Epidermolysis bullosa simplex (EBS) is an autosomal inherited mechano-bullous disease, characterized by intraepidermal blistering and skin fragility caused by mutations in the keratin (KRT) 5 or 14 genes. Despite a vast knowledge about the intermediate filament pathology in this disease, the progress in therapy has been slow. Animal models and well-characterized continuous cell culture models of EBS are needed prior to clinical testing.
Our aim was to generate immortalized cell lines as an in vitro model for the study of EBS and test a chemical chaperone, trimethylamine N-oxide (TMAO), as a putative novel therapy.
We generated four immortalized cell lines, two each from an EBS patient with a KRT5-mutation (V186L) and a healthy control, using human papillomavirus 16 (HPV16) E6E7 as transducer. Cell lines were established in serum-free and serum-containing medium and assessed for growth characteristics, keratin expression profiles, ability to differentiate in organotypic cultures, and response to heat stress with and without the presence of TMAO.
All cell lines have been expanded >160 population doublings and their cellular characteristics are similar. However, the formation of cytoplasmic keratin filament aggregates in response to heat-shock treatment differed between EBS and normal cell lines. Notably, serum-free established EBS-cell line was most vulnerable to heat shock but both cell lines exhibited significant reduction in the number of keratin aggregates containing cells by TMAO.
The immortalized cell lines represent a suitable model for studying novel therapies for EBS. TMAO is a promising new agent for future development as a novel EBS therapy.
Abbreviations:EBS (epidermolysis bullosa simplex), DAPI (4′-6-diamidino-2-phenylindole), DED (de-epidermized dermis), HPV16 E6/E7 (human papilloma virus 16 E6–E7 proteins), KRT (keratin), TGm1 (transglutaminase-1), PBS (phosphate-buffered saline), PCR (polymerase chain reaction), TMAO (trimethylamine N-oxide dihydrate)
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Published online: July 12, 2010
Accepted: November 13, 2008
Received in revised form: September 17, 2008
Received: July 24, 2008
☆This experimental work was carried out at the Uppsala University Hospital and Biomedical Centre, Uppsala, Sweden and at Centre for Cutaneous Research, ICMS, Queen Mary's School of Medicine and Dentistry, London, UK.
© 2008 Japanese Society for Investigative Dermatology. Published by Elsevier Inc. All rights reserved.