696 Three dimensional IHC characterization of aging in 3D skin models

Abstract

The type I keratin 14 (K14) is mainly expressed in the basal layer of epidermis and related stratified epithelia. Previous work revealed a role for inter-keratin disulfide bonding involving residue cysteine (Cys) 367 of human K14 toward the assembly, organization, and dynamics of keratin filaments in skin keratinocytes in culture. To define the function of K14-dependent disulfide bonding in skin epithelia in vivo, we generated Krt14C373A mice using the Crispr/ Cas9 technology (mouse Cys373 is orthologous to human Cys367). Western blotting revealed a marked decrease in disulfide-bonded K14 species in Krt14C373A ear and tail skin relative to control. Morphological analyses revealed a state of hyperproliferation and hyperkeratosis, dysregulated keratinocyte differentiation, and abnormal cornified envelopes in Krt14C373A skin. Adult Krt14C373A mice show a skin barrier defect at baseline, reflected by increased trans-epidermal water loss. The latter trait is enhanced following topical acetone irritation. Mass spectrometry-based assays identified 14-3-3 as a major K14 interacting protein, and follow-up studies confirmed that 14-3-3 interacts with both WT and C373A mutant forms. 14-3-3 forms aggregates in Krt14C373A keratinocytes in situ, accompanied by abnormal nuclear localization of Yap1, a transcriptional effector of Hippo signaling, in suprabasal differentiated keratinocytes. Mutagenesis shows that the subcellular distribution and regulation of 14-3-3 and Yap1 is specifically regulated by residue Cys373 in mouse K14. The results obtained to date establish that K14-dependent disulfide bonding plays a physiologically important role in epidermal homeostasis and barrier function by impacting the solubility, regulation and function of 14-3-3/Yap1.