Introduction

The skin forms a protective barrier not only by means of constituting an efficient physico-chemical barrier but also via its highly active and specialized participation in immune and inflammatory reactions. The major cellular constituent of the epidermis, the keratinocyte, has been described as a "cytokine factory" and plays an important role in our immune defence (reviewed by Steinhoff et al., 2001). A new concept in skin immunology is that proteolytic enzymes may act as key modulators of biological functions in a cytokine-like manner. Endogenous or exogenous proteinases may influence cells through the activation of proteinase-activated receptors (PARs) (reviewed by Steinhoff et al., 2005).

Proteinase-activated receptor-2 (PAR₂) is a member of the PAR family, which encompasses four PARs, designated PAR1–4 (Vu et al., 1991; Nystedt et al., 1995; Ishihara et al., 1997; Xu et al., 1998). These are G-protein-coupled receptors with seven transmembrane domains, which are activated by a unique mechanism dependent on proteolytic cleavage. Upon cleavage within the N-terminal extracellular part of the receptor, a peptide is released that binds to and irreversibly activates the receptor while still tethered to it. Activation leads to interaction with heterotrimeric G-proteins in the plasma membrane and downstream signalling events. Proteinases capable of activating PARs belong to the serine protease family and include thrombin, trypsin, and cathepsin G. PAR₂ is not activated by thrombin but has been shown to be activated by the following proteases in vitro: trypsin, mast cell tryptase, factor Xa, acrosin, gingipain, and neuronal serine proteases (reviewed by Dery et al., 1998; Macfarlane et al., 2001; Ossovskaya and Bunnett, 2004; Steinhoff et al., 2005). PAR₂ is expressed by keratinocytes (Santulli et al., 1995; D'Andrea et al., 1998; Steinhoff et al., 1999) where it may function as a regulator of growth and differentiation (Derian et al., 1997) and is highly implicated in inflammatory reactions (Steinhoff et al., 2005). Recently, PAR₂ was identified as a novel signalling mechanism of the epidermal permeability barrier (Hachem et al., 2006). The endogenous activator of PAR₂ in the epidermis, however, has not been identified yet. It has been suggested that mast cell tryptase may activate PAR₂ during inflammatory conditions upon mast cell infiltration and degranulation (Steinhoff et al., 1999). Other possibilities include endogenous trypsin-like proteases present in the epidermis.

We have previously purified, cloned, and characterized three epidermal serine proteases; human kallikrein-related peptidase (KLK) 5 (hK5, stratum corneum tryptic enzyme, SCTE), KLK7 (hK7, stratum corneum chymotryptic enzyme, SCCE), and KLK14 (hK14) (Hansson et al., 1994; Brattsand and Egelrud, 1999; Brattsand et al., 2005; Stefansson et al., 2006). KLK8 is another human KLK, which is abundantly present in the stratum corneum (Komatsu et al., 2005a). These four enzymes all belong to the human kallikrein gene family of 15 related serine proteases (Clements et al., 2001; Yousef and Diamandis, 2001) and have different substrate specificities. KLK5 and KLK8 have trypsin-like activity (Brattsand et al., 2005; Rajapakse et al., 2005), whereas KLK7 has chymotrypsin-like activity (Skytt et al., 1995). KLK14 has mainly trypsin-like but also a significant chymotrypsin-like activity (Brattsand et al., 2005; Felber et al., 2005). These enzymes are here denoted as human kallikrein-related peptidases according to the new nomenclature proposed by Lundwall et al. (2006).

Given the presence of both PAR₂ and KLKs in the epidermis, it is plausible that one or several KLKs may function as activators of PAR₂ and thus play a role in inflammatory skin diseases. In this work, we investigated whether KLKs 5, 7, 8, or 14 may be capable of activating PAR₂ in vitro. We selected KLKs 5, 7, and 14 because these enzymes are known to be present in the skin in catalytically active form (Egelrud, 1993; Brattsand and Egelrud, 1999; Stefansson et al., 2006). KLK8 may, besides KLK11, be the most abundant trypsin-like KLK in normal human skin (Komatsu et al., 2005a). We show that KLK5 and KLK14, but neither KLK7 nor KLK8, could activate PAR₂. We also show the colocalization of KLK14 and PAR₂ in the epidermis of inflammatory skin disorders. Thus, certain but not all KLKs expressed by keratinocytes are potential endogenous activators of PAR₂ within the epidermis, and may play a role in epidermal barrier physiology/pathophysiology and itching.

Results

Production and activation of recombinant KLKs

Production of active KLK5 and KLK14 has been published elsewhere (Brattsand et al., 2005). Purified recombinant pro^EK-KLK7 could, as expected, be activated by recombinant enterokinase (rEK). Recombinant pro-KLK8, having the native propeptide sequence, was also easily activated by rEK. Activation was verified by a mobility shift on SDS-PAGE (Figure 1) and by measuring activity toward chromogenic peptide substrates. Active KLK8 fusion protein had an apparent molecular mass of 38.6 kDa according to SDS-PAGE analysis (Figure 1). Active-site titration of enzyme preparations showed that around 30% (KLK7^EK) and 60% (KLK5^EK) of the respective enzyme was active when compared with expected activity according to protein concentration determined with the DC Protein Assay. Close to 100% of the KLK14 preparation was in active form. In Figures 2 and 3 all KLK8 was assumed to be in active form. However, as KLK8 does not react with ₁-antitrypsin, it was not possible to perform active-site titration of the enzyme in the same way as for KLKs 5, 7, and 14, but taking into account the proportion of active enzyme in the other preparations, and the fact that electrophoretic analyses showed no or negligible degradation of KLK8 (Figure 1), an assumption was made that at least 30% of the KLK8 preparation should be functional. The active KLK8 preparation also showed high activity toward chromogenic peptide substrates (see below).

Figure 1.

Preparations of recombinant human tissue KLK protein precursors and active enzymes. Coomassie brilliant blue stained SDS-PAGE.

Full figure and legend (32K)

Figure 2.

Ca²⁺ responses to trypsin and KLK in KNRK-PAR2 cells. KNRK-PAR₂ cells were treated with (a) 1 M trypsin (T), (b) 10 M KLK5^EK, (c) 10 M KLK7^EK, (d) 10 M KLK8, and (e) 1 M KLK14. Trypsin or KLK was added at indicated time points (arrows). If no response could be detected, the integrity of the cells was verified by addition of 100 nM trypsin (T).

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Figure 3.

Dose response curves for Ca²⁺ mobilization in response to trypsin and KLK. (a) KNRK-PAR₂ cells were treated with active trypsin (circles), KLK5^EK (triangles), KLK7^EK (crosses), KLK8 (diamonds) or KLK14 (squares). MeansSEM, n3 for trypsin, KLK5^EK, and KLK14. For KLK7^EK and KLK8, experiments were carried out in duplicate at 1 M, at lower concentrations single experiments were performed. ^*1: statistically significantly different from baseline (n=3, P=0.04); ^*2: statistically significantly different from baseline (n=4, P=0.003). (b) Human dermal microvascular endothelial cells (hDMEC) cells were treated with active trypsin (circles), KLK5^EK (triangles), or KLK14 (squares) with concentrations as indicated in the figure. 100% Ca²⁺ corresponds to fluorescence ratio 340/380 nm=2.

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Effects of KLKs 5, 7, 8, and 14 on Ca²⁺ mobilization

Trypsin, KLK5^EK, and KLK14 stimulated Ca²⁺ mobilization in Kirsten Murine Sarcoma Virus transformed rat kidney epithelial (KNRK)-PAR₂ cells (Figure 2a, b, and e), interpreted as PAR₂ signalling. No reaction was obtained when KLK7^EK or KLK8, in concentrations from 0.1 nM up to 10 M, was used (Figure 2c and d). However, subsequent addition of 100 nM trypsin to the same cells gave an expected reaction, confirming the integrity of cells (Figure 2c and d). Addition of rEK in concentrations similar or higher to that present in preparations of KLK5^EK, KLK7^EK, and KLK8 did not lead to any detectable Ca²⁺ mobilization (data not shown). Figure 3a shows dose response curves. Both trypsin and KLK14 were far more potent activators of PAR₂ than KLK5^EK. The lowest detectable signal was obtained at 50 nM concentration for KLK14. KLK5^EK gave a detectable signal at 0.1 M. Even at 1 M, the signal obtained with KLK5 was lower than that for KLK14 at 50 nM. Neither KLK7 nor KLK8 could activate PAR₂. The ability of KLK5 and KLK14 to induce Ca²⁺ mobilization in cells via the PAR₂ signal system was confirmed using human dermal microvascular endothelial cells naturally expressing functional PAR₂ receptors (Figure 3b; Shpacovitch et al., 2002).

Immunofluorescence analyses

By using antibodies to the Flag and the HA11 epitopes of the PAR₂ receptor, we were able to discriminate between intact and cleaved PAR₂. The antibody to the extracellular Flag would detect only uncleaved receptor, because proteolytic cleavage removes the proximal Flag epitope. Results from immunofluorescence analyses (Figure 4) were in accordance with the results obtained in Ca²⁺ mobilization assays. After treatment with 1 M recombinant KLK5^EK or KLK14, no staining of the Flag epitope could be detected around the cell membrane, indicating cleavage and loss of the extracellular part of the PAR₂ receptor. Treatment with 1 M KLK7^EK or KLK8 did not abolish staining of the Flag epitope, that is, with these enzymes results similar to the negative control with cells treated with vehicle only were obtained. rEK treatment did not have any effect on immunofluorescence patterns (results not shown).

Figure 4.

Confocal photomicrographs of KNRK-PAR2 cells stimulated with trypsin or KLK. KNRK-PAR₂ cells were stimulated with KLK5^EK, KLK7^EK, KLK8, KLK14, or trypsin, all at 1 M. Negative control shows cells incubated in DMEM/0.1% BSA only. Cells incubated with rEK looked like cells in negative control (data not shown). Cells were stained with anti-Flag antibody toward extracellular Flag epitope and anti-HA antibody toward intracellular HA11 epitope. Merged: superimposition of images in the same row. Yellow color in superimposed images indicates no degradation of extracellular parts of PAR₂.

Full figure and legend (243K)

Kinetic properties of KLK8

Despite its trypsin-like properties, KLK8 did not give any detectable PAR₂ activation. Therefore, we wanted to test this enzyme against a series of chromogenic peptide substrates and compare its kinetic properties to that of KLK14. The results with the chromogenic peptide substrates S-2251, S-2288, and S-2302 (see Materials and Methods for structures) are shown in Table 1. KLK8 had a catalytic rate (k_cat) and catalytic efficiency (k_cat/K_m) 3–5 times lower than that of KLK14 for S-2288 and S-2302. Whereas S-2251 was readily cleaved by KLK8, it was not cleaved at detectable rates by KLK14.

Table 1 - Kinetic parameters of recombinant human KLK8 and KLK14.

Full table

Immunolocalization of KLK14 and PAR₂ in inflammatory skin disorders

Staining of inflamed skin from patients with atopic dermatitis and rosacea shows immunoreactivity of both KLK14 and PAR₂ widely distributed in the upper squamous and granular layers (Figure 5).

Figure 5.

Immunoreactivity of KLK14 and PAR2 in inflamed skin tissues. Immunoreactivity of KLK14 as well as PAR₂ was found widely distributed in the upper squamous and granular layer in patients with inflammatory skin disorders. (a–d) Atopic dermatitis, (e–h) rosacea. Staining was performed with antisera specific for (a, b, e, f) KLK14 or (c, g) PAR₂. (d, h) Negative controls. Inset size bars=50 M (b, f) or 100 M.

Full figure and legend (324K)

Discussion

PAR₂ has been implicated in immunological and inflammatory responses of many tissues. This receptor may contribute to the pathophysiology of inflammatory skin diseases such as atopic dermatitis and psoriasis (Steinhoff et al., 1999, 2005). It has also been depicted as a novel pathway for pruritus in human skin (Steinhoff et al., 2003) and for events linked to lamellar body secretion in response to epidermal barrier injuries (Hachem et al., 2006). The nature of the proteinases which activate PAR₂ in the skin under physiological and pathophysiological conditions is, however, still uncertain. Therefore, the aim of this study was to identify possible endogenous activators of PAR₂ in the epidermis. Candidates were four serine proteinases within the family of KLKs. Several KLKs have been shown to be produced by epidermal cells (Sondell et al., 1994; Brattsand and Egelrud, 1999; Komatsu et al., 2003, 2005a, 2005b; Stefansson et al., 2006). In this work, we focused on KLKs 5, 7, 8, and 14. So far, epidermal KLKs 5 and 7 have been predominantly implicated in the process of desquamation (Egelrud and Lundström, 1991; Lundström and Egelrud, 1991; Egelrud, 1993; Ekholm et al., 2000; Caubet et al., 2004). However, similar as PAR₂, both KLKs 5 and 7 show the highest expression in the stratum granulosum, suggesting a possible interaction between these proteinases and PAR₂ (Sondell et al., 1994; Steinhoff et al., 1999; Ekholm et al 2000). In this context, also KLK14 may be of interest. In normal skin, the highest expression of this enzyme is seen in sweat ducts (Stefansson et al., 2006). It exhibits trypsin-like specificity and a high catalytic efficiency (Brattsand et al., 2005). Thus, KLK14 may also be associated with PAR₂ activation via a paracrine mechanism. KLK8, another trypsin-like KLK (Rajapakse et al., 2005), is abundantly present in the stratum corneum (Komatsu et al., 2005a) and may therefore also be a possible activator of PAR₂ in the epidermis.

In the dermis, a likely activator of PAR₂ is mast cell tryptase. Steinhoff et al. (1999) showed that tryptase stimulated a rapid increase of intracellular calcium concentration in cultured human keratinocytes. Mast cell tryptase may constitute as much as 20% of the total protein content of mast cells. Under inflammatory conditions such as atopic dermatitis, the number of mast cells in the dermis is dramatically increased, and mast cells can be found close to the dermal–epidermal border. Also in psoriasis, there is an increased number of dermal mast cells, sometimes apparently invading the epidermis (Steinhoff et al., 1999). Considering the fact that keratinocytes in high suprabasal epidermal layers show the highest expression of PAR₂ under normal conditions (Steinhoff et al., 1999), it seems less likely, however, that tryptase would be the only PAR₂-activating enzyme in the skin. Besides mast cell tryptase, another possible activator of PAR₂ in the epidermis is trypsin IV (Cottrell et al., 2004). Trypsin IV generated by keratinocytes in human skin can activate PAR₂ in vitro (M Steinhoff, unpublished observation). Cottrell et al. (2004) showed that trypsin IV is able to activate PAR₂ in KNRK-PAR₂ cells as well as in the epithelial cell lines PC-3 (prostate) and SW480 (colon). These results were, however, questioned by Grishina et al. (2005), who claimed that trypsin IV cannot activate PAR₂ in epithelial cells. The opposing results may be due to different glycosylation or transactivation mechanisms (Hollenberg and Compton, 2002).

In normal skin, PAR₂ is highly expressed by keratinocytes. The highest levels can be found in the stratum granulosum. PAR₂ is also localized in keratinocytes of hair follicles and sebaceous glands, as well as endothelial cells, myoepithelial cells of sweat glands, and dermal dendritic cells (Steinhoff et al., 1999). Under inflammatory conditions, staining for PAR₂ can also be observed in primary afferent nerve fibers (Steinhoff et al., 2000, 2003). In atopic dermatitis, differing from normal conditions and psoriasis, PAR₂ is expressed also by keratinocytes in lower epidermal layers (Steinhoff et al., 1999, 2003). In this work, we show that KLK14 and the PAR₂ receptor are coexpressed in the upper squamous and granular layers of inflammatory skin (Figure 5).

We found that KLK5 and KLK14, but not KLK7 nor KLK8, were able to activate PAR₂. The positive results of KLK5 and KLK14 were confirmed in a recent study of Oikonomopoulou et al. (2006), where they also found that KLK6 could activate PAR₂. Owing to its chymotrypsin-like primary substrate specificity (Skytt et al., 1995), the inability of KLK7 to activate PAR₂ was expected. Trypsin-like KLKs show different substrate specificities, which may reflect different functions among these enzymes in the skin. Considering the trypsin-like primary substrate specificity of KLK8 (Rajapakse et al., 2005), its abundance in the stratum corneum (Komatsu et al., 2005a) and the capacity of KLKs 5 and 14 to activate PAR₂, the inability of KLK8 to activate PAR₂ was unexpected. In order to confirm that our KLK8 preparation was active, we compared the catalytic properties of KLKs 8 and 14. We found that KLK8 had a catalytic efficiency 3–6 times lower than KLK14 against the chromogenic substrates S-2288 and S-2302. On the other hand, KLK8 was able to cleave S-2251, a substrate not cleaved by KLK14 (Table 1). Hence, KLK8 is active but differs in its substrate specificity from KLK14. This difference may be reflected in the inability of KLK8 to activate PAR₂. A functional difference among KLKs in the skin is supported also by the fact that KLK5, which has a catalytic efficiency around 100-times lower than KLK14 toward S-2288 (Brattsand et al., 2005), could activate PAR₂. In this context, it should be noted that an inactivation of PAR₂ by KLK7 and KLK8 could be ruled out by control experiments (cf. Figure 2c and d).

PAR₂ activation in the skin may be involved in the elicitation of itch. PAR₂ as well as tryptase has been shown to be highly expressed in lesional skin in atopic dermatitis (Steinhoff et al., 2003 and Figure 5). Neuronal PAR₂ can be activated by tryptase (Steinhoff et al., 2000), and PAR₂ agonists induce pruritus in atopic dermatitis. It has therefore been suggested that PAR₂ activation may play a major role in pruritus of atopic dermatitis (Steinhoff et al., 2003). The presence of KLKs in sweat, and the fact that sweating is the most common itch-triggering factor in atopic dermatitis, may be of relevance in this context. Komatsu et al. (2006) showed that among other KLKs, KLKs 5, 7, 8, and 14 are present in sweat. We have earlier found that KLK14 is preferentially detected in sweat ducts and glands by immunohistochemistry of normal skin (Stefansson et al., 2006). Although the concentration of KLK14 may be low in sweat (Komatsu et al., 2006), its high catalytic efficiency (Brattsand et al., 2005) and its ability to activate PAR₂ could make this protease (together with other proteases) an important sweat-mediated itch inducer. It may be speculated that skin barrier defects in atopic dermatitis (Werner and Lindberg, 1985; Linde, 1992) may provide access to pruritogenic nerve fibers for sweat-carried KLKs such as KLK14. Proteinases, as itch mediators, have been proposed for a long time (reviewed by Ständer and Steinhoff, 2002; Steinhoff et al., 2006).

In conclusion, we clearly demonstrated that at least two (but not all) KLKs present in human epidermis can act as PAR₂ activators in vitro. The situation in vivo remains to be elucidated. Our results give further evidence that epidermal proteases may play important roles not only under normal conditions but also under pathophysiological conditions including skin inflammation, epidermal barrier function, and pruritus.

Materials and Methods

All described studies were approved by the medical ethical committee of Umeå University and performed according to the Declaration of Helsinki Principles. All participants gave their written informed consent.

Cell lines

KNRK cells stably expressing human PAR₂ with N-terminal Flag epitope and C-terminal HA11 epitope (Bohm et al., 1996; Dery et al., 1999; DeFea et al., 2000) were propagated in DMEM high glucose (Invitrogen, Groningen, The Netherlands), 10% bovine calf serum, 200 g/ml geneticin (G-418) (Invitrogen, 10131). For passage of cells, cell dissociation buffer enzyme-free Hank's-based (Invitrogen, 13150-016) was used. Human dermal microvascular endothelial cells, derived from dermis at the proliferating state, expressing functional PAR₂ receptors were grown in Endothelial Cell Basal Medium (PromoCell, Heidelberg, Germany), as described (Shpacovitch et al., 2002).

Production and activation of recombinant proteins

KLK5^EK was produced as pro^EK-KLK5 in High Five insect cells and activated as described by Brattsand et al. (2005). KLK14 was produced in active form in Pichia pastoris KM71H as described by Brattsand et al. (2005).

Active KLK7^EK was produced by enterokinase treatment of pro-KLK7 with the activation site replaced by an enterokinase cleavage site (Huang et al., 1997), pro^EK-KLK7. Pro^EK-KLK7 was produced by site-directed mutagenesis, using the Quick-Change Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA) with pS500 plasmid as template (Hansson et al., 1994), and the primer pair KLK7^EKs (5'-CTGCAGGAGAAGAAGCCGATGATGACAAGATTATTGATGGCGCC-3') and KLK7^EKas (5'-GGCGCCATCAATAATCTTGTCATCATCGGCTTCTTCTCCTGCAG-3'). As this rendered an incomplete activation site (DDDK), one additional D was inserted through amplification with primers KLK7FD4K (5'-GAAGAAGACGATGATGACAAGATTA-3') and KLK7R2 (5'-TCTAGATTAGCGATGCTTTTTCATGGTGTCAT-3'). The reverse primer included a stop codon and an Xba1 site. The resulting fragment was cloned into a TOPO TA vector (Invitrogen) and then transferred into the pPICZA vector through cleavage with restriction enzymes EcoR1 and Xba1 and subsequent ligation. P. pastoris strain X-33 was transformed with the pPICZA-KLK7^EK vector using the Easy Comp Transformation kit (Invitrogen). Recombinant protein was produced essentially as described in the EasySelect Pichia Expression Kit manual, purified, and activated as described for KLK5 and KLK14 by Brattsand et al. (2005). Pro^EK-KLK7 activation was performed by incubation with 0.006 U of rEK per g recombinant proenzyme for 5 hours at room temperature (RT) under conditions as described (Brattsand et al., 2005).

(cDNA)KLK8 was cloned from total RNA prepared from human epidermis (Brattsand and Egelrud, 1999), using the primer pair KLK8F1 (5'-CAGGAGGACAAGGTGCTGGGG-3') and KLK8R1 (5'-CCCTTGCTGATGATCTTCTTG-3'). (cDNA)KLK8 fragment was ligated into a TOPO TA vector and thereafter transferred into the pPICZA vector. The construct was used to transform P. pastoris strain KM71H using the Easy Comp Transformation kit. Recombinant protein was produced according to manual. As the reverse primer does not contain a stop codon, this results in a protein containing C-terminal His- and V5 tags. Purification of pro-KLK8 was made in several steps. First, conditioned cell media were concentrated by precipitation with 80% saturated ammonium sulfate. The pellet was dissolved in 1/10 of the original volume in 20 mM Tris-HCl pH 8.0. The mixture was filtered through a 0.45 M Millipore filter and then subjected to reversed phase chromatography. The first chromatography step was followed by ion exchange chromatography and a second run of reversed phase chromatography (Brattsand and Egelrud, 1999). Recombinant pro-KLK8 was activated by incubation with rEK, 0.02 U per g protein as described for pro^EK-KLK5 by Brattsand et al. (2005).

Accuracy of desired nucleotide sequences was verified by sequencing using the Big Dye Terminator Cycle Sequencing Kit (Applied Biosystems, Foster City, CA) and analyzed using an ABI377 automated DNA sequencer (Applied Biosystems). The sequence of (cDNA)KLK8 was compared and matched to the published sequence of KLK8 transcript variant 1 (accession no. NM_007196). The sequence of (cDNA)KLK7EK was compared to the published sequence of KLK7 transcript variant 1 (GenBank NM_005046). Protein concentration was determined with the DC Protein Assay (Bio-Rad Laboratories, Hercules, CA). All active protein preparations used, with the exception of KLK8, were active-site titrated with ₁-antitrypsin (Sigma, St Louis, MO, A-9024), essentially according to Salvesen and Nagase (1994). Known amounts of bovine pancreatic trypsin (Sigma, T-8253) were used for standardization.

Measurement of intracellular calcium concentration

PAR₂ signalling was assessed by measuring cytosolic [Ca²⁺] with fura 2-AM (Molecular Probes, Eugene, OR, F-1201) (Bohm et al., 1996; Dery et al., 1999; DeFea et al., 2000). Cells growing on glass coverslips were loaded with 2.5 M fura 2-AM in Hank's buffer/0.1% BSA (PAA Laboratories GmbH, Cölbe, Germany, Fraction V K41-001-100) at 37°C for minimum 25 minutes. Extracellular fura 2-AM was removed by rinsing in Hank's buffer/0.1% BSA. Cells were transferred to a cuvette with 37°C Hank's buffer/0.1% BSA and placed in a spectrofluorometer (Fluoro-Max2, Yobin Yvon GmbH, Munich, Germany). Cells were challenged with recombinant KLK or trypsin (Sigma, T-4665) at 0.1 nM to 10 M concentrations. Fluorescence was measured at 340 and 380 nm excitation wavelengths. The ratio of the fluorescence signal at the two wavelengths, which is proportional to the intracellular calcium ion concentration, was calculated.

Immunofluorescence and confocal microscopy

Cells were grown on glass coverslips in DMEM until near confluence. The coverslips were transferred into an incubation chamber and washed once with DMEM/0.1% BSA preheated to 37°C. Cells were incubated with 1 M recombinant KLK or trypsin (Sigma, T-4665), diluted in DMEM/0.1% BSA for 10 minutes at 37°C. Negative controls were treated with rEK in amounts corresponding to rEK content in activated enzyme preparations or appropriate buffer only. Cells were fixed with ice-cold methanol for 20 minutes on ice. Cells were washed and subsequently treated with 0.3% Triton-X 100 in phosphate-buffered saline (PBS)/0.1% BSA for 15 minutes. The cells were washed before blocking in blocking solution (PBS/2% goat serum (DakoCytomation, Glostrup, Denmark, X0907)) for 60 minutes at RT. Cells were incubated with mouse anti-Flag M1 mAb (Sigma, F 3040) diluted 1:200 and rabbit anti-HA antibody (Sigma, H 6908) diluted 1:200 over night at 4°C. After washing in blocking solution, cells were incubated with secondary antibodies: FITC-conjugated AffiniPure goat anti-rabbit antibody (Jackson Immuno Research, Cambridgeshire, UK, 111-095-003) and Cy3 AffiniPure anti-mouse antibody (Jackson Immuno Research, 715-166-151) diluted 1:200 in blocking solution, for 4 hours in the dark at RT. The cells were again washed and mounted on superfrost slides using Vectashield mounting medium (Vector Laboratories, Burlingame, CA, H-1000). Specimens were observed using a Bio-Rad MRC 1.000 confocal microscope. Images were collected at 0.68 M intervals using a Zeiss ^*100 Plan Apo 1.4 NA objective and a zoom from 1.5–2X.

Enzyme kinetics

Buffer (37.5 mM Tris-HCl pH 8.0/0.1 M NaCl/0.0075% Tween-20), enzymes (final concentrations as specified in Table 1), and chromogenic peptide substrates (Haemochrom Diagnostica AB, Mölndal, Sweden) at final concentrations ranging from 50 to 900 M (S-2251 (H-D-Val-Leu-Lys-pNA 2HCl), S-2302 (H-D-Pro-Phe-Arg-pNA 2HCl)) or 1,000 M (S-2288 (H-D-Ile-Pro-Arg-pNA 2HCl)) were mixed in a 96-well plate. Total volume in each well was 77.5 l. Mixtures were incubated in duplicate at RT for 10 minutes in an ELISA reader (SpectraMAX 340, Molecular Devices, Berkshire, UK). The apparatus was calibrated using known amounts of pNA (Haemochrom Diagnostica AB). K_m and V_max values were calculated using a Lineweaver Burk plot. rEK in amounts corresponding to that used for activation of KLK8 gave no detectable activity toward any of the substrates used.

Immunohistochemistry

Tissues were fixed in 10% (w/v) buffered formalin and embedded in paraffin. Slides (5–6 m) were cut and incubated for 30 minutes at 60°C. Afterwards, sections were dewaxed, rehydrated, and heated in a steamer (MultiGourmet plus FS20, Braun, Kronberg, Germany) for 25 minutes in 0.1 M ChemMate Target Retrieval Solution (DakoCytomation) in a plastic cuvette. Sections were allowed to cool in this buffer for 20 minutes. Endogenous peroxidase activity was quenched with 100 mM NaN₃/0.1% (w/v) H₂O₂ in PBS for 20 minutes at RT. After washing with PBS, sections were blocked with 2% (w/v) BSA in PBS for 25 minutes at RT. Sections were incubated with first specific polyclonal antibody diluted in 1% (w/v) BSA overnight at 4°C in a humid chamber. For KLK14, Em-14 (1.6 g/ml; Stefansson et al., 2006) and for PAR₂, H-99 at final concentration 0.4 g/ml (Santa Cruz Biotechnology, Santa Cruz, CA) was used. Negative controls were prepared by omission of the primary antibodies. Specificity of Em-14 antibody to KLK14 has been shown through adsorption experiments (Stefansson et al., 2006). After rinsing with PBS, slides were incubated with secondary antibodies for 1 hour at RT in a humid chamber (EnVision+ System Labeled Polymer-HRP, anti-rabbit; DakoCytomation). The immunoreactivity was detected with the Liquid DAB+ Substrate Chromogen System (DakoCytomation). Sections were counterstained with Hematoxylin QS (Vector Laboratories) and mounted with Aquamount (BDH, Poole, UK). Stainings were examined using a DM LB microscope (Leica, Solms, Germany), equipped with a HV-C20M CCD camera (Hitachi, Rodgau, Germany) and Diskus 4.20 software (Carl H. Hilgers, Königswinter, Germany).

Conflict of Interest

Torbjörn Egelrud has a patent on KLK7.

References

1. Brattsand M, Egelrud T (1999) Purification, molecular cloning, and expression of a human stratum corneum trypsin-like serine protease with possible function in desquamation. J Biol Chem 274:30033–30040 | Article | PubMed | ISI | ChemPort |
2. Brattsand M, Stefansson K, Lundh C, Haasum Y, Egelrud T (2005) A proteolytic cascade of kallikreins in the stratum corneum. J Invest Dermatol 124:198–203 | Article | PubMed | ISI | ChemPort |
3. Bohm SK, Khitin LM, Grady EF, Aponte G, Payan DG, Bunnett NW (1996) Mechanisms of desensitization and resensitization of proteinase-activated receptor-2. J Biol Chem 271:22003–22016 | Article | PubMed | ISI | ChemPort |
4. Caubet C, Jonca N, Brattsand M, Guerrin M, Bernard D, Schmidt R et al. (2004) Degradation of corneodesmosome proteins by two serine proteases of the kallikrein family, SCTE/KLK5/hK5 and SCCE/KLK7/hK7. J Invest Dermatol 122:1235–1244 | Article | PubMed | ISI | ChemPort |
5. Clements J, Hooper J, Dong Y, Harvey T (2001) The expanded human kallikrein (KLK) gene family: genomic organisation, tissue-specific expression and potential functions. J Biol Chem 382:5–14 | Article | ChemPort |
6. Cottrell GS, Amadesi S, Grady EF, Bunnett NW (2004) Trypsin IV, a novel agonist of protease-activated receptors 2 and 4. J Biol Chem 279:13532–13539 | Article | PubMed | ISI | ChemPort |
7. D'Andrea MR, Derian CK, Leturcq D, Baker SM, Brunmark A, Ling P et al. (1998) Characterization of protease-activated receptor-2 immunoreactivity in normal human tissues. J Histochem Cytochem 46:157–164 | PubMed | ISI | ChemPort |
8. DeFea KA, Zalevsky J, Thoma MS, Dery O, Mullins RD, Bunnett NW (2000) Beta-arrestin-dependent endocytosis of proteinase-activated receptor 2 is required for intracellular targeting of activated ERK1/2. J Cell Biol 148:1267–1281 | Article | PubMed | ISI | ChemPort |
9. Derian CK, Eckardt AJ, Andrade-Gordon P (1997) Differential regulation of human keratinocyte growth and differentiation by a novel family of protease-activated receptors. Cell Growth Differ 8:743–749 | PubMed | ISI | ChemPort |
10. Dery O, Corvera CU, Steinhoff M, Bunnet NW (1998) Proteinase-activated receptors: novel mechanisms of signalling by serine proteases. Am J Physiol 274:C1429–C1452 | PubMed | ISI | ChemPort |
11. Dery O, Thoma MS, Wong H, Grady EF, Bunnett NW (1999) Trafficking of proteinase-activated receptor-2 and beta-arrestin-1 tagged with green fluorescent protein. Beta-arrestin-dependent endocytosis of a proteinase receptor. J Biol Chem 274:18524–18535 | Article | PubMed | ISI | ChemPort |
12. Egelrud T (1993) Purification and preliminary characterization of stratum corneum chymotryptic enzyme: a proteinase that may be involved in desquamation. J Invest Dermatol 101:200–204 | Article | PubMed | ISI | ChemPort |
13. Egelrud T, Lundström A (1991) A chymotrypsin-like proteinase that may be involved in desquamation in plantar stratum corneum. Arch Dermatol Res 283:108–112 | Article | PubMed | ISI | ChemPort |
14. Ekholm E, Brattsand M, Egelrud T (2000) Stratum corneum tryptic enzyme in normal epidermis: a missing link in the desquamation process? J Invest Dermatol 114:56–63 | Article | PubMed | ISI | ChemPort |
15. Felber LM, Borgono CA, Cloutier SM, Kundig C, Kishi T, Ribeiro Chagas J et al. (2005) Enzymatic profiling of human kallikrein 14 using phage-display substrate technology. Biol Chem 386:291–298 | Article | PubMed | ISI | ChemPort |
16. Grishina Z, Ostrowska E, Halangk W, Sahin-Toth M, Reiser G (2005) Activity of recombinant trypsin isoforms on human proteinase-activated receptors (PAR): mesotrypsin cannot activate epithelial PAR-1, -2, but weakly activates brain PAR-1. Br J Pharmacol 146:990–999 | Article | PubMed | ISI | ChemPort |
17. Hachem J-P, Houben E, Crumrine D, Man M-Q, Schurer N, Roelandt T et al. (2006) Serine protease signaling of epidermal permeability barrier homeostasis. J Invest Dermatol 126:2074–2086 | Article | PubMed | ISI | ChemPort |
18. Hansson L, Strömqvist M, Bäckman A, Wallbrandt P, Carlstein A, Egelrud T (1994) Cloning, expression, and characterization of stratum corneum chymotryptic enzyme. A skin-specific human serine proteinase. J Biol Chem 269:19420–19426 | PubMed | ISI | ChemPort |
19. Hollenberg MD, Compton SJ (2002) International union of Pharmacology. XXVIII. Proteinase-activated receptors. Pharmacol Rev 54:203–217 | Article | PubMed | ISI | ChemPort |
20. Huang C, Wong GW, Ghildyal N, Gurish MF, Sali A, Matsumoto R et al. (1997) The tryptase, mouse mast cell protease 7, exhibits anticoagulant activity in vivo and in vitro due to its ability to degrade fibrinogen in the presence of the diverse array of protease inhibitors in plasma. J Biol Chem 272:31885–31893 | Article | PubMed | ISI | ChemPort |
21. Ishihara H, Connolly AJ, Zeng D, Kahn ML, Zheng YW, Timmons C et al. (1997) Protease-activated receptor 3 is a second thrombin receptor in humans. Nature 386:502–506 | Article | PubMed | ISI | ChemPort |
22. Komatsu N, Saijoh K, Sidiropoulos M, Tsai B, Levesque MA, Elliott MB et al. (2005a) Quantification of human tissue kallikreins in the stratum corneum: dependence on age and gender. J Invest Dermatol 125:1182–1189 | Article | PubMed | ISI | ChemPort |
23. Komatsu N, Saijoh K, Toyama T, Ohka R, Otsuki N, Hussack G et al. (2005b) Multiple tissue kallikrein mRNA and protein expression in normal skin and skin diseases. Br J Dermatol 153:274–281 | Article | PubMed | ISI | ChemPort |
24. Komatsu N, Takata M, Otsuki N, Toyama T, Ohka R, Takehara K et al. (2003) Expression and localization of tissue kallikrein mRNAs in human epidermis and appendages. J Invest Dermatol 121:542–549 | Article | PubMed | ISI | ChemPort |
25. Komatsu N, Tsai B, Sidiropoulos M, Saijoh K, Levesque MA, Takehara K et al. (2006) Quantification of eight tissue kallikreins in the stratum corneum and sweat. J Invest Dermatol 126:927–931 | Article | ChemPort |
26. Linde YM (1992) Dry skin and atopic dermatitis. Acta Derm Venereol Suppl (Stockh) 177:9–13 | PubMed | ChemPort |
27. Lundström A, Egelrud T (1991) Stratum corneum chymotryptic enzyme: a proteinase which may be generally present in the stratum corneum and with a possible involvement in desquamation. Acta Derm Venereol 71:471–474 | PubMed | ISI | ChemPort |
28. Lundwall Å, Blaber M, Clements J, Courty Y, Diamandis E, Lilja H et al. (2006) A comprehensive nomenclature for serine proteases with homology to the tissue kallikrein. Biol Chem 387:637–641 | Article | PubMed | ChemPort |
29. Macfarlane SR, Seatter MJ, Kanke T, Hunter GD, Plevin R (2001) Proteinase-activated receptors. Pharmacol Rev 53:245–282 | PubMed | ISI | ChemPort |
30. Nystedt S, Emilsson K, Larsson AK, Strombeck B, Sundelin J (1995) Molecular cloning and functional expression of the gene encoding the human proteinase-activated receptor 2. Eur J Biochem 232:84–89 | Article | PubMed | ISI | ChemPort |
31. Oikonomopoulou K, Hansen KK, Saifeddine M, Vergnolle N, Tea I, Blaber M et al. (2006) Kallikrein-mediated cell signalling: targeting proteinase-activated receptor (PARs). Biol Chem 387:817–824 | Article | PubMed | ISI | ChemPort |
32. Ossovskaya VS, Bunnett NW (2004) Protease-activated receptors: contribution to physiology and disease. Physiol Rev 84:579–621 | Article | PubMed | ISI | ChemPort |
33. Rajapakse S, Ogiwara K, Takano N, Moriyama A, Takahashi T (2005) Biochemical characterization of human kallikrein 8 and its possible involvement in the degradation of extracellular matrix proteins. FEBS Lett 579:6879–6884 | Article | PubMed | ISI | ChemPort |
34. Salvesen G, Nagase H (1994) Inhibition of proteolytic enzymes. In: Proteolytic enzymes, a practical approach (Reynon RJ, Bond JS, eds), Oxford: Oxford University Press, 83–104
35. Santulli RJ, Derian CK, Darrow AL, Tomko KA, Eckardt AJ, Seiberg M et al. (1995) Evidence for the presence of a protease-activated receptor distinct from the thrombin receptor in human keratinocytes. Proc Natl Acad Sci USA 92:9151–9155 | Article | PubMed | ChemPort |
36. Shpacovitch VM, Brzoska T, Buddenkotte J, Stroh C, Sommerhoff CP, Ansel JC et al. (2002) Agonists of proteinase-activated receptor 2 induce cytokine release and activation of nuclear transcription factor B in human dermal microvascular endothelial cells. J Invest Dermatol 118:380–385 | Article | PubMed | ISI | ChemPort |
37. Skytt A, Stromqvist M, Egelrud T (1995) Primary substrate specificity of recombinant human stratum corneum chymotryptic enzyme. Biochem Biophys Res Commun 211:586–589 | Article | PubMed | ISI | ChemPort |
38. Sondell B, Thornell LE, Stigbrand T, Egelrud T (1994) Immunolocalization of stratum corneum chymotryptic enzyme in human skin and oral epithelium with monoclonal antibodies: evidence of a proteinase specifically expressed in keratinizing squamous epithelia. J Histochem Cytochem 42:459–465 | PubMed | ISI | ChemPort |
39. Stefansson K, Brattsand M, Ny A, Glas B, Egelrud T (2006) Kallikrein-related peptidase 14 may be a major contributor to trypsin-like proteolytic activity in human stratum corneum. Biol Chem 387:761–768 | Article | PubMed | ISI | ChemPort |
40. Steinhoff M, Bienenstock J, Schmelz M, Maurer M, Wie E, Bíró T (2006) Neurophysiological, neuroimmunological, and neuroendocrine basis of pruritus. J Invest Dermatol 126:1705–1718 | Article | PubMed | ISI | ChemPort |
41. Steinhoff M, Brzoska T, Luger TA (2001) Keratinocytes in epidermal immune responses. Curr Opin Allergy Clin Immunol 1:469–476 | Article | PubMed | ChemPort |
42. Steinhoff M, Buddenkotte J, Shpacovitch V, Rattenholl A, Moormann C, Vergnolle N et al. (2005) Proteinase-activated receptors: transducers of proteinase-mediated signalling in inflammation and immune response. Endocr Rev 26:1–43 | Article | PubMed | ISI | ChemPort |
43. Steinhoff M, Corvera CU, Thoma MS, Kong W, McAlpine BE, Caughey GH et al. (1999) Proteinase-activated receptor-2 in human skin: tissue distribution and activation of keratinocytes by mast cell tryptase. Exp Dermatol 8:282–294 | PubMed | ISI | ChemPort |
44. Steinhoff M, Neisius U, Ikoma A, Fartasch M, Heyer G, Skov PS et al. (2003) Proteinase-activated receptor-2 mediates itch: a novel pathway for pruritus in skin. J Neurosci 23:6176–6180 | PubMed | ISI | ChemPort |
45. Steinhoff M, Vergnolle N, Young SH, Tognetto M, Amadesi S, Ennes HS et al. (2000) Agonists of proteinase-activated receptor 2 induce inflammation by a neurogenic mechanism. Nat Med 6:151–158 | Article | PubMed | ISI | ChemPort |
46. Ständer S, Steinhoff M (2002) Pathophysiology of pruritus in atopic dermatitis: an overview. Exp Dermatol 11:12–24 | Article | PubMed | ISI | ChemPort |
47. Vu TK, Hung DT, Wheaton VI, Coughlin SR (1991) Molecular cloning of a functional thrombin receptor reveals a novel proteolytic mechanism of receptor activation. Cell 64:1057–1068 | Article | PubMed | ISI | ChemPort |
48. Werner Y, Lindberg M (1985) Transepidermal water loss in dry and clinically normal skin in patients with atopic dermatitis. Acta Derm Venereol 65:102–105 | PubMed | ISI | ChemPort |
49. Xu W-F, Andersen H, Whitmore TE, Presnell SR, Yee DP, Ching A et al. (1998) Cloning and characterization of human protease-activated receptor 4. Proc Natl Acad Sci USA 95:6642–6646 | Article | PubMed | ChemPort |
50. Yousef GM, Diamandis EP (2001) The new human tissue kallikrein gene family: structure, function, and association to disease. Endocr Rev 22:184–204 | Article | PubMed | ISI | ChemPort |

Acknowledgments

This work was supported by Umeå University, Arexis AB, the VINNOVA Foundation, Arners Foundation, the Kempe Foundation, the Welander-Finsen Foundations (M.B. and T.E.), and SFB 492 (B13), IZKF (STEI2/076/06), SFB 293, DFG (STE 1014/2–1), Galderma, and Rosacea Foundation (M.S.).