The surface-exposed epidermis, a self-renewing stratified squamous epithelium composed of several layers of keratinocytes, is most important for the barrier defense against these challenges. Keratinocytes in the outmost stratum corneumof the epidermis are shed off and replaced by newly differentiated cells originating from epidermal stem cells located in the basal layer. They undergo a specific differentiation process and form the cornified envelope, which is a rigid and insoluble protein and lipid structure with essential properties of the barrier function. Recent discoveries have highlighted the importance of proteaseinhibitors and proteases as key players in the desquamation process and in epidermal barrier function. Human tissue kallikreins, or kallikrein-related peptidases, are the largest family of trypsin or chymotrypsin-like secreted INCB28060 serine proteases encoded by 15 genes on chromosome 
region 19q13.4. At least eight KLKs are expressed in normal skin, among which KLK5, KLK7, KLK8 and KLK14 have been reported to be most important. KLKs are capable of cleaving corneodesmosomesand are thought to be key regulators of the desquamation process. Epidermal overexpression of KLK7 resulted in pathologic skin changes with increased epidermal thickness, hyperkeratosis, dermal inflammation, and severe pruritus. The activity of the KLKs is regulated by the pH and specific protease inhibitors in human skin. The importance of epithelial protease inhibitors has been revealed impressively in Netherton Syndrome, an autosomal recessive disorder caused by mutations in the serine protease inhibitor Kazal-type 5gene. NS presents as an ichthyosiform dermatosis with variable erythroderma, hair-shaft defects, atopic features, and growth retardation. Lymphoepithelial Kazal-type-related inhibitor, the product of Spink5, includes in its primary structure 15 different serine protease inhibitory domains. The inhibitory functions of LEKTI are highly diverse. Inhibitory activities are directed toward trypsin, plasmin, subtilisin A, cathepsin G, and human neutrophil elastase. Though LEKTI is absent, NS patients can still develop hyperkeratosis �C a clinical sign of inhibited desquamation. Therefore, we speculated that more KLK inhibitors are present in human skin generating a complex network of KLKs and their inhibitors to control the desquamation process. Since KLK5 is thought to be one of the most important enzymes involved in this process, we started a preparative attempt to identify KLK5 inhibitors in human stratum corneum. Herein we report the identification of a new protease inhibitor LEKTI-2 and its gene Spink9, which specifically inhibits KLK5. In this study we aimed to identify major substances that might contribute to the epithelial barrier shield by inhibiting the epidermal serine protease KLK5. We identified a new peptide termed LEKTI-2 as a specific inhibitor for KLK5, which is encoded by Spink9, a novel member of the Spink gene family. Our findings give evidence for the importance of LEKTI-2 in epidermal desquamation and provide new insight to the complex protease-protease inhibitor interaction in human skin. LEKTI-2 expression shows some similarities to the expression of LEKTI, which was demonstrated to be expressed in lamellar bodies, likely the granular-like structures in our fluorescent staining, and secreted into the intercellular space, in the uppermost stratum granulosum. BYL719 PI3K inhibitor Electron microscopy studies revealed that LEKTI and KLK7 are transported separately in the lamellar granule system and are co-localized in the extracellular spaces.