with psoriasis alone (Chandran and Gladman, 2012). These serum biomarkers tend to be overexpressed in the lesional psoriatic skin and/or the inflamed synovial fluid of psoriasis and PsA patients, respectively (Gladman, 2009; Myers et al., 2006). Herein, we examined KLK expression in inflamed PsA synovial fluids and psoriatic skin under the hypothesis that KLKs may mediate both skin and joint inflammation in PsA and hence may be useful as screening biomarkers of PsA in psoriasis patients. We then measured the levels of a panel of epidermal and synovial fluid-expressed KLKs in 152 serum samples of well-phenotyped psoriasis patients, with or without PsA, to determine the utility of KLKs as soluble biomarkers for screening PsA.

4.2 Materials and Methods

4.2.1 Collection of synovial fluids (SF) from PsA and control patients

Synovial fluids were aspirated from inflamed knee joints of three PsA patients and three non- inflammatory (early osteoarthritis) controls. Each sample was subjected to BCA total protein assay prior to loading 800 µg protein into antibody-coated plates to measure KLK levels by ELISAs.

4.2.2 Immunohistochemistry

Rabbit anti-human Kallikrein 6 and 8 antibodies were purchased from R&D Systems. Formalin- fixed paraffin sections were deparaffinized with 2x xylene, and serials dilutions of ethanol (100%, 95%, 80%, 50%) and water, 5 minutes per step. Slides were heated at 80°C for 20 minutes in autoclave, and cooled on ice for 20 minutes. Endogenous peroxidase activity was quenched with Dako peroxidase quenching buffer for 20 minutes at room temperature. After PBS washing, sections were blocked with 2% BSA in PBS for 1 hour and incubated with

113 antibodies diluted in 2% BSA (1:250, 1:400, 1:500, 1:1000 dilutions) overnight at 4°C in a humid chamber. After rinsing with PBS, slides were incubated for 1 hour at RT in a humid chamber with rabbit horseradish peroxidase-conjugated secondary antibodies diluted 1:400 in blocking buffer. Nuclei were counterstained with Hematoxylin QS (Vector Laboratories, Burlingame, CA) and mounted with Aquamount (BDH, Poole, UK). The immunoreactivity was detected with the Liquid DAB+ Substrate Chromogen System (Dako Cytomation).

4.2.3 Setting and participants

We recruited 152 age and sex-matched patients with active psoriasis, with or without psoriatic arthritis (PsA), from the University of Toronto PsA and psoriasis clinics. The study protocol and informed consent forms were approved by the University Health Network research ethics board and all patients signed a written consent form. Consenting patients were recruited into on observational cohort and evaluated according to a standard protocol every 6-12 months.

At each visit, symptoms, physical examination (including complete musculoskeletal examination and assessment of psoriasis severity), current use of medications and laboratory findings were recorded. The data were entered and stored in a computerized database. In phase I, 52 psoriasis patients and 26 healthy controls were recruited. Of the 52 psoriasis patients, 26 were diagnosed with PsA by a rheumatologist and satisfied the ClASsification of Psoriatic ARthritis (CASPAR) criteria. PsA was excluded by a rheumatologist in the remaining 26 psoriasis patients. Psoriasis severity was evaluated using the psoriasis area and severity index (PASI) score. Phase I patients had mild-to-moderate psoriasis as indicated by PASI < 8. Controls were recruited from healthy volunteers who did not have psoriasis or inflammatory arthritis. Patients with psoriasis and PsA were group-matched for age, sex and psoriasis duration, while controls were matched for age and sex. In phase II of the study, KLKs showing promise, were further investigated in a second independent cohort of 100 patients with moderate-to-severe psoriasis (PASI scores >8), 50 of whom had PsA. None of the 152 patients were treated with TNF inhibitors at the time of study participation. All recruited patients reported European ethnicity. Blood samples were drawn at the time of clinical assessment, processed immediately, and serum aliquots stored at -80°C until laboratory analysis.

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4.2.4 Enzyme linked immunosorbent assays (ELISAs)

Serum levels of KLK5, KLK6, KLK7, KLK8, KLK10, KLK11 and KLK13 were determined using KLK-specific and sensitive ELISAs developed in-house. The assays were performed on stored serum samples linked to phenotypic information collected prospectively. The reader of the laboratory tests were blinded to the diagnosis and clinical information.

4.2.5 Statistical Analysis

In Phase I, logistic regression models were fit with disease classification as the outcome, using KLKs as explanatory variables while controlling for age and sex. Univariate, full multivariate and reduced multivariate logistic regression models were used to identify KLKs that are independently associated with disease class. Patients with PsC and PsA were grouped into one group to identify biomarkers for ‘psoriatic disease’. Subsequently, patients with PsC, PsA, and controls were compared using polychotomous logistic regression. Finally, biomarkers that differentiate PsA from psoriasis were investigated by comparing patients with PsA to those with psoriasis alone. Since phase I of the study was exploratory, results were considered to be statistically significant at p<0.05 and correction for multiple testing was not done. In phase II logistic regression models were fit to examine the relationship between serum KLK6 and 8 levels and disease class. In order to determine the association between disease activity and KLK6 and 8 levels, correlation analyses and linear regression analyses were done with PASI score and joint counts as outcome and KLK6 and 8 levels, age, sex and disease duration as explanatory variables.

4.3 Results

4.3.1 KLK6 and KLK8 are elevated in PsA synovial fluids and lesional psoriatic skin

Synovial fluid samples were aspirated from the knee joints of three PsA patients and three patients with early osteoarthritis (OA). OA samples are commonly used as controls in rheumatology studies, since synovial fluid is not usually available from healthy individuals. We explored the presence of KLK proteases in the inflammatory synovial fluid from PsA patients, compared to non-inflammatory fluid from patients with OA. As shown in Figure 4.1, many of the epidermal KLKs (such as KLK5, 6, 7, 8, 13 and 14) were detected in the synovial fluids of

115 all three PsA patients, with significant elevation of KLK6 (p=0.039) and KLK8 (p=0.013) in PsA compared to OA. To our knowledge, this is the first report of KLK serine protease expression in human synovial fluids, although metalloproteases (MMPs) were previously detected in PsA synovial fluids and have been investigated as serum PsA biomarkers (Chandran et al., 2010). Since KLK6 and KLK8 were significantly elevated in the synovial fluids of PsA patients among the KLKs tested, we next investigated their expression in psoriatic skin and confirmed the overexpression of these two KLKs in lesional psoriatic skin tissues by immunohistochemistry. Compared to normal skin, where KLKs are normally expressed in the uppermost stratum corneum (SC) layer, KLK6 and KLK8 expression expanded below the SC into the spinous layer of the elongated rete ridges of psoriatic skin, Figure 4.2.

4.3.2 PsA and PsC Patients

PsC and PsA patients had well established disease and were matched for age, sex and psoriasis duration. The patients’ demographics and clinical characteristics are summarized in Table 4.1.

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Figure 4.1. Expression of KLK proteases in PsA inflamed joint synovial fluids and control (osteoarthritis) synovial fluids. Asterisk denotes statistically significant differences between groups (p<0.05)

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Figure 4.2. Immunohistochemical expression of KLK6 and KLK8 in lesional psoriatic skin. (a) Normal skin. (b) Psoriatic skin. SC: stratum corneum, RG: rete ridges. Note expression of both KLK6 and KLK8 below the SC into the rete ridges (RG) of psoriatic skin indicated with an arrow.

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Table 4.1. Demographics and clinical characteristics of psoriatic disease patients

Phase I

Characteristic PsA1 (N=26) PsC 2 (N=26) Controls (N=26)

Females/males 14/12 14/12 14/12

Age (years) 46.9 ± 10.4 3 45.0 ± 12.1 42.5 ± 14.1

Duration of psoriasis (years) 16.9 ± 13.8 16.7 ± 13.8 -

Duration of PsA (years) 13.4 ± 10.7 - -

Psoriasis Area and Severity Index 3.70 ± 3.50 4.90 ± 5.20 - score (PASI)

Number of tender or swollen joints 15.7 ± 12.3 - -

Number of Swollen joints 4.90 ± 4.20 - -

Phase II

Characteristic PsA (N=50) PsC (N=50) Controls (N=26) 4

Females/males 18/32 17/33 14/12

Age (years) 51.8 ± 12.2 45.9 ± 13.0 42.5 ± 14.1

Duration of psoriasis (years) 23.0 ± 11.7 17.4 ± 12.4 -

Duration of PsA (years) 16.9 ± 11.7 - -

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Psoriasis Area and Severity Index 18.1 ± 10.0 16.5 ± 9.40 - score (PASI)

Number of tender and/or swollen 11.4 ± 12.0 - - joints

Number of Swollen joints 2.20 ± 3.0 - -

1. PsA: psoriasis arthritis. 2. PsC: cutaneous psoriasis without arthritis 3. For all continuous variables mean ± standard deviation is reported. 4. Controls are the same for Phase I (mild to moderate psoriasis) and PhaseII (Moderate to severe psoriasis) patients.

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4.3.3 KLK8 is independently elevated in sera of patients with psoriatic disease

We investigated KLKs as soluble biomarkers of psoriatic disease by measuring their concentration in the serum of psoriasis patients (PsC and PsA) and healthy controls in two subsequent phases (Phase I and Phase II). In phase I pilot study, epidermal and synovial fluid- expressed KLK5, 6, 7, 8, 11, 13 were measured in 52 psoriatic disease serum samples compared to 26 healthy controls, as shown in Table 4.2. Among all the KLKs tested, only KLK8 levels were significantly elevated in psoriatic disease patients compared to controls in univariate logistic regression analyses adjusted for age and sex. Increased serum levels of KLK8 associated with psoriatic disease in a multivariate reduced model adjusted for age and sex [Odds ratio per unit increase (OR) 2.56, 95% CI (1.08, 6.12), p = 0.03]. Polychotomous logistic regression analysis showed that only KLK8 had significantly different effects when modelling PsC and PsA separately, controlling for age, sex and the other KLKs, as shown in Table 4.3. Binomial logistic regression analyses did not demonstrate a difference in serum KLK levels between PsC and PsA.

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Table 4.2. KLK levels in the serum of plaque-type cutaneous psoriasis (PsC) and psoriasis arthritis (PsA) patients

Phase I samples 1 PsA PsC Controls

(N=52) (N=26) (N=26) (N=26)

KLK5 0.26 ± 0.09 3 0.30 ± 0.17 0.29 ± 0.16

KLK6 1.85 ± 0.55 1.95 ± 0.49 1.78 ± 0.45

KLK7 8.00 ± 22.6 7.43 ± 7.93 4.37 ± 5.68

KLK8 2.03 ± 0.95 2.20 ± 0.93 1.67 ± 0.50

KLK10 1.08 ± 0.57 1.03 ± 0.47 1.12 ± 0.43

KLK11 0.64 ± 0.45 0.53 ± 0.12 0.53 ± 0.15

KLK13 0.20 ± 0.0 0.21 ± 0.07 0.20 ± 0.02

Phase II samples 2 PsA PsC Controls

(N=100) (N=50) (N=50) (N=26)

KLK6 5.48 ± 2.64 4.68 ± 1.78 1.78 ± 0.45

KLK8 4.74 ± 4.19 3.89 ± 2.15 1.67 ± 0.50

1. Mild to moderate psoriasis with mean PASI scores 3.7-4.9. 2. Severe psoriasis with mean PASI scores 16.5 – 18.1 3. For all KLK concentrations measured in µg/L, the mean ± standard deviation is reported

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4.3.4 KLK8 serum levels in PsA correlate with PASI score but not inflamed joint counts

As mentioned above, we detected significant elevation of KLK8 in the sera of a small set of PsC and PsA patients with mild-to-moderate plaque-type psoriasis. Furthermore, KLK6 and KLK8 were significantly elevated in the synovial fluids of PsA patients and lesional psoriatic skin. Thus, we subsequently aimed to re-examine and validate KLK6 and KLK8 as PsA screening biomarkers in a larger patient cohort consisting of 100 patients with moderate-to-severe psoriasis (PASI >8), with or without PsA (Phase II). Both KLK6 and KLK8 were elevated in the sera of these patients (Figure 3a). As listed in Table 4.2, mean KLK6 level in patients with mild-to- moderate psoriasis (1.95 ± 0.49 µg/L) was similar to age and sex-matched healthy controls (1.78 ± 0.45 µg/L), but was significantly higher in the sera of moderate-to-severe psoriasis patients (4.68 ± 1.78 µg/L). Alternatively, mean KLK8 level was increased in both PsA patients with mild-to-moderate psoriasis (2.20 ± 0.93 µg/L) and patients with moderate-to-severe psoriasis (3.89 ± 2.15 µg/L), compared to healthy controls (1.67±0.5 µg/L). A similar trend was observed for mean KLK6 and KLK8 serum levels in PsA patients. Logistic regression analysis showed that neither KLK8 nor KLK6 levels distinguished PsA from PsC, as indicted in Table 4.3 and Figure 4.3.

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Table 4.3. Polychotomous logistic regression analysis to identify biomarkers associated with patients having psoriasis alone and psoriatic arthritis

Homogeneity Psoriatic Arthritis (PsA) Cutaneous Psoriasis (PsC)

Covariate P value 1 OR 2 95% CI 3 P-value OR 95% CI P-value

KLK5 0.36 0.01 (<0.001, 5.3) 0.16 0.071 (<0.001, 16.0) 0.34

KLK6 0.47 1.36 (0.321, 5.7) 0.67 2.490 (0.566, 10.9) 0.23

KLK7 0.70 1.06 (0.928, 1.2) 0.40 1.054 (0.926, 1.2) 0.43

KLK8 0.03 4.27 (1.024, 17.8) 0.05 6.623 (1.62, 26.9) 0.01

KLK10 0.10 0.46 (0.099, 2.1) 0.33 0.143 (0.024. 0.85) 0.03

KLK11 0.49 1.88 (0.102, 34.6) 0.67 0.284 (0.006, 12.5) 0.51

1. The homogeneity p values indicate whether the markers have significantly different effects when modelling psoriasis and PsA separately, controlling for age, sex and the other KLKs listed. The p values associated with PsA and psoriasis indicates the significance of difference between either patient group compared to controls. KLK13 levels were un-measurable and are not reported. Only KLK8 was statistically significant, for discussion see text. 2. OR: odds ratio 3. CI: confidence interval

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Furthermore, we investigated the association between KLK6 and KLK8 serum levels with clinical parameters of PsA skin and joint activity, such as PASI scores and actively inflamed (tender and/or swollen) and swollen joint counts. As shown in Figure 4.4, both KLK6 (r = 0.52) and KLK8 (r = 0.42) serum levels correlated with the PASI scores of all patients (p <0.0001). KLK8 correlated positively with PASI scores when patients with PsA (r=0.60, p <0.0001) and PsC (r= 0.43, p=0.001) were considered separately. KLK6 correlated with PASI scores in PsA patients (r=0.63, p <0.0001) but not in PsC patients (r=0.036, p=0.8). Only KLK8 association with the PASI score was significant (β=1.153, p=0.0003) after adjusting for age, sex, psoriasis duration and disease group (PsA and PsC) in a linear regression model with PASI score as the outcome. However, there was no correlation between KLK8 and actively inflamed joint count (p=0.35) or swollen joint count (p=0.12) in PsA patients. Thus, KLK8 serum level in PsA correlates with skin, but not arthritis, activity.

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Figure 4.3. KLK6 and KLK8 cannot function as screening biomarkers for arthritis in psoriasis patients. (A) KLK6 and KLK8 levels are elevated in moderate-to-severe psoriatic disease patients (combined PsC and PsA) compared to healthy controls. (B) KLK6 and KLK8 serum levels do not distinguish PsA from PsC patients.

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Figure 4.4. KLK8 correlates positively with the PASI scores in psoriatic disease patients

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4.4 Discussion

Psoriasis (PsC) and psoriatic arthritis (PsA) are relatively common inflammatory diseases of the skin and joints, respectively. Psoriatic arthritis (PsA) is an inflammatory arthritis that develops in up to one-third of patients with psoriasis (Langley et al., 2005). The varied manifestations of PsA make it sometimes difficult to recognize. Dermatologists managing a patient’s psoriasis often do not inquire about symptoms of arthritis. Thus, the presence of PsA in psoriasis patients is often overlooked in dermatology clinics. Early diagnosis of PsA is essential to prevent joint damage progression and disability. The key to early diagnosis of PsA is better recognition of the presence of PsA in patients with psoriasis (Chandran et al., 2010), but the diagnosis of PsA is difficult due to the lack of specific diagnostic tests. Soluble biomarkers have the potential to provide means for screening PsA in psoriasis patients so that appropriate referral to a rheumatologist for early diagnosis is made. Candidate serum PsA biomarkers are likely to originate from target tissues such as inflamed skin and joints. To date, few potential PsA screening biomarkers have been identified, none validated and additional biomarkers await discovery. The main aim of this study was to evaluate various kallikreins (KLKs) as potential PsA serum biomarkers in a cohort of psoriasis patients with or without PsA, with special emphasis on the relationships between KLKs and clinical parameters of PsA skin and joint activity.

Given that the majority of PsA patients initially present with cutaneous psoriasis, we hypothesized that kallikrein proteases may be overexpressed in the inflamed skin, joints and sera of PsA patients and may thus function as serum markers of PsA. Kallikreins are secreted serine proteases that are expressed in a wide range of tissues, including skin epidermis. They regulate skin barrier integrity via their ability to degrade adhesion molecules and activate antimicrobial peptides and the immune system (Borgono et al., 2007b; Briot et al., 2009; Eissa and Diamandis, 2008; Yamasaki et al., 2006). KLK upregulation has been implicated in a number of inflammatory skin diseases including psoriasis (Komatsu et al., 2007b; Kuwae et al., 2002), but their involvement in inflammatory joint arthritis, including PsA, has not been studied. The potential role of serum Kallikreins as soluble biomarkers of psoriasis severity and PsA has not been examined.

We detected multiple KLKs in synovial fluids, with increased expression of KLK6 and KLK8 in PsA compared to non-inflammatory fluid (osteoarthritis). The expression of multiple KLKs in

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PsA synovial fluids has not been previously reported. In PsA, the synovial membrane becomes inflamed in response to proinflammatory cytokines, such as TNF-α, leading to the secretion of cartilage-digesting enzymes, such as matrix metalloproteinases (MMPs) by synovial fibroblasts (Chandran and Gladman, 2012 ; Ribbens et al., 2002). Similarly, KLK6 and KLK8 may be induced by cytokines to degrade collagenous and non-collagenous structural molecules in the joint.This hypothesis requires further scrutiny. These two KLKs were also elevated in lesional psoriatic skin in which they expanded lower into the spinous layer of the elongated rete ridges. Our immunohistochemistry results are consistent with previous reports of elevation of KLK6 and KLK8 transcripts within two separate gene clusters in a large-scale psoriasis gene expression analysis (Bowcock et al., 2001).

After examining KLK expression in the synovial fluids and lesional psoriatic skin, we measured the concentrations of KLK5, 6, 7, 8, 10, 11 and 13 in the sera of a cohort of PsA and PsC patients (N=52) with mild-to-moderate psoriasis (PASI <8). Among all the KLKs tested, patients with PsC (N=26) and PsA (N=26) displayed significantly higher KLK8 serum levels compared to healthy controls (N=26). The remaining tested KLKs did not significantly vary in these patients. Thus, our initial exploratory analysis of KLK expression in the synovial fluids, lesional skin and serum of patients with psoriatic disease suggested KLK6 and KLK8 as candidate psoriatic disease biomarkers. Therefore, we next measured the levels of these two KLKs in a larger independent cohort of serum samples of moderate-to-severe psoriasis patients, with or without arthritis (N=100, PASI >8). Although both KLK6 and KLK8 were elevated in the sera of these patients compared to healthy controls, the increase in KLK6 serum levels was not significant when we controlled for sex, age and disease duration as well as KLK8.

Given that KLK8 was independently and significantly elevated in the sera of PsC and PsA patients, we next examined its correlation with clinical parameters of skin and joint activity. KLK8 correlated positively with the PASI score, but there was no significant correlation between KLK8 levels in the serum and actively inflamed joint or swollen joint counts, indicating that KLK8 is a soluble marker of skin, but not arthritis, activity in PsA patients. We recently showed that KLK8 is a physiologically-active trypsin-like serine protease in normal skin epidermis. KLK8 protein levels are elevated in skin extracts from lesional psoriatic skin compared to non- lesional and normal skin, whereby the lesional skin exhibits significantly higher trypsin-like activity (Komatsu et al., 2007b). Hence, the significant correlation of KLK8 serum levels with

129 both PsC and PsA patients’ PASI scores reported herein indicates that KLK8 trypsin-like activity is related to the progressive skin barrier dysfunction in both PsC and PsA.

To summarize, our study illuminates the importance of KLK8 as a marker of cutaneous psoriasis severity. We demonstrate that although some KLKs are elevated in PsA synovial fluid, lesional psoriatic skin and serum, none of these KLKs, including KLK8, can distinguish PsA from PsC patients. Identifying PsA-specific soluble biomarkers remains a challenge to date. For instance, biomarkers from genetic and genomic studies such as HLA-Cw06, IL12B and IL23R are associated with PsA, but their primary association is with psoriasis susceptibility (Chandran and Gladman, 2012; Gladman and Farewell, 2003; Veale et al., 2005). TNF-α is present in the sera and skin of psoriasis patients and in the synovial fluids of PsA patients (Slobodin et al., 2009; Veale et al., 2005). However, TNF-α serum levels are not useful for identifying PsA in psoriasis patients. Nonetheless, anti-TNF agents are effective in controlling skin and joint manifestations in PsA patients and preventing progression of joint damage (Weger, 2010). Since we demonstrate that KLK8 can act as a surrogate serum biomarker of PASI, KLK8 may hold promise as a biomarker for monitoring response to psoriasis therapies and relapse. Furthermore, the detection of higher levels of KLK8 in the sera of both PsC and PsA patients, as well as in the skin and synovial fluid of PsA patients, suggests that blockade of this protease may be beneficial in both skin and musculoskeletal manifestations of psoriasis.

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Chapter 5 Ongoing Studies, General Discussion and Future Directions

Sections of this chapter were reproduced from the following manuscript with copy right permission:

Ramachandran R., Eissa, A. et al. (2012). Proteinase-activated receptors (PARs): differential signalling by kallikrein-related peptidases KLK8 and KLK14. Biological Chemistry. 393(5):421

Eissa, A., ……, Diamanids E.P. High through put screening of large small molecule compound libraries reveals novel KLK8-specific inhibitors which exhibit potential as topical drug targets in psoriasis. (Unpublished data, ongoing studies, manuscript in preparation)

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5 Ongoing Studies, General Discussion and Future Directions

5.1 Introduction

We and others have recently begun to challenge the prevailing dogma that trypsin-like KLKs play similar roles in the skin. The lack of KLK8 inhibition by all three physiological serine protease inhibitors from the LEKTI family (LEKTI-1, LEKTI-2, LEKTI-3) attests to the fact that trypsin-like KLKs may be regulated differently to play overlapping and/or distinct dermatological functions (Brattsand et al, 2009). One of the important functional pathways KLKs target in the skin is the activation of the keratinocyte cell surface-expressed receptors known as the proteinase-activated receptors or PARs. KLK5, KLK6 and KLK14 are able to cleave specific recognition sites in the extracellular amino termini of PARs to reveal a motif known as the tethered ligand (TL). The exposed tethered ligand then interacts with the extracellular loops of the receptor to initiate the recruitment of G-proteins and other signaling molecules to the intracellular domains of the receptors (Oikonomopoulou et al., 2006). Both PAR1, PAR2 are expressed in epidermal keratinocytes (Santulli et al., 1995), and PAR2 has been heavily studied in the context of keratinocyte proliferation and differentiation, skin hydration and inflammation (Steinhoff et al., 2005; Rattenholl et al., 2008). Recent seminal studies enhanced our understanding of KLK-PAR2 signaling pathways in inflamed skin of Netherton syndrome human patients and mouse models, but the main focus of these studies was KLK5 (Briot et al., 2009). A study by Stefansson et al, indicated that KLK5 and KLK14 can induce a calcium signal through PAR2, while KLK7 and KLK8 can not (Stefansson et al., 2008). In terms of signaling, PAR2 is able to activate multiple downstream pathways including MAPKinase, as well as stimulating elevations of intracellular calcium. It was recently shown that PAR2 signaling can occur by a G-protein-independent mechanism to activate MAPKinase by interacting with - arrestins (Ramachandran et al., 2011). Whether KLK8 is able to signal through PAR2 via calcium/G-protein independent mechanisms remains unknown.

The limitations in our current understanding of KLK8 signaling mechanisms in the skin is partly due to the lack of KLK-specific inhibitors or activity-based probes to distinguish its proteolytic activity from other epidermal trypsin-like KLKs in vivo. Identification of KLK8-specific inhibitors is of major interest as they may serve as important biochemical tools in functional assays and may have a future therapeutic potential in reducing psoriasis flares.

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Hence, the ongoing studies described below address the missing gaps in KLK8-skin research by (1) investigating KLK8-mediated proteinase-activated receptors (PARs) signaling and (2) searching for KLK8 inhibitors by high throughput screening of small molecule compound libraries.

5.2 Materials and Methods

5.2.1 KLK8-mediated PAR2 signaling in cell-based assays

Active recombinant KLK8 and KLK14 were prepared as previously documented (Eissa et al, 2011; Oikonomopoulou et al., 2006) and the specific activities in standard samples of each enzyme U/ml (micromoles substrate cleaved/min/ml) were determined using the synthetic substrate: VPR-aminomethylcoumarin. Both KLK8 and KLK14 were used at comparable levels of enzyme catalytic activity. Suspensions of human embryonic kidney (HEK) cells were harvested from cultured monolayers and calcium signalling was measured as documented previously for studies of KLK14 (Oikonomopoulou et al., 2006). Calcium assays were performed as previously described (Ramachandran et al, 2009).

To investgate if KLK8 can induce PAR2 and β-arrestin interactions, Bioluminescence Resonance Energy Transfer (BRET) between YFP-tagged human PAR2 and renilla luciferase (rLuc)-tagged -arrestin1 and -arrestin2 was measured as described in detail previously (Ramachandran et al., 2009; Ramachandran et al., 2011) following a 20 minute incubation with the indicated agonists. Data show the mean ± s.e.m. (bars) for triplicate measurements in two different cultures of cells transfected with PAR2 and arrestins. To visualize receptor internalization, YFP-tagged human PAR2 (C-terminal YFP) was transfected in HEK cells in glass bottom petridishes. 48 hrs after transfection cells were treated with proteases for 20 minutes at 37 C. Cells were fixed with 10% formalin in phosphate-buffered isotonic saline, pH7.4 and the localization of YFP-tagged receptor was determined by confocal microscopy. Receptor activation is indicated by the formation of punctate YFP fluorescent speckles both internalized and adjacent to the plasma membrane. Activation/internalization was quantified by morphometric analysis of speckle formation (speckles/cell). For MAPK activation studies, YFP-tagged human PAR2 (C-terminal YFP) was transfected in KRNK cells in six well plates. Fourty-eight hours after transfection, cells were quiesced in serum-free media for 2 h and either treated or exposed to trypsin (10nM),

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2f-LIGRLO-NH2 (10 mM), KLK14 (10 U/ml) or KLK8 (10 U/ml). MAPK activation was monitored as described previously (Ramachandran et al, 2009).

5.2.2 Search for KLK8 inhibitors by high throughput screening of small molecule compound libraries

A library of 13,569 small molecule compounds were screened against KLK5, KLK8 and KLK14, including pharmacologically-active, FDA-approved, natural and synthetic small molecules from 5 different libraries as indicated in Table 5.1, below. Our previously reported protease assays (100 μl) (Eissa et al, 2011) were adapted for a 384-well plate format (50 μl). The primary screens for each enzyme were performed in 384-well plates (catalog no. 3370; Corning, NY) in duplicates using a fully automated Beckman/Coulter Sagian core system in high throughput screening facility. Microtiter plates were loaded sequentially with small molecule compounds first (10µM final concentration in 0.2% DMSO), followed by the reaction buffer 100 mM Sodium Phosphate +0.1% Tween (pH 8.5) containing 0.2 mM of the fluorogneic substrate VPR-AMC, and finally by adding the KLK protease of interest (20 nM KLK5, 10 nM KLK8 or 10 nM KLK14 in 3 parallel screens). Fluorescence was measured immediately at excitation and emission wavelengths of 385 nm and 465 nm, respectively, every 30 seconds for the first 7 minutes in an Envision plate reader. Assay mixtures containing DMSO only were used as controls. The average of fluorescence values in duplicate wells for a given compound was used to determine the residual activity by taking the values obtained with DMSO controls as 100%. Compounds that reduced the protease activity by ≥50% were selected for further analysis.

After applying filtering criteria such as excluding compounds with reactive groups, and including compounds that inhibited only one KLK but not the other two, a total of 59 putative KLK-specific inhibitors with RA <20% as well as 13 general inhibitory compounds were subjected to a secondary dose-dependent validation screen to confirm findings and determine the compounds’ IC50. Known general serine protease inhibitors acted as pan-KLK inhibitors validating the sensitivity of our HTS-assays. Compound-specific IC50 values were determined by nonlinear regression to the following four-parameter equation, as previously described.

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According to the multi-parametric equation (1), dose-response relationships were classified into one of 4 categories: no-inhibition (NI: little to no decrease in activity), good dose-response inhibition (GI: Y-range close to 100, slope factor close to 1, background close to zero), moderate dose-response inhibition (MI: A good sigmoidal curve seen, but either with a Y-range significantly less than 100, or with a slope factor significantly higher or lower than 1) and poor dose-response inhibition (PI: low fit curve parameters, inhibition seen only in the highest doses).

Table 5.1. Libraries selected for KLK-inhibitor screening

Compound Distributor Description

Library

DIVERSet Chembridge Corp., San Diego,USA 9,989 synthetic small molecules

PRESTWICK Prestwick Chemical, Illkirch,France 1,214 off-patent small molecules

SPECTRUM Microsource Discovery System, 1,120 natural products and

Gaylordsville, USA bioactives

LOPAC Sigma-Aldrich, Canada Ltd., Oakville, 885 pharmacologically active

Canada

Natural Biomol International L.P., Plymouth 361 natural products

Products Meeting, USA

Library

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5.3 Results

5.3.1 KLK8 displays differential PAR2 signaling compared to KLK14

Recent studies indicated that PAR2 signaling can occur without calcium induction. We hypothesized here that KLK8 may exhibit differential PAR signaling compared to KLK14, giving support to our hypothesis that trypsin-like epidermal KLKs are not redundant. Potential KLK8-mediated and KLK14-mediated PAR2 signaling was examined in collaboration with the Dr. Hollenberg lab, in terms of calcium release in human embryonic kidney cells (HEK) cells, PAR2 and -arrestins interaction and receptor internalization, and MAPK activation.

As shown in Figure 5.1, we confirmed that KLK8 does not induce calcium release through PAR2, while KLK14 induces calcium release via PAR2, in a different cell background from the one used by Stefansson et al (Stefansson et al., 2008).

Since PAR2 signaling can occur by a G-protein-independent mechanism to activate MAPKinase by interacting with -arrestins, we next aimed to determine if PAR2 activation by KLKs 14 and 8 could trigger interactions of PAR2 with -arrestins, as does trypsin. Our results showed that KLK14 is able to promote the interaction of the receptor with both -arrestin1 and 2. However, KLK8 was unable to promote interaction of PAR2 with neither -arrestin1 or 2, even at high enzyme concentrations, Figure 5.2. In terms of PAR2 internalization, KLK14, like trypsin and the PAR-activating peptide, 2-furoyl-LIGRLO-NH2, was able to stimulate PAR2 clustering and internalization, as illustrated by the formation of visible membrane-associated and internalized fluorescent speckles, but KLK8 was unable to do so, as shown in Figure 5.2. Thus, our results confirm that KLK8 is a unique trypsin-like epidermal KLK that cannot signal through PAR2 via calcium dependent or independent mechanisms. This was further confirmed as we observed no downstram MAPK activation upon treating cells with KLK8, Figure 5.3.

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Figure 5.1: KLK8 does not cause calcium signalling via either human PAR1 or PAR2, but disarms thrombin-mediated human PAR1 signalling. The effects of thrombin and KLK8 on calcium signalling in human HEK cell suspensions (E530 fluorescence emission) was measured as outlined in the materials and methods. KLK8 at 4 U/ml did not cause a calcium signal, as would be expected for the activation of either PAR1 or PAR2 in the HEK cells. The signal generated by 1 U/ml thrombin either before (right-hand-tracing) or after exposure of cells to 4 U/ml KLK8 (left-hand tracing) shows a reduction in fluorescence signal due to receptor dis-arming (double arrow pointing to the thrombin-generated signal prior to (right) and after (left) KLK8 treatment).

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Figure 5.2. KLK8 does not trigger human PAR2 and -arrestins interaction, or PAR2 internalization, unlike KLK14. YFP-tagged human PAR2 (C-terminal YFP) was transfected in HEK cells in glass bottom petridishes. 48 hrs after transfection cells were either (A) untreated (No Treatment,

NT) or exposed to (B) 2f-LIGRLO-NH2 (50M), (C) Trypsin (20nM), (D) KLK14 (20U/ml) or (E) KLK8 (20U/ml) for 20 minutes at 37 C. Receptor interaction is indicated by the BRET ratio and receptior internatlization is indicated by the formation of punctate YFP fluorescent speckles both internalized and adjacent to the plasma membrane (A-E). Internaliztion was quantified by morphometric analysis of speckle formation (speckles/cell) (F). * indicates a significant increase in the number of internalized receptors (speckles/cell) over the untreated (NT) cells.

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Figure 5.3. Unlike KLK14, KLK8 does not activate P42/44 MAP kinase-signalling in human PAR2-expressing cells. The densitometry intensities representing ctivated/phosphorylated MAPK (p-P42/44) were normalized to the intensity of total MAPK for each sample. Histograms represent the averages +/- SEM for data obtained from two separately transfected and stimulated cells.

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5.3.2 Identification of KLK8-specific inhibitors by high throughput screens (HTS) of small molecule compounds

Our primary screens of ~13,000 compound libraries resulted in 181, 226 and 196 small molecules that reduced residual activity (RA %) of KLKs 5, 8 and 14 to less than 50%, respectively, as shown in Table 5.2. After applying filtering criteria such as excluding compounds with reactive groups, and including compounds that inhibited only one KLK but not the other two, a total of 59 putative KLK-specific inhibitors with RA <20% as well as 13 general inhibitory compounds were subjected to a dose-dependent validation screen to confirm findings and determine the compounds’ IC50. Known general serine protease inhibitors acted as pan- KLK inhibitors validating the sensitivity of our HTS-assays. Our secondary screen results indicate that the three epidermal trypsin-like KLKs exhibited differential inhibition profiles despite their significant sequence homology. Our dose-dependent secondary screens identified 1 KLK5-specific inhibitor (IC50: 17.298 µM), 9 KLK8-specific inhibitors (IC50s: 0.08-1.6 µM) and 7 KLK14-specific inhibitors (IC50s: 0.08-1.7µM), as listed in Table 5.3. An example of a good KLK8-specific inhibitor is shown in Figure 5.4.

Moreover, there was a clear functional classification among the inhibitors specific for each enzyme. For instance, we discovered that several XXX natural derivatives as KLK14-specific, while they exhibited no inhibition against the two other KLKs. Furthermore, there was a high structural homology among potent KLK8-specific inhibitors. Differences in the 3D structures of these enzymes might account for the formation of distinct active site cleavages with unique steric properties. Further structure-activity relationship (SAR) studies are required to understand the structure-based inhibition mechanism of these compounds. Fortunately, the crystal structure of KLK8 was resolved very recently, although not published yet. We obtained the crystal structure by collaboration with Dr. Peter Goettig and docked our top 9 compound hits into it. Interestingly, the active site pockets of KLK8 versus KLK5 exhibited different electrostatic potentials, which may explain their different inhibition profiles towards some small molecules. The majority of the novel KLK8-specific small molecule inhibitors we identified docked into KLK8’s active site pocket, as expected. These compounds represent the starting point for further biochemical kinetic characterization assays and development into potential therapeutic targets.

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Table 5.2. Primary HTS assays identify potential KLK5, KLK8 and KLK14-specific small molecule inhibitors

Total Statistical Unique Active against 1 Active against actives actives other KLK 2 other KLK's

KLK5 181 (1.4%) 113 38 30

KLK8 226 (1.7%) 115 81 30

KLK14 196 (1.5%) 99 67 30

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Table 5.3. KLK-specific inhibitors IC50’s

A. KLK5-specific Compound Name A) KLK5 Inhibition (IC50) KLK8 Inhibition (IC50) KLK14 Inhibition (IC50) CHEMBRIDGE (17.29 uM) NI NI

B. KLK8-specific Compound Name B) KLK5 Inhibition (IC50) KLK8 Inhibition (IC50) KLK14 Inhibition (IC50) CHEMBRIDGE 6627234 W (0.08 uM) NI CHEMBRIDGE 6625809 W (0.08 uM) NI CHEMBRIDGE 5757756 W (0.09 uM) NI CHEMBRIDGE 6623448 W (0.49 uM) W CHEMBRIDGE 6623548 NI (0.65 uM) NI CHEMBRIDGE 6631370 NI (0.66 uM) NI CHEMBRIDGE 5653470 NI (1.01 uM) NI CHEMBRIDGE 6625888 NI (1.09 uM) NI CHEMBRIDGE 6625410 NI (1.68 uM) NI

C. KLK14-specific Compound Name C) KLK5 Inhibition (IC50) KLK8 Inhibition (IC50) KLK14 Inhibition (IC50) Thiaflavin Digallate NI NI (0.08 uM) CHEMBRIDGE 5965041 W W (0.08 uM) Hematein NI NI (0.09 uM) Epigallocatechin NI NI (0.66 uM) CHEMBRIDGE 5318527 NI NI (1.01 uM) Gossypol NI NI (1.68 uM) Teaflavin Monogallate NI NI (1.97 uM)

D. general-KLK Compound Name D) KLK5 Inhibition (IC50) KLK8 Inhibition (IC50) KLK14 Inhibition (IC50) Compound 40 (5.53 uM) (2.28 uM) (6.76 uM) CHEMBRIDGE 5578375 (8.89 uM) (9.59 uM) (4.52 uM) Gabexate Mesylate (16.08 uM) (0.57 uM) (4.12 uM)

(A) KLK5-specific, (B) KLK8-specific, (C) KLK14-specific, (D) pan-inhibitors. NI: No inhibition. W:Weak inhibition.

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40 Parameter Value Std. Error

20 Y Range 96.6313 4.7118 IC 50 0.0812 0.0104

% Residual Activity Residual % 0 Slope factor 1.5064 0.2505 Background -8.7613 2.0557 -20

0.001 0.01 0.1 1 10 100 [cmd] (uM)

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120 100 100 100 80 8080 60 60 60 142 40 40

% Residual Activity Residual % 40

20 Activity Residual % 20

% Residual Activity Residual % 200 0 0.001 0.01 0.1 1 10 100 0 [cmd] (uM) 0.001 0.01 0.1 1 10 100 0.001 0.01 0.1 1 10 100 KLK8 [cmd] (uM) [cmd] (uM) 100 120 80 120

10060 100

4080 Parameter80 Value Std. Error

2060 Y Range60 98.5004 4.9437 IC 50 0.0887 0.0110 % Residual Activity Residual % 400 40

Slope factor 1.8011 0.3549

% Residual Activity Residual % % Residual Activity Residual % -2020 Background20 -6.3812 2.3187

0 00.001 0.01 0.1 1 10 100 0.001 0.01 0.1[cmd] (uM)1 10 100 0.001 0.01 0.1 1 10 100 [cmd] (uM) [cmd] (uM) KLK5 KLK14 100 100 80 80 60 60 40 40

% Residual Activity Residual % 20 % Residual Activity Residual % 20 0 0 0.001 0.01 0.1 1 10 100 0.001 0.01 0.1 1 10 100 [cmd] (uM) [cmd] (uM)

Figure 5.3. An example of a KLK8-specific inhibitor identified from the high throughput screen. Small molecule compound displays no significant inhibitory effect on KLK5 and KLK14

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5.4 General Discussion

Our data shed light, at the molecular level, on the activity, regulation and putative role of Kallikrein-related peptidase-8 (KLK8) in normal and psoriatic skin. In Chapter 2, recombinant human KLK8 proteases were produced in active and latent zymogen forms to perform in vitro biochemical kinetic assays to: (1) understand pro-KLK8 activation by other epidermal KLKs known to be present in active forms in human stratum corneum, (2) understand regulation by important epidermal ions such as calcium and by the epidermal pH gradient, which regulate keratinocyte differentiation and healthy skin barrier function, and to (3) identify KLK8 enzymatic substrate specificity and potential substrate targets in the skin surface. In order to confirm the physiological relevance of this protease in human skin surface, we designed a sandwich-type KLK8-specific immunocapture-activity assay that elucidated the presence of KLK8 as a physiologically active trypsin-like serine protease in normal human epidermal stratum corneum extracts and sweat ex vivo. Our analysis of the immunocaptured skin and sweat KLK8 demonstrated that it exhibits optimal activity at pH 8.5 and retains activity at pH 5, within the physiological pH gradient of upper human epidermis. Our data showed that KLK8 in vitro expression and activity is enhanced by calcium-induced terminal keratinocyte differentiation and cornification in culture. Hence, KLK8 can be considered a marker of terminal keratinocyte differentiation with an active innate immune role in normal human skin surface and sweat. Our in vitro biochemical assays placed KLK5 as an activator of pro-KLK8 and pro-KLK1, pro- KLK11 and LL-37 antimicrobial peptide as downstream targets of active KLK8, augmenting its functional involvement in a proteolytic cascade regulating skin desquamation and antimicrobial peptide activation. Interestingly, a recent study showed that the C-terminal peptide of KLK8 has an antimicrobial and antifungal function against P. aeruginosa, S.aureus and C. albicans and a positive effect on an LPS-treated mouse model (Kasetty et al., 2011). Thus, in addition to regulating terminal keratinocyte differentiation, KLK8 most likely plays a role in modulating the skin’s antimicrobial function through LL37 cathelicidins and/or human beta-defensins.

Identifying KLK8-specific activity in healthy skin surface and sweat indicated potential important implications for inflammatory skin diseases, such as psoriasis. For one, unlike healthy skin surface which has a slightly acidic pH of ~5, psoriatic patients’ skin surface has an elevated pH of ~8 near the pH optimum of KLK serine proteases, suggesting enhanced activity of physiologically-active KLK5, KLK7, KLK8 or KLK14 in normal skin surface. Secondly, KLK8

144 mRNA expression levels are dramatically elevated in hyperkeratotic skin of psoriasis vulgaris, followed by seborrheic keratosis, lichen planus, and squamous cell carcinoma patients, compared to normal and basal cell carcinoma skin suggesting KLK8 involvement in abnormal and excessive keratinocyte differentiation (Kuwae et al., 2002). Since KLK8 is not inhibited by LEKTI or any other known epidermal serine protease inhibitors, unlike other trypsin-like KLKs, then this barrier repair protease is more likely to play a distinct epidermal role compared to other KLKs, which is yet to be specified. We demonstrated here that KLK8 cannot signal through PAR2 via calcium dependent or independent means, yet it can process LL-37 antimicrobial peptide. KLK8 inhibition profile is consistent with the results obtained by Yamasaki et al when they first identified KLK5 and KLK7 as regulators of cathelicidin LL37 processing (Yamasaki et al, 2006), suggesting that its endogenous activity is linked in skin innate immunity. In this thesis, we identified specific elevation of KLK8 in lesional skin and serum of psoriasis patients, further suggesting an innate immune role for this protease.

KLK8 overexpression and hyperactivity in normal skin can be a trauma-induced epidermal stress signal that aims to restore the barrier by inducing terminal keratinocyte differentiation and desquamation to enhance cell turnover and barrier recovery. Consistently, KLK8 knock-out mice skin suffers from delayed recovery after chemical, physical, and UV-induced barrier impairment (Kirihara et al., 2003; Kitayoshi et al., 1999). Studies have shown that serine protease activity increases in the uppermost stratum corneum following barrier disruption, consistent with KLK8 role in wound healing (Hachem et al., 2005; Kishibi et al., 2012). Paradoxically, inhibition of serine proteases, but no other protease types, was also shown to accelerate barrier recovery (Hachem et al., 2006). This improvement in barrier function can be due to inhibition of KLK8- mediated accelerated differentiation program. Hence, KLK8 seems to play opposing protective and damaging roles during barrier breaches. It is likely that initial increases in KLK8 activity upon acute barrier breaches are beneficial as a stress response or repair mechanism. However, sustained KLK hyperactivity is likely to cause the skin to enter a pathological state as alluded to by our investigation of KLK8 regulation and role in psoriasis.

In Chapter 3 of this thesis, we hypothesized that KLK8 overexpression and hyperactivity is induced and sustained by Th1 and/or Th17 immune cell-mediated effects on epidermal keratinocytes. Since psoriasis and atopic dermatitis have opposing polarization of T-cell subpopulations, we hypothesized that KLK8 is distinctly regulated by the immune subsets

145 governing these two common skin diseases. In particular, psoriasis was an interesting model to study KLK8 function in the skin, given the scaly lesions appearance and their intensified innate immune nature. Psoriatic lesions are characterized by absence of a functional granular layer which normally secretes KLK8 and absence of cornification-related proteins (such as caspase-14 and loricin). Yet, KLK8 is abundant in psoriatic lesions indicating its secretion by non-granular keratinocytes. Limited attention has been given to investigating the mechanisms leading to KLK8 upregulation in psoriatic lesions. Thus, we tested the hypothesis that secreted immune- related factors in psoriatic skin induce KLK8 overexpression by keratinocytes in lower stratum spinosum. Chapter 3 results supported the inside-outside theory of psoriasis pathogenesis, where ‘inside’ T-cell immune aberrations result in ‘outside’ epidermal barrier dysfunction. We show that synergy of TNFα and IL17A cytokines induces dramatic morphological changes in epidermal polarity and keratinocytes cell differentiation, indicated by the TNFα+IL17A-induced ‘web-like’ structures and ‘stratification domes’. TNFα and IL17A treatment of cultured keratinocytes mimicked psoriatic keratincoytes, as it induced synergistic upregulation of the psoriasis-related innate immune psoriasin (S100A7) and human beta-defensin-4 (hBD4) gene expression, as well as IL6. Our results support a recent study reporting S100A7 and hBD4 as synergistic IL-17A and TNF-α response genes in human keratinocytes (Chiricozzi et al., 2011). Although this study demonstrated a list of key synergistic/additive TNFα and IL17A response epidermal genes, it did not investigate IL-17A and TNF-α effect on keratinocyte differentiation in culture and did not point KLKs as potential key pathogenic players in psoriasis circuits.

Our data show that TNFα and IL17A induce significant KLK8 hypersecretion by cultured keratinocytes, suggesting that KLK8 overexpression in lesional psoriatic skin is an immune- related response that contributes to the development and/or maintenance of psoriatic lesions. We confirmed this notion upon examining the effect of KLK8 treatment on full-thickness 3D epidermis tissue equivalents’ expression of keratinocyte differentiation and proliferation markers and innate host defense genes. KLK8 induced keratinocyte differentiation leading to increased stratum corneum and full epidermis thickness, as well as enhanced involucrin terminal differentiation marker expression in all layers. However, adding a 10-fold higher KLK8 dose resulted in dramatic changes that mimic lesional psoriatic skin, such as stratum corneum detachment, epidermal thickening, retention of keratinocytes’ nuclei in the stratum corneum (parakeratosis) and elongation of dermal fiboblasts. KLK8 also induced significant upregulation

146 of the psoriasis-related genes S100A7, hBD4 and IL6. Our study extends data from recent genomic findings of KLK8 gene specific involvement in psoriasis (Ainali et al., 2012) to KLK8 protein levels and activity. We link KLK8-mediated induction of hyperkeratosis in inflamed mouse skin to psoriasis skin disease in humans, and provide a possible explanation for KLK8 epidermal overexpression and induction of hyperkeratosis in psoriatic lesions.

The mechanisms by which KLK8 can cause a thickened epidermis may involve inhibition of AP- 2α protein expression resulting in keratinocyte hyperproliferation, induction of IL6-mediated keratinoytes growth and differentiation, or increased proteolytic degradation of structures responsible for basal cell cohesion in the basement membrane such as collagen IV, as discussed in chapter 3. Further studies are required to dissect KLK8 signaling mechanisms in psoriasis. For instance, our data pointed a dramatic KLK8 overexpression by dermal cells in psoriatic, but not atopic dermatitis, lesions compared to matched non-lesional psoriatic and normal skin. However, the identity of these cells remains unclear. Potential candidates include T17-cells and/or neutrophils known to secrete serine proteases. KLK8 was previously localized in mast cells of mouse skin (Wong et al., 2003) but was not studied in any other immune cell types. Virtually all mast cells in the skin are known to secrete tryptase and IL17A-positive mast cells and neutrophils are found in high levels at sites of skin and joint disease in humans (Kirkham et al., 2013). Thus, it is possible that immune cells express KLK8 in psoriatic lesions. Interestingly, we noted KLK8 overexpression in synovial fluids of psoriatic arthritis patients, suggesting a possibly similar TNFα and IL17A regulation of KLK8 expression in the skin and joint of psoriatic patients.

This thesis also highlights KLK8 potential as a therapeutic target for psoriasis topical treatment. We show that current TNFα and IL17A blocking biologic therapies used to treat psoriasis in the clinic reduce KLK8 expression in the skin and serum significantly. However, immune-based therapies are often used intermittently due to their tendency to cause immunosuppression in psoriatic patients. Thus, hampering KLK8-specific activity in the skin surface of psoriatic lesions may be a beneficial alternative for psoriasis patients to use as a topical agent during the intermittent halt periods of TNFα and IL17A immune-blocking therapeutic use. The KLK8- specific small inhibitors we identified in Chapter 5 represent the starting points for future studies aiming at developing and fine-tuning KLK8-inhibitors as topical psoriasis therapeutic targets.

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Figure 5.5. KLK8 in normal and psoriatic skin. KLK8 is secreted by granular keratinocytes in normal upper skin epidermis as indicated by the packman. (1) KLK8 is an active trypsin-like serine protease in stratum corneum extracellular environment. (2-3) In psoriatic skin, the abundance of immune cells secreting TNFα and IL17A induces KLK8 hypersecretion (and increased trypsin-like activity) by keratinocytes in lower layers. KLK8 protein overexpression correlates with the accelerated keratinocyte differentiation program, epidermal scaling, area and severity of skin lesions in psoriasis patients. Inhibition of TNFα and IL17A (by biologic drugs, such as etanercept which inhibits TNFα-binding to its receptor), reduces psoriasis scaling and KLK8 expression. (4) Future studies should investigate the effect of topical application of KLK8-specific inhibitors to lesional psoriatic skin as an adjuvant and/or an alternative to the use of TNFα and IL17A-blockers. Figure is adapted by permission from Macmillan Publishers Ltd: Nature (Crow, 2012), copyright (2012).

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5.5 Future Directions

Our knowledge of KLK8 activity and functionality in normal human skin and psoriasis is emerging. This work has contributed to a significant improvement in the understanding of KLK8 characteristics and functions in normal and psoriatic skin, and has identified KLK8 as an attractive therapeutic target in psoriasis. Despite considerable progress, many important questions remain elusive. For instance, what are the physiological substrate targets of KLK8 in normal and psoriatic skin?? It is clear that this protease is unique compared to other KLKs in terms of its PAR-signaling and inhibition properties. Thus, future directions should apply unbiased advanced quantitative mass-spectrometry-based proteomic methods, in the field of ‘degradomics’, to identify natural KLK8-specific skin substrates and pathways, in comparison to KLK5, KLK7 and KLK14. A recent approach known as terminal amine isotopic labeling of substrates (TAILS) was developed to identify natural substrates of orphan proteases in tissues and conditioned cell culture media. The recombinant human KLK proteases produced in this thesis are available reagents that can be used to treat cultured keratinocytes and 3D skin equivalents, prior to applying the TAILS protocol to identify KLK substrates by differential display, as described previously (Kleifeld et al, 2010).

Seminal research in the KLK world focused on their roles as epidermal proteases, but limited consideration was given to KLKs as immune proteases. Future research should characterize KLK protein expression by immune cells in skin diseases and investigate their in vivo roles as innate immune proteases. In the case of KLK8, immunofluorescence and flow cytometry studies investigating KLK8 co-localization with immune cell markers in psoriatic lesions and sera are warranted.

Finally, in the drug development arena, more research is needed in terms of fine-tunning the small molecule compounds we identified here as KLK8 inhibitors. When the peptide inhibitor leupeptin is docked into KLK5 and KLK8 active site pockets, these two trypsin-like proteases exhibited different electorstatic potentials in their active site pockets, as shown in Appendix Figure 5.1. Thus, molecular docking studies may reveal subtle differences between trypsin-like KLK5 and KLK8. Furthermore, biochemical kinetic analyses of the inhibition mechanisms of the top-identified KLK8 inhibitors are underway. Inhibitors’ specificity and selectivity towards other epidermal proteases will also be investigated, prior to characterizing the inhibitor’s biological

149 effect in normal and psoriatic 3D skin epidermis models. The KLK8 recombinant proteases and KLK8-immunocapture activity assays designed in this thesis are valuable biochemical tools to test inhibitors’ potency in inhibiting physiological KLK8 activity in SC tissue extracts and sweat.

In conclusion, this work raises several important questions, such as what is the natural inhibitor of KLK8 activity in the skin? What are the downstream targets of KLK8 and which in vivo proteolytic cascades is it involved in? What are KLK8 epidermal and immune functions? How will the structural information brought by the recent crystallisation of the protein help in understanding its function and in fine-tunning inhibitors? Inhibitors of active skin-surface KLKs are attractive targets for development of cosmetic agents that improve normal barrier function and targeted topical therapies of chronic skin diseases that evade systemic immunosuppression. The future of KLK8 dermatological research is promising with specific and intriguing basic questions to explore and clinical applications to unravel.

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Appendices

Appendix Table 2.1. Steady-state kinetic parameters for the hydrolysis of synthetic AMC substrates by mat-KLK8 in optimal activity buffer (100 mM Na2HPO4 pH 8.5)

Substrate Vmax a Km kcat kcat/Km Normalized Activity c (µmol/min/ (mM) (min-1) ( mM-1 min-1) (%) mg) Trypsin-like Boc-VPR-AMC 13.08 0.0238 ± 405.38 17004.31 100 0.025 Boc-QAR-AMC 11.14 0.1844 ± 345.24 1872.253 11 0.009 H-PFR-AMC 8.610 0.1533 ± 266.89 1740.994 10 0.017 Boc-QRR-AMC 6.566 0.2247 ± 203.53 905.7995 5.0 0.017 Boc-LRR-AMC 3.136 0.1165 ± 97.216 834.4745 4.9 0.004 Boc-FSR-AMC 6.903 0.2983 ± 213.98 717.3365 4.2 0.071 Boc-LKR-AMC 3.028 0.1407 ± 93.881 667.2431 3.9 0.017 Boc-VLK-AMC 7.039 0.3326 ± 218.21 656.0810 3.8 0.023 Boc-QGR-AMC 2.766 0.3699 ± 85.751 231.8222 1.4 0.018 Benzlyoxy-GGR-AMC 2.881 0.4312 ± 89.307 207.1133 1.2 0.060 Tos-GPR-AMC 4.076 0.6897 ± 126.36 183.2210 1.0 0.125 Tos-GPK-AMC NR b NR b Boc-EKK-AMC NR b NR b Chymotrypsin-like AAPF-AMC NR b NR b LLVY-AMC NR b NR b Negative Control AAPV-AMC NR b NR b a Specific activity, Vmax (µmol/min/mg) = [Adjusted Vmax (FU/min) x Calibration factor (µmol AMC/FU)] ÷ Amount of enzyme added (mg) b NR indicates no reaction c Normalized activity = Kcat/Km (substrate) ÷ Kcat/Km (Boc-VPR-AMC) × 100

173

Appendix Table 3.1. Demographics and disease characteristics of psoriasis patients pre and post-treatment

Characteristics of patients at study entry Total number of patients

(Pre-treatment sample 1) (n = 60)

Males/ Females 42 (70.0%)/18 (30.0%)

Age (years) 47.2 (11.7)

Age at diagnosis of Psoriasis (years) 27.5 (12.4)

Age at diagnosis of PsA (years) 33.3 (11.2)

Psoriasis duration (years) 20.4 (12.4)

PsA duration (years) 14.6 (9.7)

Actively inflamed joint count 10.5 (9.9)

Swollen joint count 3.7 (4.6)

PASI score 6.5 (9.4)

Mean duration from Sample1 to Sample2 1.49 (0.76)

174

Appendix Table 3.2. KLK serum levels pre and post psoriasis treatment with TNFα- blockers

Variable Mean Sd Reduction P-value Reduction

KLK5 Reduction -0.075 0.336 0.1672

KLK6 Reduction 0.1228 0.896 0.3916

KLK7 Reduction 0.2312 3.197 0.6500

KLK8 Reduction 1.629 4.484 0.0066

KLK10 Reduction 0.0471 0.7011 0.6736

KLK11 Reduction 0.0345 0.1479 0.1483

KLK13 Reduction -0.0455 0.291 0.3289

hsCRP Reduction 9.543 16.717 <0.0001

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KLK5 KLK8

Appendix Figure 5.1. Differences in KLK5 and KLK8 active site pockets. Docking of the general serine protease inhibitor leupeptin into active site pockets is shown indicating orientation differences. Electrostatic potential (ESP) calculated on the surface of the catalytic site of trypsin- like KLK5 and KLK8 clearly displays differences in the active site pocket.