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Characterization of immortalized human epidermolysis bullosa simplex (KRT5) cell lines: trimethylamine N-oxide protects the keratin cytoskeleton against disruptive stress condition
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Dermatology and Venereology. (Dermatology and Venereology)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Dermatology and Venereology. (Dermatologi och venereologi, Dermatology and Venereology)
Centre for Cutaneous Research, ICMS, Bart's and London Queen Mary's School of Medicine and Dentistry, London, United Kingdom.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Dermatology and Venereology.
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2009 (English)In: Journal of dermatological science (Amsterdam), ISSN 0923-1811, E-ISSN 1873-569X, Vol. 53, no 3, 198-206 p.Article in journal (Refereed) Published
Abstract [en]

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.

OBJECTIVES: 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.

CONCLUSION: 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.

Place, publisher, year, edition, pages
Elsevier , 2009. Vol. 53, no 3, 198-206 p.
Keyword [en]
Chemical chaperone, Cytoprotection, EBS-cell lines, Genetic skin disorder, Human papilloma virus 16 E6/E7, Heat stress, Keratin, Organotypic epidermis, TMAO
National Category
Medical and Health Sciences
Research subject
Dermatology and Venerology
URN: urn:nbn:se:uu:diva-88245DOI: 10.1016/j.jdermsci.2008.11.003ISI: 000263766300005PubMedID: 19157792OAI: oai:DiVA.org:uu-88245DiVA: diva2:139869
Available from: 2010-05-03 Created: 2009-01-27 Last updated: 2010-08-25Bibliographically approved
In thesis
1. Disease-causing Keratin Mutations and Cytoskeletal Dysfunction in Human Skin: In vitro Models and new Pharmacologic Strategies for Treating Epidermolytic Genodermatoses
Open this publication in new window or tab >>Disease-causing Keratin Mutations and Cytoskeletal Dysfunction in Human Skin: In vitro Models and new Pharmacologic Strategies for Treating Epidermolytic Genodermatoses
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Epidermolysis bullosa simplex (EBS) and epidermolytic ichthyosis (EI) are rare skin fragility diseases characterized by intra-epidermal blistering due to autosomal dominant-negative mutations in basal (KRT5 or KRT14) and suprabasal (KRT1 or KRT10) keratin genes,  respectively. Despite vast knowledge in the disease pathogenesis, the pathomechanisms are not fully understood, and no effective remedies exist. The purpose of this work was to search for keratin gene mutations in EBS patients, to develop in vitro models for studying EBS and EI, and to investigate novel pharmacological approaches for both diseases.

We identified both novel and recurrent KRT5 mutations in all studied EBS patients but one which did not show any pathogenic keratin mutations. Using cultured primary keratinocytes from EBS patients, we reproduced a correlation between clinical severity and cytoskeletal instability in vitro. Immortalized keratinocyte cell lines were established from three EBS and three EI patients with different phenotypes using HPV16-E6E7. Only cell lines derived from severely affected patients exhibited spontaneous keratin aggregates under normal culture conditions. However, heat stress significantly induced keratin aggregates in all patient cell lines. This effect was more dramatic in cells from patients with a severe phenotype. In organotypic cultures, the immortalized cells were able to differentiate and form a multilayered epidermis reminiscent of those observed in vivo. Addition of two molecular chaperones, trimethylamine N-oxide dihydrate (TMAO) and sodium 4-phenylbutyrate (4-PBA), reduced the keratin aggregates in both stressed and unstressed EBS and EI keratinocytes, respectively. The mechanism of action of TMAO and 4-PBA was shown to involve the endogenous chaperone system (Heat shock proteins e.g. Hsp70). Besides, MAPK signaling pathways also seemed to be incriminated in the pathogenesis of EBS. Furthermore, depending on which type of keratin is mutated, 4-PBA up-regulated Hsp70 and KRT4 (possibly compensating for mutated KRT1/5), and down-regulated KRT1 and KRT10, which could further assist in protecting EBS and EI cells against stress.

In conclusion, novel and recurrent pathogenic keratin mutations have been identified in EBS. Immortalized EBS and EI cell lines that functionally reflect the disease phenotype were established. Two pharmacologic agents, TMAO and 4-PBA, were shown to be promising candidates as novel treatment of heritable keratinopathies in this in vitro model.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2010. 85 p.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 564
epidermolysis bullosa simplex, epidermolytic ichthyosis, genodermatoses, keratin, keratin mutation, keratinocytes, gene therapy, pharmacological therapy, immortalization, gene regulation, trimethylamine N-oxide (TMAO), sodium 4-phenylbutyrate (4-PBA), tissue engineering, cell culture, heat shock proteins, MAP kinases
National Category
Dermatology and Venereal Diseases
Research subject
Dermatology and Venerology
urn:nbn:se:uu:diva-123071 (URN)978-91-554-7816-2 (ISBN)
Public defence
2010-06-04, Rosénsalen, AS Akademiska sjukhuset, ingång 95/96, Uppsala, 13:15 (English)
Available from: 2010-05-12 Created: 2010-04-23 Last updated: 2011-03-15Bibliographically approved

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