Keratin filaments constitute the major component of the epidermal cytoskeleton from heterodimers of type I and
type II keratin subunits. Missense mutations in
keratin 5 or
keratin 14, highly expressed in the basal epidermis, cause the severe skin blistering disease
epidermolysis bullosa simplex (EBS) in humans by rendering the
keratin cytoskeleton sensitive to mechanical stress; yet, the mechanisms by which individual mutations cause cell fragility are incompletely understood. Here, we compared the K14p.Arg125Pro with the K5p.Glu477Asp mutation, both giving rise to severe
generalized EBS, by stable expression in
keratin-free keratinocytes. This revealed distinctly different effects on
keratin cytoskeletal organization, in agreement with in vivo observations, thus validating the cell system. Although the K14p.Arg125Pro mutation led to impaired desmosomes, downregulation of desmosomal
proteins, and weakened epithelial sheet integrity upon shear stress, the K5p.Glu477Asp mutation did not impair these functions, although causing EBS with
squamous cell carcinoma in vivo. Atomic force microscopy demonstrated that K14 mutant cells were even less resistant against deformation compared with
keratin-free keratinocytes. Thus, a
keratin mutation causing EBS compromises cell stiffness to a greater extent than the lack of
keratins. Finally, re-expression of K14 in K14 mutant cells did not rescue the above defects. Collectively, our findings have implications for EBS
therapy approaches.