(2007) Human tissue kallikrein expression in the stratum corneum and serum of atopic dermatitis patients

(2007) Human tissue kallikrein expression in the stratum corneum and serum of atopic dermatitis patients. both the intermediate form and the mature form of caspase-14. Immunohistochemical analysis demonstrated that the intermediate form Mianserin hydrochloride was localized at the granular layer. Our results indicate that regulation of procaspase-14 maturation during terminal differentiation is a unique two-step process involving KLK7 and an activation intermediate of caspase-14. apoptosome, death-inducing signaling complex, Mianserin hydrochloride and inflammasome (3, 4). Apoptosome activates caspase-9, and death-inducing signaling complex induces activation of caspase-8 and -10 (4). Inflammatory caspases, caspase-1 and -5, are similarly activated via inflammasome (5). Subsequently these initiator caspases activate executioner caspase-3 and caspase-7. In some cases, a maturation process involving cleavage of propeptides and removal of the linker region follows (6). Among the caspases, caspase-14 shows restricted expression, becoming found only in epidermis and its appendages and thymic Mianserin hydrochloride Hassall’s body (7C9). Although it is definitely structurally much like caspase-1, it is thought to function in keratinocyte terminal differentiation (10, 11). We have recently purified active caspase-14 from draw out of human being cornified cells (12), and found that it consists of a large subunit (p17) and a small subunit (p11) generated by the removal of a 6-amino acid linker peptide. The C-terminal of the large subunit was identified as Asp-146. This is identical with the granzyme B-cleaved activation site, although granzyme B is not the physiological activation enzyme of caspase-14 (12, 13). A cleavage site-directed antibody, h14D146, was prepared and found to react only with active caspase-14. This form is present in the stratum corneum (SC),2 indicating that maturation of caspase-14 happens during the transition from stratum granulosum (SG) to SC. To keep up epidermal barrier function and homeostasis, older and nonfunctional corneocytes should be eliminated promptly by dropping. This process, desquamation, is definitely accomplished via degradation of corneodesmosomes by a proteolytic cascade of human being kallikrein-related peptidases (KLKs) (14). It is thought that the cascade is initiated by activation of trypsin-like pro-KLK5, either by autoactivation or by matriptase in Netherton syndrome (15). Subsequently KLK5 activates additional trypsin-like KLKs and chymotrypsin-like KLK7 by proteolytic launch of the amino-terminal propeptide. Although most of the 15 kallikreins are indicated in pores and skin (16), KLK5 and KLK7 are highly indicated in differentiated keratinocytes (17). On the other hand, the activation and maturation mechanisms Mianserin hydrochloride of caspase-14 remain to be fully founded. Cleavage of procaspase-14 FLICE happens only in the transition stage from SG to SC. Many proteolytic enzymes could be activated at this stage. Interestingly activation of caspase-14 requires a high concentration of kosmotropic salt (13), although it is not obvious whether this condition is definitely also required for maturation, in other words, cleavage of caspase-14 at Asp146. In the present study, we investigated the mechanism of caspase-14 maturation. We display that it is completely different from those of additional caspases, being controlled by KLK7 and an intermediate form of caspase-14. EXPERIMENTAL Methods Cell Culture Human being keratinocytes derived from normal foreskin (Kurabo, Osaka, Japan) were cultured in Humedia KG2 supplemented with epidermal growth element (0.1 ng/ml), insulin (10 g/ml), hydrocortisone (0.5 g/ml), bovine pituitary extract (0.4%), gentamicin (50 g/ml), and amphotericin B (50 ng/ml). All experiments were carried out on cells at the third or fourth passages. Tissue Specimens Human being skin specimens were acquired, with prior educated consent, from individuals who underwent plastic surgery. The study was authorized by the Honest Committee of Tokyo Medical University or college and the Shiseido Committee on Human being Ethics. Tissue samples were fixed with acetone and inlayed in paraffin. Preparation of Pro-KLK7 and Its Activation Pro-KLK7 cDNA was PCR cloned into the BamHI and SalI sites of the pQE-100 vector (Qiagen Inc., Valencia,.