Attenuated kallikrein‐related peptidase activity disrupts desquamation and leads to stratum corneum thickening in human skin equivalent models

• Published in: British journal of dermatology (1951) Vol. 176; no. 1; pp. 145 - 158
• Main Authors: McGovern, J.A., Meinert, C., de Veer, S.J., Hollier, B.G., Parker, T.J., Upton, Z.
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 01.01.2017
• Subjects: Adult; Cell Differentiation - physiology; Cell Proliferation - physiology; Cells, Cultured; Desmocollin; Desmoglein 1; Desmosomes - genetics; DNA microarrays; Enzymes; Epidermis; Epidermis - cytology; Epidermis - metabolism; Epidermis - pathology; Gene Expression; Genome, Human - genetics; Homeostasis; Humans; Immunohistochemistry; Kallikrein; Kallikreins - metabolism; Keratinocytes - drug effects; Keratinocytes - metabolism; Ki-67 Antigen - metabolism; Localization; Membrane Proteins - metabolism; Microarray Analysis - methods; Models, Biological; Peptidase; Polymerase chain reaction; Proteinase inhibitors; Proteins; Proteins - metabolism; Proteolysis; Real-Time Polymerase Chain Reaction; Skin; Stratum corneum; Studies; Translation
• ISSN: 0007-0963; 1365-2133
• DOI: 10.1111/bjd.14879

Summary Background Epidermal homeostasis is maintained through the balance between keratinocyte proliferation, differentiation and desquamation; however, human skin equivalent (HSE) models are known to differentiate excessively. In native tissue, proteases such as kallikrein‐related peptidase (KLK) 5 and KLK7 cleave the extracellular components of corneodesmosomes; proteins corneodesmosin, desmocollin 1 and desmoglein 1, loosening the cellular connections and enabling desquamation. The actions of KLK7 are tightly controlled by protease inhibitors, skin‐derived antileucoproteinase (SKALP) and lymphoepithelial Kazal‐type‐related inhibitor (LEKTI), which also inhibits KLK5, localizing protease activity to the stratum corneum. Objectives To investigate the mechanisms that inhibit the desquamation cascade in HSE models. Methods Human skin tissue and HSE models were investigated using gene microarray, real‐time polymerase chain reaction (PCR), immunohistochemistry and Western blot analysis to examine key components of the desquamation pathway. To elucidate proteolytic activity in HSEs and native skin, in situ and gel zymography was performed. Results Histological analysis indicated that HSE models form a well‐organized epidermis, yet develop an excessively thick and compact stratum corneum. Gene microarray analysis revealed that the desquamation cascade was dysregulated in HSE models and this was confirmed using real‐time PCR and immunohistochemistry. Immunohistochemistry and Western blot indicated overexpression of LEKTI and SKALP in HSEs. Although KLK7 was also highly expressed in HSEs, zymography indicated that protease activation and activity was lower than in native skin. Conclusions These findings demonstrate that stratum corneum thickening is due to inhibited KLK5 and KLK7 activation and a subsequent lack of corneodesmosome degradation in the HSE model epidermis. What's already known about this topic? Corneocytes are sloughed from the skin surface in a process known as desquamation; this process is impaired in human skin equivalent (HSE) models. The key enzymes involved in desquamation are kallikrein‐related peptidase (KLK)5 and KLK7, which cleave the extracellular cell–cell junction proteins corneodesmosin (CDSN), desmocollin 1 and desmoglein 1. KLK5 and KLK7 are inhibited by lymphoepithelial Kazal‐type‐related inhibitor (LEKTI) and secreted antileucoprotease (SKALP). What does this study add? Altered desquamation is accompanied by aberrant localization of cell–cell junction proteins and protease inhibitors in HSE models. KLK7 protein is overexpressed, neither KLK5 nor KLK7 are activated and overall epidermal proteolytic activity is lower in HSE models than native skin. SKALP and LEKTI are present in the stratum corneum in HSEs, potentially inhibiting KLK7. CDSN protein persists in the stratum corneum, which suggests that it is not degraded. What is the translational message? These results provide a mechanistic insight into excess stratum corneum thickening in HSE models. Epidermal homeostasis must be restored to generate HSE models with physiological expression, localization and activity of desquamation‐associated proteins for future translational studies. Linked Comment: Brattsand. Br J Dermatol 2017; 176:11–12.