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Epidermolysis bullosa simplex due to KRT5 mutations: mutation-related differences in cellular fragility and the protective effects of trimethylamine N-oxide in cultured primary keratinocytes
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Dermatology and Venereology. (Dermatologi och venereologi, 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 School of Medicine and Dentistry, Queen Mary University of London. (ICMS, Centre for Cutaneous Research)
Cardiff University, School of Medicine, Department of Dermatology.
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2010 (English)In: British Journal of Dermatology, ISSN 0007-0963, E-ISSN 1365-2133, Vol. 162, no 5, 980-989 p.Article in journal (Refereed) Published
Abstract [en]

Summary Background Epidermolysis bullosa simplex (EBS) is a mechanobullous skin fragility disease characterized by cytolysis of basal keratinocytes and intraepidermal blistering often caused by mutations in keratin genes (KRT5 or KRT14). No remedies exist for these disorders presenting a need for development of novel therapies. Objectives To identify new genotype-phenotype relationships in vivo and in cultured primary EBS keratinocytes in vitro, and to study the cytoskeletal stabilizing effects of trimethylamine N-oxide (TMAO) in heat-stressed EBS cells. Methods Genomic DNA and cDNA samples from three Swedish patients with EBS were analysed for keratin mutations. Primary EBS keratinocyte cultures were established, heat stressed with and without added TMAO, followed by evaluation of cellular fragility. Results In addition to the previously reported KRT5 mutation (V186L) in one patient, two patients were found to have a novel I183M and recurrent E475G replacements in KRT5. Cultured EBS keratinocytes did not exhibit keratin aggregates or cell loss, except in the patient with the p.I183M mutation who showed 3% aggregates and 2% cell loss. Upon transient heat stress the number of aggregate-containing cells increased to 21%, 27% and 13%, respectively, in the p.I183M, p.E475G and p.V186L mutant cells. Interestingly, pretreatment with TMAO prior to heat stress, dose dependently reduced the number of aggregate-containing cells and cell loss. Conclusion These results revealed a genotype-phenotype correlation in EBS keratinocytes upon heat stress and suggest protein stabilization as a new therapeutic strategy.

Place, publisher, year, edition, pages
Wiley InterScience , 2010. Vol. 162, no 5, 980-989 p.
Keyword [en]
chemical chaperones, heat stress, keratin filament aggregates, keratin mutation, keratinocytes, protein stability
National Category
Medical and Health Sciences
Research subject
Dermatology and Venerology
Identifiers
URN: urn:nbn:se:uu:diva-114415DOI: 10.1111/j.1365-2133.2009.09615.xISI: 000276853600006PubMedID: 20128788OAI: oai:DiVA.org:uu-114415DiVA: diva2:293979
Available from: 2010-05-03 Created: 2010-02-15 Last updated: 2017-12-12Bibliographically 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.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 564
Keyword
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
Identifiers
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)
Opponent
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Available from: 2010-05-12 Created: 2010-04-23 Last updated: 2011-03-15Bibliographically approved

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Chamcheu, Jean ChristopherTörmä, Hans

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