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Immortalized keratinocytes derived from patients with epidermolytic ichthyosis reproduce the disease phenotype: A useful in vitro model for testing new treatments
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. (Dermatology and Venereology)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Dermatology and Venereology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Dermatology and Venereology.
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2011 (English)In: British Journal of Dermatology, ISSN 0007-0963, E-ISSN 1365-2133, Vol. 164, no 2, 263-272 p.Article in journal (Refereed) Published
Abstract [en]

Background: Epidermolytic ichthyosis (EI) is a skin fragility disorder caused by mutations in genes encoding suprabasal keratins 1 and 10. While the aetiology of EI is known, model systems are needed for pathophysiological studies and development of novel therapies. Objectives To generate immortalized keratinocyte lines from patients with EI for studies of EI cell pathology and the effects of chemical chaperones as putative therapies. Methods We derived keratinocytes from three patients with EI and one healthy control and established immortalized keratinocytes using human papillomavirus 16-E6/E7. Growth and differentiation characteristics, ability to regenerate organotypic epidermis, keratin expression, formation of cytoskeletal aggregates, and responses to heat shock and chemical chaperones were assessed. Results The cell lines EH11 (K1-p.Val176-Lys197del), EH21 (K10-p.156Arg>Gly), EH31 (K10-p.Leu161-Asp162del) and NKc21 (wild-type) currently exceed 160 population doublings and differentiate when exposed to calcium. At resting state, keratin aggregates were detected in 9% of calcium-differentiated EH31 cells, but not in any other cell line. Heat stress further increased this proportion to 30% and also induced aggregates in 3% of EH11 cultures. Treatment with trimethylamine N-oxide and 4-phenylbutyrate (4-PBA) reduced the fraction of aggregate-containing cells and affected the mRNA expression of keratins 1 and 10 while 4-PBA also modified heat shock protein 70 (HSP70) expression. Furthermore, in situ proximity ligation assay suggested a colocalization between HSP70 and keratins 1 and 10. Reconstituted epidermis from EI cells cornified but EH21 and EH31 cells produced suprabasal cytolysis, closely resembling the in vivo phenotype. Conclusions These immortalized cell lines represent a useful model for studying EI biology and novel therapies.

 

Place, publisher, year, edition, pages
British Association of Dermatologists , 2011. Vol. 164, no 2, 263-272 p.
National Category
Medical and Health Sciences
Research subject
Dermatology and Venerology
Identifiers
URN: urn:nbn:se:uu:diva-123067DOI: 10.1111/j.1365-2133.2010.10092.xISI: 000286668300009PubMedID: 20977447OAI: oai:DiVA.org:uu-123067DiVA: diva2:311772
Available from: 2010-04-23 Created: 2010-04-23 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)
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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 ChristopherMoustakas, AristidisTörmä, Hans

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