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-1β 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.

Human papillomaviruses (HPV) are causative agents of various tumours such as cervical cancer. HPV binding to the cell surface of keratinocytes leads to virus endocytosis at tetraspanin enriched microdomains. Complex interactions of the capsid proteins with host proteins as well as ADAM17-dependent ERK1/2 signal transduction enable the entry platform assembly of the oncogenic HPV type 16. Here, we studied the importance of tetraspanin CD9, also known as TSPAN29, in HPV16 infection of different epithelial cells. We found that both overexpression and loss of the tetraspanin decreased infection rates in cells with low endogenous CD9 levels, while reduction of CD9 expression in keratinocytes that exhibit high-CD9 protein amounts, led to an increase of infection. Therefore, we concluded that low-CD9 supports infection. Moreover, we found that changes in CD9 amounts affect the shedding of the ADAM17 substrate transforming growth factor alpha (TGFα) and the downstream phosphorylation of ERK. These effects correlate with those on infection rates suggesting that a specific CD9 optimum promotes ADAM17 activity, ERK signalling and virus infection. Together, our findings implicate that CD9 regulates HPV16 infection through the modulation of ADAM17 sheddase activity.

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-2',4',-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.

Human CD137 (4-1BB), a member of the TNF receptor family, and its ligand CD137L (4-1BBL), are expressed on immune cells and tumor cells. CD137/CD137L interaction mediates bidirectional cellular responses of potential relevance in inflammatory diseases, autoimmunity and oncology. A soluble form of CD137 exists, elevated levels of which have been reported in patients with rheumatoid arthritis and various malignancies. Soluble CD137 (sCD137) is considered to represent a splice variant of CD137. In this report, however, evidence is presented that A Disintegrin and Metalloproteinase (ADAM)10 and potentially also ADAM17 are centrally involved in its generation. Release of sCD137 by transfected cell lines and primary T cells was uniformly inhibitable by ADAM10 inhibition. The shedding function of ADAM10 can be blocked through inhibition of its interaction with surface exposed phosphatidylserine (PS), and this effectively inhibited sCD137 generation. The phospholipid scramblase Anoctamin-6 (ANO6) traffics PS to the outer membrane and thus modifies ADAM10 function. Overexpression of ANO6 increased stimulated shedding, and hyperactive ANO6 led to maximal constitutive shedding of CD137. sCD137 was functionally active and augmented T cell proliferation. Our findings shed new light on the regulation of CD137/CD137L immune responses with potential impact on immunotherapeutic approaches targeting CD137.