Characterization of Spink6 in mouse skin: the conserved inhibitor of kallikrein-related peptidases is reduced by barrier injury.

The proteolytic regulation of the desquamation process by kallikrein-related peptidases (KLKs) is crucial for epidermal barrier function, and elevated KLK levels have been reported in atopic dermatitis. KLKs are controlled by specific inhibitors of the serine protease inhibitor of Kazal-type (Spink) family. Recently, SPINK6 was shown to be present in human stratum corneum. In order to investigate its role in epidermal barrier function, we studied mouse Spink6. Sequence alignment revealed that the Kazal domain of Spink6 is highly conserved in animals. Recombinant Spink6 efficiently inhibited mouse Klk5 and human KLK2, KLK4, KLK5, KLK6, KLK7, KLK12, KLK13, and KLK14, whereas human KLK1 and KLK8 were not inhibited. Spink6 was expressed in mouse epidermis mainly in the stratum granulosum, and the inner root sheath of hair follicles. Stimulation with flagellin, EGF, and IL-1ß did not alter Spink6 expression, whereas stimulation with tumor necrosis factor-α (TNFα)/IFNγ and all-trans retinoic acid resulted in a significant downregulation of Spink6 expression in cultured primary mouse keratinocytes. Mechanically and metabolically induced skin barrier dysfunction resulted both in a downregulation of Spink6 expression. Our study indicates that Spink6 is a potent inhibitor of KLKs and involved in skin barrier function.

Cross-linking of SPINK6 by transglutaminases protects from epidermal proteases.

Extracellular kallikrein-related peptidases (KLKs) are involved in the desquamation process and the initiation of epidermal inflammation by different mechanisms. Their action is tightly controlled by specific protease inhibitors. Recently, we have identified the serine protease inhibitor of Kazal-type (SPINK) 6 as a selective inhibitor of KLKs in human stratum corneum extracts. As SPINK6 is expressed in the same localization as transglutaminases (TGM) and contains TGM substrate motifs, SPINK6 was tested to be cross-linked in the epidermis. Recombinant SPINK6 was shown to be cross-linked to fibronectin (FN) by TGM1 by western blot analyses. Moreover, SPINK6 was cross-linked in epidermal extracts and cultured keratinocytes by immunoblotting analyses. The use of TGM1 and TGM3 resulted in different immunoreactivities in western blot analyses of SPINK6 and epidermal extracts, suggesting substrate specifities of different TGMs for SPINK6 cross-linking in the epidermis. Conjugated SPINK6 exhibited protease inhibitory activity in keratinocytes and stratum corneum extracts; cross-linked SPINK6 protected FN from KLK5-mediated cleavage, whereas a lower KLK-inhibiting SPINK6-GM mutation did not. In conclusion, we demonstrated that SPINK6 is cross-linked in keratinocytes and human epidermis and remains inhibitory active. Thus, cross-linked SPINK6 might protect specific substrates such as FN from KLK cleavage and contributes to the regulation of proteases in the epidermis.

Flagellin delivery by Pseudomonas aeruginosa rhamnolipids induces the antimicrobial protein psoriasin in human skin.

The opportunistic pathogen Pseudomonas aeruginosa can cause severe infections in patients suffering from disruption or disorder of the skin barrier as in burns, chronic wounds, and after surgery. On healthy skin P. aeruginosa causes rarely infections. To gain insight into the interaction of the ubiquitous bacterium P. aeruginosa and healthy human skin, the induction of the antimicrobial protein psoriasin by P. aeruginosa grown on an ex vivo skin model was analyzed. We show that presence of the P. aeruginosa derived biosurfactant rhamnolipid was indispensable for flagellin-induced psoriasin expression in human skin, contrary to in vitro conditions. The importance of the bacterial virulence factor flagellin as the major inducing factor of psoriasin expression in skin was demonstrated by use of a flagellin-deficient mutant. Rhamnolipid mediated shuttle across the outer skin barrier was not restricted to flagellin since rhamnolipids enable psoriasin expression by the cytokines IL-17 and IL-22 after topical application on human skin. Rhamnolipid production was detected for several clinical strains and the formation of vesicles was observed under skin physiological conditions. In conclusion we demonstrate herein that rhamnolipids enable the induction of the antimicrobial protein psoriasin by flagellin in human skin without direct contact of bacteria and responding cells. Hereby, human skin might control the microflora to prevent colonization of unwanted microbes in the earliest steps before potential pathogens can develop strategies to subvert the immune response.