View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector ORIGINAL ARTICLE Activation of Proteinase-Activated Receptor-2 by Human Kallikrein-Related Peptidases Kristina Stefansson1, Maria Brattsand1, Dirk Roosterman2, Cordula Kempkes2, Georgeta Bocheva2, Martin Steinhoff2 and Torbjo¨rn Egelrud1 Proteinase-activated receptor-2 (PAR2) is a seven transmembrane spanning, G-protein-coupled receptor, present on the membrane of many cell types including keratinocytes. In skin, PAR2 is suggested to play a regulatory role during inflammation, epidermal barrier function, and pruritus. PAR2 is activated by trypsin-like proteases by a unique mechanism where cleavage of the receptor leads to the release of a small peptide, which activates the receptor as a tethered ligand. The endogenous activators of PAR2 on keratinocytes have not been identified as of yet. Potential candidates are kallikrein-related peptidases (KLKs) expressed by epidermal cells. Therefore, the ability of four human skin-derived KLKs was examined with regard to their capacity to activate PAR2 in vitro.PAR2 cleavage was followed by immunofluorescence analysis and functional activation by measurements of changes in intracellular calcium levels. We found that KLK5 and KLK14, but neither KLK7 nor KLK8, induced PAR2 signalling. We conclude that certain, but not all, epidermal KLKs are capable of activating PAR2. We could also show the coexpression of KLK14 and PAR2 receptor in inflammatory skin disorders. These in vitro results suggest that KLKs may take part in PAR2 activation in the epidermis and thereby in PAR2-mediated inflammatory responses, including epidermal barrier repair and pruritus. The role of KLKs in PAR2 activation in vivo remains to be elucidated. Journal of Investigative Dermatology (2008) 128, 18–25; doi:10.1038/sj.jid.5700965; published online 12 July 2007 INTRODUCTION (Vu et al., 1991; Nystedt et al., 1995; Ishihara et al., 1997; Xu The skin forms a protective barrier not only by means of et al., 1998). These are G-protein-coupled receptors with seven constituting an efficient physico-chemical barrier but also via transmembrane domains, which are activated by a unique its highly active and specialized participation in immune and mechanism dependent on proteolytic cleavage. Upon cleavage inflammatory reactions. The major cellular constituent of the within the N-terminal extracellular part of the receptor, a epidermis, the keratinocyte, has been described as a peptide is released that binds to and irreversibly activates the ‘‘cytokine factory’’ and plays an important role in our immune receptor while still tethered to it. Activation leads to interaction defence (reviewed by Steinhoff et al., 2001). A new concept in with heterotrimeric G-proteins in the plasma membrane and skin immunology is that proteolytic enzymes may act as key downstream signalling events. Proteinases capable of activat- modulators of biological functions in a cytokine-like manner. ing PARs belong to the serine protease family and include Endogenous or exogenous proteinases may influence cells thrombin, trypsin, and cathepsin G. PAR2 is not activated by through the activation of proteinase-activated receptors (PARs) thrombin but has been shown to be activated by the following (reviewed by Steinhoff et al., 2005). proteases in vitro: trypsin, mast cell tryptase, factor Xa, acrosin, Proteinase-activated receptor-2 (PAR2) is a member of the gingipain, and neuronal serine proteases (reviewed by Dery PAR family, which encompasses four PARs, designated PAR1–4 et al., 1998; Macfarlane et al., 2001; Ossovskaya and Bunnett, 2004; Steinhoff et al., 2005). PAR2 is expressed by keratino- 1Department of Public Health and Clinical Medicine, Dermatology and cytes (Santulli et al., 1995; D’Andrea et al., 1998; Steinhoff Venereology, Umea˚ University, Umea˚, Sweden and 2Department of et al., 1999) where it may function as a regulator of growth and Dermatology, University of Mu¨nster, Mu¨nster, Germany differentiation (Derian et al., 1997) and is highly implicated in Correspondence: Dr Maria Brattsand, Department of Public Health and inflammatory reactions (Steinhoff et al., 2005). Recently, PAR2 Clinical Medicine, Dermatology and Venereology, Umea˚ University, Build 6M, 3rd floor, SE-901 85 Umea˚, Sweden. E-mail: was identified as a novel signalling mechanism of the [email protected] epidermal permeability barrier (Hachem et al., 2006). The Abbreviations: KLK, human kallikrein-related peptidase gene; KLK, human endogenous activator of PAR2 in the epidermis, however, has kallikrein-related peptidase protein; KNRK, Kirsten Murine Sarcoma Virus not been identified yet. It has been suggested that mast cell transformed rat kidney epithelial; PAR2, proteinase-activated receptor-2; tryptase may activate PAR during inflammatory conditions PBS, phosphate-buffered saline; rEK, recombinant enterokinase; RT, room 2 temperature upon mast cell infiltration and degranulation (Steinhoff et al., Received 14 March 2007; revised 7 May 2007; accepted 17 May 2007; 1999). Other possibilities include endogenous trypsin-like published online 12 July 2007 proteases present in the epidermis. 18 Journal of Investigative Dermatology (2008), Volume 128 & 2007 The Society for Investigative Dermatology K Stefansson et al. PAR2 Activation by Human KLKs We have previously purified, cloned, and characterized three epidermal serine proteases; human kallikrein-related EK -KLK7 peptidase (KLK) 5 (hK5, stratum corneum tryptic enzyme, EK kDa pro SCTE), KLK7 (hK7, stratum corneum chymotryptic enzyme, KLK7 pro-KLK8 KLK8 204 SCCE), and KLK14 (hK14) (Hansson et al., 1994; Brattsand 116 and Egelrud, 1999; Brattsand et al., 2005; Stefansson et al., 92 2006). KLK8 is another human KLK, which is abundantly 50 present in the stratum corneum (Komatsu et al., 2005a). These four enzymes all belong to the human kallikrein gene 37 family of 15 related serine proteases (Clements et al., 2001; 29 Yousef and Diamandis, 2001) and have different substrate specificities. KLK5 and KLK8 have trypsin-like activity 20 (Brattsand et al., 2005; Rajapakse et al., 2005), whereas KLK7 has chymotrypsin-like activity (Skytt et al., 1995). 6 KLK14 has mainly trypsin-like but also a significant chymo- trypsin-like activity (Brattsand et al., 2005; Felber et al., Figure 1. Preparations of recombinant human tissue KLK protein precursors 2005). These enzymes are here denoted as human kallikrein- and active enzymes. Coomassie brilliant blue stained SDS-PAGE. related peptidases according to the new nomenclature proposed by Lundwall et al. (2006). Given the presence of both PAR2 and KLKs in the account the proportion of active enzyme in the other epidermis, it is plausible that one or several KLKs may preparations, and the fact that electrophoretic analyses function as activators of PAR2 and thus play a role in showed no or negligible degradation of KLK8 (Figure 1), an inflammatory skin diseases. In this work, we investigated assumption was made that at least 30% of the KLK8 whether KLKs 5, 7, 8, or 14 may be capable of activating preparation should be functional. The active KLK8 prepara- PAR2 in vitro. We selected KLKs 5, 7, and 14 because these tion also showed high activity toward chromogenic peptide enzymes are known to be present in the skin in catalytically substrates (see below). active form (Egelrud, 1993; Brattsand and Egelrud, 1999; Stefansson et al., 2006). KLK8 may, besides KLK11, be the Effects of KLKs 5, 7, 8, and 14 on Ca2 þ mobilization most abundant trypsin-like KLK in normal human skin Trypsin, KLK5EK, and KLK14 stimulated Ca2 þ mobilization in (Komatsu et al., 2005a). We show that KLK5 and KLK14, Kirsten Murine Sarcoma Virus transformed rat kidney but neither KLK7 nor KLK8, could activate PAR2. We also epithelial (KNRK)-PAR2 cells (Figure 2a, b, and e), interpreted EK show the colocalization of KLK14 and PAR2 in the epidermis as PAR2 signalling. No reaction was obtained when KLK7 of inflammatory skin disorders. Thus, certain but not all KLKs or KLK8, in concentrations from 0.1 nM up to 10 mM, was used expressed by keratinocytes are potential endogenous activa- (Figure 2c and d). However, subsequent addition of 100 nM tors of PAR2 within the epidermis, and may play a role in trypsin to the same cells gave an expected reaction, epidermal barrier physiology/pathophysiology and itching. confirming the integrity of cells (Figure 2c and d). Addition of rEK in concentrations similar or higher to that present in RESULTS preparations of KLK5EK, KLK7EK, and KLK8 did not lead to any Production and activation of recombinant KLKs detectable Ca2 þ mobilization (data not shown). Figure 3a Production of active KLK5 and KLK14 has been published shows dose response curves. Both trypsin and KLK14 were far EK elsewhere (Brattsand et al., 2005). Purified recombinant more potent activators of PAR2 than KLK5 . The lowest EK pro -KLK7 could, as expected, be activated by recombinant detectable signal was obtained at 50 nM concentration for EK enterokinase (rEK). Recombinant pro-KLK8, having the native KLK14. KLK5 gave a detectable signal at 0.1 mM. Even at propeptide sequence, was also easily activated by rEK. 1 mM, the signal obtained with KLK5 was lower than that for Activation was verified by a mobility shift on SDS-PAGE KLK14 at 50 nM. Neither KLK7 nor KLK8 could activate PAR2. (Figure 1) and by measuring activity toward chromogenic The ability of KLK5 and KLK14 to induce Ca2 þ mobilization peptide substrates. Active KLK8 fusion protein had an in cells via the PAR2 signal system was confirmed using apparent molecular mass of 38.6 kDa according to SDS- human dermal microvascular endothelial cells naturally PAGE analysis (Figure 1). Active-site titration of enzyme expressing functional PAR2 receptors (Figure 3b; Shpacovitch preparations showed that around 30% (KLK7EK) and 60% et al., 2002). (KLK5EK) of the respective enzyme was active when compared with expected activity according to protein Immunofluorescence analyses concentration determined with the DC Protein Assay. Close By using antibodies to the Flag and the HA11 epitopes of the to 100% of the KLK14 preparation was in active form.