A cell-based drug discovery assay identifies inhibition of cell stress responses as a new approach to treatment of epidermolysis bullosa simplex.

Tong San Tan,E Birgitte Lane,Muhammad Jasrie Firdaus,John S Y Lim,Steven S Leonardi,John E A Common,Cedric Badowski

doi:10.1242/jcs.258409

Abstract

ABSTRACTIn the skin fragility disorder epidermolysis bullosa simplex (EBS), mutations in keratin 14 (K14, also known as KRT14) or keratin 5 (K5, also known as KRT5) lead to keratinocyte rupture and skin blistering. Severe forms of EBS are associated with cytoplasmic protein aggregates, with elevated kinase activation of ERK1 and ERK2 (ERK1/2; also known as MAPK3 and MAPK1, respectively), suggesting intrinsic stress caused by misfolded keratin protein. Human keratinocyte EBS reporter cells stably expressing GFP-tagged EBS-mimetic mutant K14 were used to optimize a semi-automated system to quantify the effects of test compounds on keratin aggregates. Screening of a protein kinase inhibitor library identified several candidates that reduced aggregates and impacted on epidermal growth factor receptor (EGFR) signalling. EGF ligand exposure induced keratin aggregates in EBS reporter keratinocytes, which was reversible by EGFR inhibition. EBS keratinocytes treated with a known EGFR inhibitor, afatinib, were driven out of activation and towards quiescence with minimal cell death. Aggregate reduction was accompanied by denser keratin filament networks with enhanced intercellular cohesion and resilience, which when extrapolated to a whole tissue context would predict reduced epidermal fragility in EBS patients. This assay system provides a powerful tool for discovery and development of new pathway intervention therapeutic avenues for EBS.

Highlights

Epidermolysis bullosa simplex (EBS) is a rare skin condition usually caused by mutations in the keratin intermediate filament proteins keratin 5 (K5, known as KRT5) or keratin 14 (K14, known as KRT14) (Bonifas et al, 1991; Coulombe et al, 1991; Lane et al, 1992), leading to skin blistering and wound development
NEB-1 keratinocytes engineered to express plasmid-derived GFP–K14 R125P mutant protein to mimic EBS-gs show mutation-related elevation of activated ERK1/2 when compared to NEB-1-based keratinocytes expressing wild-type GFP–K14 protein (GFP–K14 WT) (Fig. 1A), similar to the patient-derived KEB-7 keratinocytes that we reported previously (Morley et al, 2003; Russell et al, 2010)
These results suggest that these mutant keratinocytes and their control wild-type keratinocyte counterparts, when grown in keratinocyte serum-free medium (K-SFM), constitute a robust system in which to investigate the mechanism of keratin aggregate formation and their relationship to the clinical EBS-gs phenotype

Summary

Introduction

Epidermolysis bullosa simplex (EBS) is a rare skin condition usually caused by mutations in the keratin intermediate filament proteins keratin 5 (K5, known as KRT5) or keratin 14 (K14, known as KRT14) (Bonifas et al, 1991; Coulombe et al, 1991; Lane et al, 1992), leading to skin blistering and wound development. Keratinocytes with the keratin mutations found in severe EBS patients [generalized severe EBS (EBS-gs), called EBS Dowling–Meara], are under intrinsic stress, mediated by constitutive mitogen-activated protein kinase (MAPK) pathways through JNK1 and JNK2 (JNK1/2, known as MAPK8 and MAPK9, respectively), p38 proteins, and ERK1 and ERK2 (ERK1/2, known as MAPK3 and MAPK1, respectively) (D’Alessandro et al, 2002; Morley et al, 2003; Liovic et al, 2008; Russell et al, 2010; Chamcheu et al, 2011a; Wagner et al, 2013) This stress has the features of a misfolded protein response (Chamcheu et al, 2009; Russell et al, 2010), and protein aggregates formed by the mutant keratin are hallmarks of severe EBS (Anton-Lamprecht and Schnyder, 1982). Such profound cytoskeletal differences would render the EBS cells less adhesive to one another, as would be required in wound healing activation

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Conclusion