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

Abstract

In 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.

Detrimental Effects of IFN-γ on an Epidermolysis Bullosa Simplex Cell Model and Protection by a Humanized Anti–IFN-γ Monoclonal Antibody

“…K14 seen in cell lysates of subconfluent N/TERT-1 KCs stably expressing GFP-tagged K14 WT or GFP-tagged K14 R125P. Actin as loading control (a similar but nonidentical immunoblot is shown in Tan et al. [2021] wherein these lines are described.)…”

“…Using this tissue culture disease model, we asked whether IFN-γ itself could have an effect on any of the phenotypic characteristics that define EBS. Using a semiautomated image analysis algorithm ( Tan et al., 2021 ), we assessed keratin aggregate formation in the presence and absence of human recombinant IFN-γ added to the culture medium. When subconfluent EBS KCs were treated with IFN-γ, the cells retained abundant keratin aggregates for at least 7 days ( Figure 2 a and b), that is, well beyond confluence.…”

“…Protein concentration of the supernatants was then determined by BCA assay (Thermo Fisher Scientific) and standardized using BSA. SDS-PAGE and immunoblotting were performed as previously described ( Tan et al., 2021 ). Protein levels were quantified from images of immunoblots using ImageJ software (National Institutes of Health, Bethesda, MD) ( Schneider et al., 2012 ).…”

“…Fluorescent images of EBS cells with keratin aggregates were acquired using the Olympus IX-81 high content screening inverted microscope (Olympus, Tokyo, Japan), attached with a Marzhauser Scan IM (120 × 100) motorized XY stage controlled by Ludl Mac5000 and a Photometrics CCD camera (CoolSNAP HQ2) with DAPI, EGFP, and TRITC filters. Fluorescent images were analyzed with customized algorithms written with ImageJ software for quantitative analysis of keratin aggregates in mutant cells ( Tan et al., 2021 ). Alternatively, these images were analyzed using CellProfiler free open-source software ( cellprofiler.org ) to count keratin aggregates in mutant cells.…”

“…Briefly, the wells were washed once with PBS, before a solution containing 2 μM calcein AM, 1 μM ethidium homodimer-1, and 1 μM Hoechst 33342 (Thermo Fisher Scientific) in PBS was added to each well and cells were incubated for 45 minutes in the dark at room temperature. Fluorescent images in each well were then acquired using the Olympus IX-81 high content screening inverted microscope, and postanalyzed using ImageJ software as previously described ( Tan et al., 2021 ).…”