View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector ORIGINAL ARTICLE Keratinocyte-Specific Mesotrypsin Contributes to the Desquamation Process via Kallikrein Activation and LEKTI Degradation Masashi Miyai1,3, Yuuko Matsumoto1,3, Haruyo Yamanishi1, Mami Yamamoto-Tanaka1,2, Ryoji Tsuboi2 and Toshihiko Hibino1 Kallikrein-related peptidases (KLKs) have critical roles in corneocyte desquamation and are regulated by lymphoepithelial Kazal-type inhibitor (LEKTI). However, it is unclear how these proteases are activated and how activated KLKs are released from LEKTI in the upper cornified layer. Recently, we reported cloning of a PRSS3 gene product, keratinocyte-specific mesotrypsin, from a cDNA library. We hypothesized that mesotrypsin is involved in the desquamation process, and the aim of the present study was to test this idea by examining the effects of mesotrypsin on representative desquamation-related enzymes pro-KLK5 and pro-KLK7. Incubation of mesotrypsin and these zymogens resulted in generation of the active forms. KLK activities were effectively inhibited by recombinant LEKTI domains D2, D2–5, D2–6, D2–7, D5, D6, D6–9, D7, D7–9, and D10–15, whereas mesotrypsin activity was not susceptible to these domains, and in fact degraded them. Immunoelectron microscopy demonstrated that mesotrypsin was localized in the cytoplasm of granular cells and intercellular spaces of the cornified layer. Proximity ligation assay showed close association between mesotrypsin and KLKs in the granular to cornified layers. Age-dependency analysis revealed that mesotrypsin was markedly down- regulated in corneocyte extract from donors in their sixties, compared with younger donors. Collectively, our findings suggest that mesotrypsin contributes to the desquamation process by activating KLKs and degrading the intrinsic KLKs’ inhibitor LEKTI. Journal of Investigative Dermatology (2014) 134, 1665–1674; doi:10.1038/jid.2014.3; published online 30 January 2014 INTRODUCTION chymotrypsin-like enzyme (stratum corneum chymotryptic Epidermal differentiation is precisely controlled, and the thick- enzyme) (Lundstrom and Egelrud, 1991) have roles in the ness of the epidermis is maintained through constant shedding detachment of superficial corneocytes. Later, these proteases of the outermost layers of corneocytes, a process known as were identified as KLK5 and KLK7, respectively (Caubet et al., desquamation (Watt, 1989). Desquamation is accomplished by 2004). Recent investigations have suggested that other KLK– the degradation of corneodesmosomes, which are involved in related enzymes, including KLKs 6, 8, 10, 11, 13, and 14, are corneocyte–corneocyte adherence (Caubet et al., 2004; Ishida- also expressed in human epidermis, and it was suggested that Yamamoto and Kishibe, 2011). Corneodesmosomes are com- they may form a proteolytic cascade that results in desquama- posed of three major proteins: desmoglein 1, desmocollin, and tion (Komatsu et al., 2007). All KLKs are synthesized as corneodesmosin. These proteins are proteolytically hydrolyzed proenzymes, and subsequently activated through mechanisms by a family of kallikrein-related peptidases (KLKs). Earlier that have not yet been established. KLK5 is thought to be the studies demonstrated that a trypsin-like enzyme (stratum initiator of this cascade, as it is capable of activating most of the corneum tryptic enzyme) (Suzuki et al., 1993) and a family members (Ishida-Yamamoto et al., 2011). Desquamation is regulated not only by proteases involved 1Shiseido Research Center, Kanazawa-ku, Yokohama, Japan and 2Department in the activation cascade but also by inhibitors such as of Dermatology, Tokyo Medical University, Shinjuku-ku, Tokyo, Japan lymphoepithelial Kazal-type related inhibitor (LEKTI), a pro- 3These authors contributed equally to this work. duct of the serine protease inhibitor Kazal-type 5 gene SPINK5 Correspondence: Toshihiko Hibino, Shiseido Research Center, 2-12-1 Fukuura, (Borgono et al., 2007; Roelandt et al., 2009). LEKTI consists of Kanazawa-ku 236-8643, Yokohama, Japan. 15 serine protease-inhibitory domains and is believed to be E-mail: [email protected] processed to single or multiple domains to facilitate specific Abbreviations: KLK, kallikrein-related peptidase; LEKTI, lymphoepithelial inhibition of particular proteases. Recently, LEKTI2 encoded Kazal-type inhibitor; PBS, phosphate-buffered saline; PLA, proximity ligation by SPINK9 was identified in the palmoplantar epidermis and assay was found to show KLK5–specific inhibition (Brannstrom Received 12 June 2013; revised 15 October 2013; accepted 15 October 2013; accepted article preview online 3 January 2014; published online et al., 2012). SPINK6 was isolated from human plantar 30 January 2014 stratum corneum extracts as a selective inhibitor of KLK5, 7, & 2014 The Society for Investigative Dermatology www.jidonline.org 1665 MMiyaiet al. Involvement of Mesotrypsin in Desquamation and 14 (Meyer-Hoffert et al., 2010). Thus, KLK activities seem mesotrypsin (Figure 1b). In addition, incubation of pro-KLK5 to be strongly suppressed under physiological conditions. with mesotrypsin resulted in a time-dependent linear increase It is suggested that active KLKs are dissociated from various of KLK5 activity (Figure 1c). Similar results were obtained for SPINK gene products under acidic conditions at the skin pro-KLK7 (Figure 1d–f). These results clearly indicate that surface (Pampalakis and Sotiropoulou, 2007). However, mesotrypsin generates active KLKs through limited proteolysis Ishida-Yamamoto et al. (2012) clearly demonstrated that of pro-KLKs. degradation of corneodesmosomes starts deeper in the corni- fied layers. Thus, it is plausible that dissociation of active KLKs Inhibition of KLK5 by LEKTI domains and the effect of pH on the from their inhibitors is not a cell-surface event, but is interaction controlled at least in part by mechanisms other than pH. Next, we generated various fragments of LEKTI (D2, D2–5, On the other hand, we have cloned an epidermis-specific D2–6, D2–7, D5, D6, D7, D7–9, D8–9, and D10–15), splicing variant of PRSS3 gene, trypsinogen 5, from a kerati- covering the whole length of the LEKTI protein (Figure 2a). nocyte cDNA library (Nakanishi et al., 2010). Trypsinogen 5 All of these LEKTI fragments were able to inhibit KLK5 activity possesses a very short N-terminal propeptide followed by the (Figure 2b). The inhibitory concentration 50 (IC50) values are enterokinase recognition sequence DDDDK-I. After cleavage shown in Figure 2c. D2 showed a high IC50 value for KLK5 by enterokinase, the same mesotrypsin would be produced as compared with other domains, indicating that D2 may not be from pancreatic trypsinogen 3 and brain-type trypsinogen 4. an effective inhibitor of KLK5. Similarly, all LEKTI fragments We confirmed that epidermal mesotrypsin is expressed in the were able to inhibit KLK7 activity (Supplementary Figure S1 upper epidermis, whereas its activating enzyme, enterokinase, online). Each domain construct showed somewhat weaker shows a more confined localization in the boundary area inhibition of KLK7, compared with KLK5, and none of these between the granular layer and cornified layer. Characteristic LEKTI domains showed a clear preference for KLK7 inhibition. features of mesotrypsin are as follows: (1) it cannot digest high- Next, we tested whether inhibition of KLK5 by LEKTI domains molecular-weight proteins, in contrast to anionic trypsin and was abrogated under acidic conditions (Figure 2d). KLK5 was cationic trypsin, the PRSS1 and PRSS2 gene products, incubated with D5 or D6 in buffers of different pH values. respectively, and (2) it is not susceptible to naturally KLK5 was active between pH 5.5 and pH 9, and D5 and D6 occurring trypsin inhibitors (Nyaruhucha et al., 1997). Thus, completely suppressed KLK5 activity in this pH range. These the physiological role of mesotrypsin is likely to be completely results indicate that loss of LEKTI inhibition of KLKs may not different from those of trypsins. Instead, it seems likely to be occur simply as a result of the acidic pH typically observed at involved in limited proteolysis, such as zymogen activation. the skin surface. Furthermore, it is capable of inactivating naturally occurring trypsin inhibitors by proteolytic cleavage. These unique pro- Mesotrypsin degrades various LEKTI domains perties of mesotrypsin led us to hypothesize that epidermal Next, we tested the effect of these LEKTI constructs on the mesotrypsin is involved in the desquamation process. To test activity of mesotrypsin (Figure 3a). None of these LEKTI this idea, we focused on two critical processes. One is the constructs inhibited mesotrypsin activity even in the presence activation of pro-KLK5 and the other is dissociation of active of 1,000-fold excess of recombinant LEKTIs. These results KLKs from LEKTI proteins. Furthermore, we examined the indicate that mesotrypsin is resistant to intrinsic serine pro- localization of mesotrypsin in human epidermis using tease inhibitors, including LEKTI. We then examined whether immunoelectronmicroscopy. Further, in situ proximity ligation the LEKTI constructs were degraded by mesotrypsin, using assay (PLA) was used to assess the interaction of mesotrypsin newly prepared antibodies against D2 and D12, and commer- with KLKs or LEKTI proteins in human epidermis. cially available anti-D4–8 antibody (F2). The N-terminal domains of LEKTI (D2 and D2–7), the inner domains (D5, RESULTS