P14 Targeting nuclear architecture in epidermolysis bullosa simplex

Abstract Introduction and aims Epidermolysis bullosa simplex (EBS) is a severe blistering skin disease caused by mutations in K5 and K14 which lead to keratin filament instability and protein aggregation. Currently, there is no cure for EBS and treatment is focused on relieving symptoms such as pain and infections. The keratin cytoskeleton is an important mediator of mechanotransduction to the nucleus and its destabilization can impair mechanosensing via the actin cytoskeleton. Altered nuclear architecture could be an important feature of EBS. Therefore, our aim is to investigate mechanically mediated nuclear and epigenetic changes in EBS and their role in pathogenesis. Methods Keratinocyte cell lines stably expressing wildtype (WT), R416P and R125H mutant K14 were generated and used to examine K14, lamin A/C and H3K27ac expression by immunofluorescence staining and Western blotting. Immunofluorescence staining and Western blotting were used to assess, nuclear morphology, γH2AX, and cGAS induction in mutant cell lines following exposure to 20 mJ cm−2 ultraviolet (UV)B or disruption of actomyosin contractility with 10 µmol L−1 Y27632 and 25 µmol L−1 blebbistatin. Results In mutant K14 N/TERT, keratin aggregates formed at the cell periphery, alongside increased nuclear deformation (circularity < 0.7) (n = 7). In response to UVB, both WT and mutant K14 N/TERT displayed increased expression of the DNA damage marker, γH2AX, at 3 h, which recovered after 24 h (n = 3). In preliminary studies, R416P N/TERT had higher baseline expression of cGAS (cyclic GMP-AMP synthase, a sensor of cytosolic DNA) compared with their WT counterparts, and in response to UVB, R416P N/TERT displayed a sustained elevated cGAS expression (n = 1). Furthermore, mutant K14 N/TERT are more sensitive to inhibition of actomyosin contractility than WT N/TERT, as measured by reduced cell density (n = 1). Conclusions These findings suggest that keratinocytes with EBS-causing K14 mutations have deformed nuclei and altered DNA damage responses.