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Background  Terminally differentiated keratinocytes acquire corneocyte protein envelopes (CPE) complexed with corneocyte lipid envelopes (CLE). These two structural components of the corneocyte envelopes (CEs) undergo maturation by gaining in hydrophobicity, rigidity and surface area. Linoleoyl acylceramides are processed by 12R‐lipoxygenase (12R‐LOX) and other enzymes before transglutaminase (TG) attaches ω‐hydroxyceramides to involucrin in the CPE. Concurrently, structural proteins are cross‐linked by TG that has been activated by cathepsin D (CathD).    Objectives  The primary aim of this work was to demonstrate the impact of relative humidity (RH) during  ex vivo  CE maturation. Low, optimal and high RH were selected to investigate the effect of protease inhibitors (PIs) on CE maturation and TG activity; in addition, 12R‐LOX and CathD activity were measured at optimal RH. Finally, the effect of glycerol on  ex vivo  CE maturation was tested at low, optimal and high RH.    Methods  The first and ninth tape strip of photo‐exposed (PE) cheek and photo‐protected (PP) post‐auricular sites of healthy volunteers were selected.  Ex vivo  CE maturation was assessed via the relative CE maturity (RCEM) approach based on CE rigidity and hydrophobicity. The second and eighth tapes were exposed to RH in the presence of inhibitors.    Results  Irrespective of tape stripping depth, CEs from PE samples attained CE rigidity to the same extent as mature CEs from the PP site, but such improvement was lacking for CE hydrophobicity. 70% RH was optimal for  ex vivo  CE maturation. The inhibition of 12R‐LOX activity resulted in enhanced CE rigidity which was reduced by the TG inhibitor. CE hydrophobicity remained unchanged during  ex vivo  maturation in the presence of TG or 12R‐LOX inhibition. CE hydrophobicity was enhanced in the presence of glycerol at 44% RH and 100% RH but not at 70% RH. Furthermore, TG activity was significantly diminished at 100% RH compared to the commercial inhibitor LDN‐27219. However, a protease inhibitor mix reversed the negative effect of overhydration.    Conclusion  The study adds to the understanding of the roles of 12R‐LOX and TG activity in CE maturation and gives further insight into the effect of glycerol on the SC.

The importance of 12R‐lipoxygenase and transglutaminase activities in the hydration‐dependent ex vivo maturation of corneocyte envelopes