Kallikrein-related peptidases (KLKs) have critical roles in corneocyte desquamation and are regulated by lymphoepithelial Kazal-type inhibitor (LEKTI). However, it is unclear how these proteases are activated and how activated KLKs are released from LEKTI in the upper cornified layer. Recently, we reported cloning of a PRSS3 gene product, keratinocyte-specific mesotrypsin, from a cDNA library. We hypothesized that mesotrypsin is involved in the desquamation process, and the aim of the present study was to test this idea by examining the effects of mesotrypsin on representative desquamation-related enzymes pro-KLK5 and pro-KLK7. Incubation of mesotrypsin and these zymogens resulted in generation of the active forms. KLK activities were effectively inhibited by recombinant LEKTI domains D2, D2-5, D2-6, D2-7, D5, D6, D6-9, D7, D7-9, and D10-15, whereas mesotrypsin activity was not susceptible to these domains, and in fact degraded them. Immunoelectron microscopy demonstrated that mesotrypsin was localized in the cytoplasm of granular cells and intercellular spaces of the cornified layer. Proximity ligation assay showed close association between mesotrypsin and KLKs in the granular to cornified layers. Age-dependency analysis revealed that mesotrypsin was markedly downregulated in corneocyte extract from donors in their sixties, compared with younger donors. Collectively, our findings suggest that mesotrypsin contributes to the desquamation process by activating KLKs and degrading the intrinsic KLKs' inhibitor LEKTI.
Hair follicle-associated-pluripotent (HAP) stem cells are located in the bulge area of the hair follicle, express the stem-cell marker nestin, and have been shown to differentiate to neurons, glial cells, keratinocytes, smooth muscle cells, melanocytes and cardiac muscle cells. Transplanted HAP stem cells promoted the recovery of peripheral nerve and spinal cord injuries and have the potential for heart regeneration as well. In the present study, we implanted mouse green fluorescent protein (GFP)-expressing HAP stem-cell spheres captured on polyvinylidene fluoride membranes (PFM) into the severed thoracic spinal cord. of nude mice. Eight weeks after implantation, immunofluorescence staining showed that HAP stem cells differentiated into neurons and glial cells. Fluorescence microscopy showed that the PFMcaptured HAP stem-cell hair spheres promoted rejoining of the thoracic spinal cord of the nude mice. Hematoxylin and eosin (H&E) staining also showed that the severed thoracic spinal cord had rejoined. Quantitative walking analysis showed a significant difference between the implanted mice and non-implanted mice with a several spinal cord. HAP stem cells are readily accessible from everyone, do not form tumors, and can be cryopreserved without loss of differentiation potential. These results suggest that HAP stem cells may have greater potential than iPS or ES cells for regenerative medicine, such as for spinal cord repair.
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