Roles of Kallikrein-Related Peptidase in Epidermal Barrier Function and Related Skin Diseases

Jiao-Quan Chen, Bi-Huang Liang, Hua-Ping Li, Zi-Yin Mo, Hui-Lan Zhu,?

1Institute of Dermatology, Guangzhou Medical University, Guangzhou, Guangdong 510095, China, 2Department of Dermatology,Guangzhou Institute of Dermatology, Guangzhou, Guangdong 510095, China.

Introduction

The skin barrier usually refers to the physical barrier formed by the cuticle and sebum membrane of the skin.This barrier not only protects against the onslaught of microorganisms and percutaneous penetration of chemicals and allergens but also prevents the loss of nutrients and moisture from the body.Corneodesmosomes play an important role in maintaining the function of the keraphyllous skin barrier. Desmogleins, desmocollin,and corneodesmosin are examples of corneodesmosomes.The breakdown of these proteins causes the degradation of corneodesmosomes, leading to keratinocyte exfoliation.Kallikrein-related peptidases (KLKs) are serine proteases secreted from granular keratinocytes to the upper granular layer and cuticular space,and they play important roles in the process of keratinocyte exfoliation.1By degrading corneodesmosomes,KLK5,KLK7,and KLK14 can cause keratinocyte exfoliation.2-3KLK8 may also participate in de-keratinization by cooperating with other kallikreins.4Furthermore, the expression of KLKs is regulated by protease inhibitors, including serine protease inhibitors,lymphoepithelial Kazal-type inhibitor (LEKTI), serine protease inhibitor Kazal-type 6 (SPINK6), and SPINK9.Moreover,abnormal expression of KLKs may destroy the skin barrier homeostasis, causing a range of skin diseases such as Netherton syndrome(NS),atopic dermatitis(AD),rosacea, and psoriasis.

Expression and physiological activity of KLKs in skin

KLK5,KLK7,KLK8,and KLK14 are mainly found in the epidermis and the appendages of the skin (Table 1).5-8Both active KLK and inactive pro-KLK can be detected in the skin. The enzyme cascade of proteases plays a dominant role in protein activation.Previous studies have indicated that members of the KLK family are involved in the protease cascade. The relationships among KLK5,KLK7, KLK8, and KLK14 are shown in Figure 1.

As an inert precursor produced by keratinocytes, pro-KLK5 can be activated by itself or by proteolytic enzymes and KLK14. Miyaiet al.9also found that trypsin can activate both pro-KLK5 and pro-KLK7. Because KLK5 can activate other KLKs(pro-KLK7,pro-KLK8,and pro-KLK14), it is considered to be the initiator of the KLK activation cascade.1Yoonet al.10conducted pro-KLK activation experiments and found that pro-KLK14 was activated by KLK11. Previous report has shown that besides KLK5, pro-KLK8 can also be activated by lysine endopeptidase.KLK8 has also been found to participate in the enzyme cascade by activating KLK11.11

KLKs participate in homeostatic regulation of the skin barrier function through their involvement in various physiological activities of the skin, including skin desquamation, the skin permeability barrier function,and inflammation regulation.

Skin desquamation

The self-activation of pro-KLK5 is considered a downstream proteolytic cascade activator. Through its selfactivation,pro-KLK5 also activates KLK7 and KLK14 and degrades desmogleins 1 and 4 and corneodesmosin,causing skin desquamation.12However, the participation of KLK8 and mKLK8 in this process has two possible explanations: KLK8 and mKLK8 may be involved by activating KLK6 and KLK7 or by activating KLK11,which further activates KLK14.11

Permeability barrier function

KLKs can disturb the formation of the lamellar membrane by regulating the degradation of lipid-forming enzymes inthe cuticle. KLKs can also interfere with the secretion of components such as ceramides, thus destroying the function of skin as the permeability barrier. Filaggrin,also a source of ceramides, is essential for skin hydration and barrier function. After observing a high level of profilaggrin in human epidermal keratinocytes by knocking out KLK5, Sakabeet al.8reported that KLK5 plays a significant role in the formation of filaggrin. KLK7 is a physiological activator of caspase 14,which is involved in the first step of the filaggrin degradation pathway. Thus,defects in the procaspase 14 maturation pathway may lead to downregulation of natural moisturizing factors,including ceramides.13Studies have shown that an increase in the pH of the skin may elevate the expression of KLK5 and KLK7. The latter affects the function of the lamella and lamellar membrane by regulating protease-activated receptor 2(PAR2),thus affecting the permeability barrier of the skin.14By comparing wound healing of wild-type andKLK8-gene-disrupted mice,Kishibeet al.15found that KLK8 is involved in the late phase of wound healing by upregulating KLK6 and activating PAR2. However, the mechanisms of KLK6 upregulation and PAR2 activation remain unclear.

Table 1 Distribution of KLK5, KLK7, KLK8, and KLK14 in skin

Inflammation regulation

Cathepsin inhibitors belong to the main family of antimicrobialpeptidesexpressed by keratinocytes. LL-37,an active form of a cathepsin inhibitor, can cause accumulation of inflammatory cells.Increases in Toll-like receptor 2(TLR-2)and matrix metalloproteinases (MMPs) by various factors will lead to the upregulation of both KLK5 and KLK7,which will in turn increase the expression of LL-37,leading to an inflammatory reaction of host cells.16By irradiating human epidermal keratinocytes and rosacea-like mouse skin with light-emitting diodes of 630 and 940nm, respectively, Leeet al.17discovered that the expression of both KLK5 and LL-37 was downregulated. Thibautet al.18subsequently evaluated the ability of ivermectin to inhibit expression of theKLK5gene in the epidermis and reported that ivermectin could prevent the rosacea-associated inflammation caused by the abnormal LL-37 processing.

Figure 1. Interrelation and function of KLK5, KLK7, KLK8, and KLK14. KLK: Kallikrein-related peptidases; DSG: desmoglein; CDSN:corneodesmosin; PRA2: protease-activated receptor 2; TLR-2: Toll-like receptor-2; MMPs: matrix metalloproteinases; IL-37: interleukin-37.

KLKs and skin diseases

Netherton syndrome

NS is caused by a mutation in theSPINK5gene, which encodes LEKTI.LEKTI deficiency leads to activation of the protein hydrolysis cascade mediated by KLKs, causing corneodesmosome degradation and accelerated skin shedding. LEKTI deficiency also induces inflammation and atopic lesions by activating PAR219and elevating the expression of cathepsin.Abnormal expression of KLK5 or KLK7 is considered one of the main pathological causes of NS. One study showed that the proteolytic activity of KLK5 and KLK7 increased in SPINK5-deficient mice,corresponding to the increase in trypsin and chymotrypsin activity described in patients with NS.20In cultures of SPINK-deficient skin, knockout of KLK5 and KLK7 caused the cuticle to bind the lower epidermis more firmly or restore the thickness of the epidermis.21Therefore,inhibition of protease activity(KLK5 or KLK7)may be an effective treatment for NS. Interestingly, in 2017,Kaspareket al.22used a set of mouse models individually or simultaneously deficient in KLK5 and KLK7 and found that the defective skin barrier was only partially rescued in individuals with inactivation of only KLK5 or KLK7. In contrast,lethality could only be rescued when both KLK5 and KLK7 were simultaneously missing completely,allowing adult mice to survive to adulthood with a fully functional skin barrier.

Atopic dermatitis

KLKs are involved in exfoliation of the cuticle, and the expressionofKLKsisupregulatedinpatientswithAD.KLK7 is highly expressed in the damaged skin of patients with AD.23Igawaet al.24analyzed the protease activity on the extracellular surface and found that although KLK7 was overexpressed in the extracellular space, it was not completely activated.The main pathway for KLK5 to induce dermatitis is to activate inflammatory factors through the KLK5-PAR2-thymic stromal lymphopoietin-T helper 2 cell pathway. However, Zhuet al.2raised questions about this pathway,because they found that the AD-like skin structure induced by persistent activation of KLK5 was not dependent on PAR2 activity.This might be explained by the tolerance to PAR2 after long-term stimulation.In summary,regulation of the expression of KLK5 and KLK7 represents a potential treatment strategy for AD.

Rosacea

The pathogenesis of rosacea is not fully understood,but genetics, immune factors, neurovascular disorders,microbes,and environmental factors have been considered to play a role.25High levels of expression of cathepsin inhibitors and KLK5 have been detected in rosacea lesions.26Additionally, TLR-2 and MMPs are highly expressed in rosacea lesions. This increase in TLR-2 and MMP expression,which may be caused by various factors,leads to the upregulation of KLK5 expression,causing high expression of LL-37 and thus leading to the degranulation of cells and the release of inflammatory mediators.16Lightemitting diode therapy, pulsed laser therapy, ivermectin,and other treatments are currently used to downregulate the expression of KLK5 and LL-37 and thus help cure rosacea.

Psoriasis

Psoriasis is considered a multifactorial skin lesion, and KLKs are assumed to be involved in the pathogenesis of psoriasis.Komatsuet al.27detected the expression of KLKs in patients with psoriasis and normal controls. The expression of KLK7, KLK8, and KLK14 in keratinocytes was found to be significantly increased in psoriatic lesions.KLK7 and KLK8 are involved in the pathogenesis of psoriasis by activating interleukin-1β and inhibiting transcription factor activator protein-2α. About 30% of patients with psoriasis have arthritis. Eissaet al.28found that KLK8 was highly expressed in skin lesion in patients with psoriasis and psoriatic arthritis.However,an increase in the KLK8 level only reflects the activity of skin disease,not the activity of joint disease.

KLK inhibitors

Since KLKs are active in skin, their inhibitors will be the most effective strategies for management of skin barrier diseases caused by abnormal expression of KLKs. The KLK inhibitors that have been the most thoroughly studied to date are those related to the inhibition of KLK5,KLK7,KLK8, and KLK14 (Table 2).

Irreversible inhibitors

Serine protease inhibitors (SERPINs) are the main irreversible inhibitors of KLKs, and they irreversibly destroy the protein activity of most KLKs by placing their reaction ring into the active site of KLKs.The inhibition of KLKs by SERPINs was summarized by Goettig29and Swedberget al.30as follows:SERPINA1,SERPINA3(β1-chymotrypsin), and SERPINA4 (follistatin) can inhibit KLK7 and KLK14.SERPINA5,protease C inhibitor,and SERPINF2 (α2-antifibrinolytic enzyme) are stronger inhibitors of KLK5, KLK7, KLK8, and KLK14. SERPINE1 (plasminogen activator inhibitor 1) can inhibit KLK14.31A later study showed that SERPINA12(vaspin)could inhibit KLK7 and KLK14.32-33

Reversible inhibitors

Zinc ion

The zinc ion (Zn2+) can be combined with KLKs to regulate its proteolysis function, and the combination isreversible. Zn2+can inhibit KLK5, KLK7, KLK8, and KLK14; additionally,Zn2+and LEKTI,a serine protease inhibitor, are involved in the physiological regulation of epidermal KLKs.31One study showed that when KLKs were mixed with Zn2+at a proportion of 1:10,the rate of inhibition of KLK5 and KLK14 by Zn2+were 97.5%and 95.0%,respectively,while that of KLK8 was 0.0%.11

Table 2 Protein inhibitors classification

LEKTI

LEKTI is a reversible inhibitor that comprises 1,064 amino acids.It is encoded by theSPINK5gene and can be divided into 15 serine protease inhibitory regions (D1-D15).Different inhibitory regions of LEKTI impose different degrees of inhibitory effects on KLK5, KLK7, KLK8, and KLK14.34Table 2 shows that the inhibition ability of LEKTI fragment and LEKTI domain are not necessarily related to the number of domains contained in each fragment. Moreover, Fortugnoet al.35identified a 420K LEKTI variant associated with AD in 2012 and discovered that this variant can increase the activity of KLK5,KLK7,and elastase 2 in epidermal models in vitro. Miyaiet al.9subsequently reported that by adding active mesotrypsin into a mixture of KLK5 and LEKTI,the activity of KLK5 gradually recovered; the same result was obtained using KLK7.

LEKTI-2

LEKTI-2,encoded by SPINK9 and known as SPINK9,has long been thought to be predominantly composed of normal granular layers on the soles of the hands and feet.Notably,Redelfset al.36found that SPINK9 was expressed not only in normal palmoplantar skin but also in skin affected by diseases such as lichen simplex chronicus,photokeratosis, and squamous cell carcinoma. SPINK9 can selectively inhibit KLK5,and this effect is strengthened in an acidic environment.37

SPINK6

Both SPINK6 and SPINK9 have a typical Kazal domain.SPINK6 is found in the granular layer, sebaceous glands,sweat glands, and other appendages of normal human skin. However, the expression of SPINK6 is decreased in AD.38The inhibitory function of SPINK6 on the KLK family is inconsistent and can be divided into high inhibition (KLK5 and KLK14) and low inhibition(KLK7)39; however, human KLK8 is not inhibited by SPINK6.38

Other inhibitors

Sunflower trypsin inhibitor 1 (SFTI-1), a serine protease inhibitor extracted from sunflower seeds, can inhibit the activity of KLK7.40Shariffet al.41produced a stronger inhibitor of KLK5 by substituting histidine for glycine at the 10th position of the natural SFTI-1 sequence. By substituting and ranking the SFTI-1 sequence again, the authors found analog 6,which can serve as a substitute for STFI-1. They also discovered that analog 6 can inhibit KLK5,KLK7,KLK8,and KLK14.Moreover,SFTI-1 can delay the secretion of interleukin-8 and completely inhibit the KLK5-induced dependent intracellular calcium mobilization caused by PAR2.42

Summary

KLKs, especially KLK5, KLK7, KLK8, and KLK14, are proteases that play a critical role in the physiological and pathological processes of the skin barrier. Abnormal expression of KLKs may cause various skin diseases,including NS, AD, rosacea, and psoriasis. The recently discovered KLK inhibitors are expected to be among the most effective strategies for management of skin barrier diseases. Further research on the mechanism of KLKs is needed for additional developments in disease diagnosis,monitoring of prognosis,and new treatment methods such as KLK-targeted vaccines, immunotherapy, and KLKactivated precursor medications.