Keratin 5 knockout dysregulates keratinocyte differentiation and epidermal morphogenesis and reveals potential pathogenic drivers of epidermolysis bullosa simplex

Summary: Abstract Body: Epidermolysis bullosa simplex (EBS) is a rare genetic disorder characterized by skin fragility and painful blistering due to shearing of the epidermis. This disease has been linked to a variety of mutations in the keratin genes KRT5 or KRT14, which encode major cytoskeletal components of basal layer epidermal keratinocytes. While the genetic etiology of EBS is well-known, there is currently no cure for this disease and treatments are focused on pain management, infection risk, and wound care. Since KRT5 loss-of-function was not viable in mouse or macaque models, we engineered human in vitro cellular and tissue models to determine how KRT5 depletion compromises epidermal integrity and to elucidate the pathogenesis of EBS. Leveraging CRISPR/Cas9, we generated TERT-immortalized KRT5-deficient human keratinocytes and organotypic epidermis. KRT5-deficient cells exhibited irregular morphology and dysfunctional intercellular junctions along with differential expression of epidermal maturation markers. Utilizing a mechanical dissociation assay, we found that KRT5 depletion weakened intercellular adhesion of highly differentiated keratinocyte sheets. Moreover, KRT5-deficient organotypic cultures exhibited stunted epidermal development with basal cell fragility, diminished intermediate layers, and disruption of desmosomal proteins. To delineate the mechanisms underlying this phenotype, we performed RNA sequencing on KRT5-deficient cultures, which revealed altered expression of key regulators of cytokine signaling, epidermal differentiation, and cell stress pathways. Overall, our model indicates that KRT5 is essential for orchestrating proper human keratinocyte differentiation and epidermal morphogenesis. Importantly, we identified multiple druggable pathways that may drive EBS pathogenesis and could represent novel therapeutic targets. Karina N. Schmidt¹, Afua Tiwaa¹, Anthony M. Coon², Mrinal K. Sarkar², Johann E. Gudjonsson², Cory L. Simpson¹ 1. Dermatology, University of Washington, Seattle, WA, United States. 2. Dermatology, University of Michigan, Ann Arbor, MI, United States. Epidermal Structure and Barrier Function