ORIGINAL ARTICLE SPINK9 Stimulates Metalloprotease/EGFR–Dependent Keratinocyte Migration via Purinergic Receptor Activation Maria Sperrhacke1, Jan Fischer1, Zhihong Wu1, Sarah Klu¨nder1,3, Radislav Sedlacek2,Jens-MichaelSchroeder1, Ulf Meyer-Hoffert1 and Karina Reiss1 Serine protease inhibitors of the Kazal-type 9 (SPINK9) is a keratinocyte-derived cationic peptide that is found most abundantly in the upper layers of the palmar–plantar epidermis. In vitro, the peptide displays the capacity to inhibit specifically kallikrein-related peptidase 5 (KLK5). Here, we report that cells expressing SPINK9 secrete the peptide constitutively. Recombinant SPINK9 (rSPINK9) provoked transactivation of the EGFR in human keratinocytes, resulting in efficient downstream triggering of cell migration. Transactivation occurred via functional upregulation of a disintegrin and metalloproteases (ADAMs), as evidenced by suppression with a metalloproteinase inhibitor and an EGFR–blocking antibody. SPINK9 preparations isolated from human skin also displayed EGFR–transactivating capacity. The classical purinergic receptor antagonists oxidized ATP and pyridoxalphosphate-6-azophenyl-20,40,-disulfonic acid effectively suppressed EGFR transactivation by rSPINK9, indicating that in analogy to what has recently been reported for the cationic antimicrobial peptides cathelicidin LL-37 and bee venom melittin, purinergic receptors have an essential bridging role in promoting the upregulation of ADAM function by the cationic peptide. SPINK9 could represent an example of how a cationic peptide may subserve multiple and interrelated functions that contribute to the maintenance of the physical and immunological barrier of the skin. Journal of Investigative Dermatology (2014) 134, 1645–1654; doi:10.1038/jid.2014.23; published online 6 February 2014 INTRODUCTION derived serine proteases such as proteinase 3 (Sorensen et al., The two major families of cationic antimicrobial peptides 2001). In the skin, however, cathelicidin is processed mainly (AMPs) in epithelial tissues are human b-defensins and by kallikrein-related peptidase 5 (KLK5) that is constitutively cathelicidins (Bowdish et al., 2006; Gallo and Hooper, secreted by keratinocytes and present in tissues (Yamasaki 2012). The parent cathelicidin molecule hCAP18 is et al., 2006). This serine protease cleaves LL-37 further to produced mainly by leukocytes, and is also expressed in generate a wide array of smaller degradation products with keratinocytes. hCAP18 must be cleaved to generate the active varying antimicrobial activity, which can naturally be found in AMP LL-37. This can occur through the action of leukocyte- the epidermis. LL-37 has important roles in skin physiology (Nijnik et al., 2012; Vandamme et al., 2012). In addition to its AMP 1Department of Dermatology, University Hospital Schleswig-Holstein, properties, the peptide promotes IL-18 release from keratino- 2 Christian-Albrecht University Kiel, Kiel, Germany and Department of cytes, is chemotactic, and provokes EGFR transsignaling Transgenic Models of Diseases, Institute of Molecular Genetics of the ASCR, Prague, Czech Republic (Tjabringa et al., 2003; Tokumaru et al., 2005; Niyonsaba 3Current address: Department of Biochemistry, Children’s Hospital, University et al., 2005; Carretero et al., 2008). The latter is in turn because Medical Center Hamburg-Eppendorf, Hamburg, Germany. of the activation of shedding metalloproteinases of a disintegrin- Correspondence: Karina Reiss, Department of Dermatology, University like and metalloproteinases (ADAMs) family, which release Hospital Schleswig-Holstein, Christian-Albrecht University Kiel, Kiel, EGFR ligands from their membrane anchors, allowing them to Germany. E-mail: [email protected] bind to the receptor (Blobel, 2005). EGFR transactivation is Abbreviations: ADAM, a disintegrin and metalloprotease; AMP, antimicrobial pivotally important for triggering keratinocyte proliferation and peptide; AREG, amphiregulin; Cetux, cetuximab; eGFP, endothelial green fluorescent protein; ERK, extracellular signal–regulated kinase; HB-EGF, migration. The link between ADAM17 and EGFR transsignaling heparin binding-EGF; HEK, human embryonic kidney; KLK5, kallikrein-related is well established. Deletion of ADAM17 mimics EGFR defi- peptidase 5; LEKTI, lymphoepithelial Kazal-type related inhibitor; MM, ciency, causing defects in hair follicle development, immature marimastat; NHEK, normal human epidermal keratinocyte; pERK, tissue differentiation, and embryonic death (Sibilia and phosphorylated ERK; SC, stratum corneum; SG, stratum granulosum; SPINK, serine protease inhibitors of the Kazal type Wagner, 1995; Peschon et al., 1998; Franzke et al., 2012). Received 17 September 2013; accepted 10 December 2013; accepted article A centrally important function of skin kallikreins is the preview online 17 January 2014; published online 6 February 2014 degradation of keratinocyte corneodesmosomes, enabling & 2014 The Society for Investigative Dermatology www.jidonline.org 1645 MSperrhackeet al. SPINK9 Stimulates EGFR Signaling desquamation to take place. Maintenance of dermal home- finding was made with SPINK9, this could set an example of ostasis requires strict control over this process (Fischer and how cationic peptides might exert multiple and interrelated Meyer-Hoffert, 2013). The lymphoepithelial Kazal-type related functions in the skin. The affirmative results reported herein inhibitor (LEKTI) encoded by serine protease inhibitor Kazal- further tempt speculation that a common general pattern type 5 (spink5) assumes a pivotal role in this context (Magert involving purinergic receptor activation with ensuing upregu- et al., 1999, 2002). The 120 kDa LEKTI precursor is cleaved lation of ADAM function underlies important responses in intracellularly by furin into 15 smaller fragments with highly mammalian cells provoked by cationic peptides of widely differing inhibitory activities. Only two of these domains (2 differing origin. and 15) contain the characteristic six cysteine motif of Kazal- type related inhibitors. Spink5 loss-of-function mutations RESULTS cause the Comel–Netherton syndrome, a severe disease Expression and secretion of SPINK9 characterized by increased KLK activity and altered desqua- LEKTI is localized in lamellar granules of keratinocytes and is mation, keratinization disorder, and skin barrier defects transported in cargo vesicles, in which it is subject to furin (Chavanas et al., 2000). processing, and is then secreted to the extracellular space in SPINK9, also referred to as LEKTI2, shares high homology to the SG. Immunohistochemical analyses of SPINK9 expression the LEKTI Kazal-type domains, and the 7.7 kDa peptide also similarly suggested a vesicular localization in the SG and SC, displays specific inhibitory activity against KLK5 in vitro as demonstrated by a punctate staining pattern (Figure 1a and (Brattsand et al., 2009; Meyer-Hoffert et al., 2009). SPINK9 b). When an SPINK9 expression vector with a C-terminal is detectable in the stratum granulosum (SG) and stratum enhanced green fluorescent protein (eGFP) tag and an corneum (SC) of palmar and plantar skin, and is thus assumed N-terminal hemagglutinin tag was transfected in HaCaT to participate in overall control of epidermal turnover. The keratinocytes, vesicular localization of the peptide was noted peptide shares its strongly cationic properties with AMPs such clearly (Figure 1c). Anti-GFP immunoblot analyses of HaCaT as LL-37 and melittin. keratinocytes and human embryonic kidney (HEK) 293T cells The impetus for the present investigation stemmed from revealed double bands of approximately 37 and 41 kDa in the our recent observation that melittin, the evolutionarily distant pellets of both cell types and one band in the conditioned AMP of bee venom, shared with LL-37 the remarkable medium (Figure 1d and e). Secretion of SPINK9 was com- capacity of provoking ADAM–dependent EGFR transsignaling pletely abrogated in the presence of the classical secretory (Niyonsaba et al., 2007; Sommer et al., 2012). If a similar pathway inhibitor brefeldin A (data not shown). Immunoblot Lysate Supernatant Lysate Supernatant SPINK9- SPINK9- SPINK9- SPINK9- kDa Mock eGFP Mock eGFP kDa Mock eGFP Mock eGFP 55 55 35 35 HaCaT HEK 293T Figure 1. Serine protease inhibitors of the Kazal-type 9 (SPINK9) is secreted as a soluble molecule. Immunohistochemical staining of SPINK9 in normal human plantar skin. (a, b) SPINK9 is expressed in stratum granulosum and stratum corneum of plantar skin (black arrow). Bars ¼ 50 mm. (c) HaCaT cells were transfected with enhanced green fluorescent protein (eGFP)–tagged SPINK9 and visualized under the microscope. Bar ¼ 10 mm. (d and e) Western blot analysis of cell lysates and supernatants of HaCaT and human embryonic kidney (HEK) 293T cells transfected with SPINK9-eGFP. One representative immunoblot of three independent experiments is shown, respectively. 1646 Journal of Investigative Dermatology (2014), Volume 134 MSperrhackeet al. SPINK9 Stimulates EGFR Signaling analyses using an anti-HA antibody did not clearly identify stimulated with rSPINK9 in the presence of the broad-spec- SPINK9, possibly indicating that the majority of SPINK9 is trum metalloprotease inhibitor marimastat (MM) or the EGFR– stored as a mature protein without the signal peptide. blocking antibody cetuximab (Cetux). As shown in Figure 2b and d, rSPINK9–dependent phosphorylation of ERK1/2 was SPINK9 provokes metalloprotease-dependent EGFR activation completely