Characterization of immortalized human epidermolysis bullosa simplex (KRT5) cell lines: trimethylamine N-oxide protects the keratin cytoskeleton against disruptive stress condition.
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.
Chamcheu
Jean Christopher
JC
Department of Medical Sciences/Dermatology and Venereology, Uppsala University Hospital, SE-751 85 Uppsala, Sweden.
Lorié
Elizabeth Pavez
EP
Akgul
Baki
B
Bannbers
Elin
E
Virtanen
Marie
M
Gammon
Luke
L
Moustakas
Aristidis
A
Navsaria
Harshad
H
Vahlquist
Anders
A
Törmä
Hans
H
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
C503940:KRT5 protein, human; D053555:Keratin-5; D008744:Methylamines; D016877:Oxidants; D007633:Keratins; C005855:trimethyloxamine