Grant: Travel Awards - Institute Community Support

Epidermolysis bullosa simplex (EBS) is a genetic disorder characterized by recurrent skin blister formation from cytolysis of basal epidermal keratinocytes in response to minor injury or friction. Most cases of EBS are associated with mutations in the genes encoding keratin proteins K5 and K14 that form a filamentous cytoskeletal network in basal keratinocytes. In spite of the tight correlation between EBS phenotypes and mutations in keratin (and keratin associated) genes, the exact mechanisms by which these mutations lead to cell fragility in EBS patients are unknown. We recently showed that cells expressing a mutant EBS K14 protein are not more fragile than WT cells when subjected to extreme uniaxial stretches. Here we tested the hypothesis that EBS cells are more fragile when exposed to shear stress than WT cells. We subjected a wild type keratinocyte line (NEB-1), a mutant K14-R125P EBS cell line (KEB-7), and a K14-null keratinocyte cell line (KEB-11) to shear stresses within microfluidic devices and found that mutants displayed more shear-induced cell necrosis than WT. Moreover, the preliminary cell morphology analysis showed that mutant cells deform less than WT cells when subjected to high shear stress. We also subjected NEB-1, KEB-7 and KEB-11 cell lines to large scale stretching and found that NEB-1 and KEB-7 cells exhibited almost no fragility, whereas KEB-11 cells displayed significant amounts of necrosis. This is the first study to demonstrate the fragility of EBS cell monolayers in culture and also the first to demonstrate that EBS cells are particularly fragile when loaded in shear.