title: Characterization of immortalized human epidermolysis bullosa simplex (KRT5) cell lines: trimethylamine N-oxide protects the keratin cytoskeleton against disruptive stress condition.
abstract: BACKGROUND
  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.
  
  
  OBJECTIVE
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Medical Sciences/Dermatology and Venereology, Uppsala University Hospital, SE-751 85 Uppsala, Sweden.
authors.forename: Jean Christopher
authors.initials: JC
authors.lastname: Chamcheu
authors.forename: Elizabeth Pavez
authors.initials: EP
authors.lastname: Lorié
authors.forename: Baki
authors.initials: B
authors.lastname: Akgul
authors.forename: Elin
authors.initials: E
authors.lastname: Bannbers
authors.forename: Marie
authors.initials: M
authors.lastname: Virtanen
authors.forename: Luke
authors.initials: L
authors.lastname: Gammon
authors.forename: Aristidis
authors.initials: A
authors.lastname: Moustakas
authors.forename: Harshad
authors.initials: H
authors.lastname: Navsaria
authors.forename: Anders
authors.initials: A
authors.lastname: Vahlquist
authors.forename: Hans
authors.initials: H
authors.lastname: Törmä
mesh_terms: D017209:Apoptosis / Q000187:drug effects; D001706:Biopsy; D016022:Case-Control Studies; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D003599:Cytoskeleton / Q000187:drug effects* / Q000378:metabolism; D016110:Epidermolysis Bullosa Simplex / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D006358:Hot Temperature / Q000009:adverse effects*; D006801:Humans; D066298:In Vitro Techniques; D053555:Keratin-5 / Q000235:genetics* / Q000378:metabolism; D015603:Keratinocytes / Q000378:metabolism / Q000473:pathology*; D007633:Keratins / Q000378:metabolism*; D008744:Methylamines / Q000494:pharmacology*; D008954:Models, Biological; D009154:Mutation / Q000235:genetics; D016877:Oxidants / Q000494:pharmacology; D012867:Skin / Q000378:metabolism / Q000473:pathology
chemical_list: C503940:KRT5 protein, human; D053555:Keratin-5; D008744:Methylamines; D016877:Oxidants; D007633:Keratins; C005855:trimethyloxamine