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Chemical chaperones protect epidermolysis bullosa simplex keratinocytes from heat stress-induced keratin aggregation::  Involvement of heat shock proteins and MAP kinases
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences. (Dermatologi och venereologi, Dermatology and Venereology)
Bart's and London Queen Mary's University School of Medicine and Dentistry . (ICMS, Centre for Cutaneous Research)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Dermatology and Venereology. (Dermatology and Venereology)
University of Ljubjana, Institute of Chemistry and Centre for Molecular Biology.
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(English)In: Journal of Investigative Dermatology, ISSN 0022-202X, E-ISSN 1523-1747Article in journal (Refereed) Submitted
Abstract [en]

Epidermolysis bullosa simplex (EBS) is an inherited epithelial tissue fragility disorder due to mutations in keratin genes (KRT5 or KRT14), with no existing therapies. Aggregates of misfolded mutant keratins are seen in cultured keratinocytes from severe EBS patients. In some protein folding disorders such as cystic fibrosis and Alzheimer’s disease, chaperones and the ubiquitin-proteasome system modify disease severity, suggesting a novel therapeutic approach even for EBS. In this study, the effects of two chemical chaperones (trimethylamine-N oxide (TMAO) and 4-phenylbutyrate (4-PBA)) on heat stress-induced keratin aggregation responses were examined in newly and previously established immortalized control and EBS patient-derived keratinocyte cell lines. Heat-induced keratin-positive aggregates were observed in all EBS cells, which were most prominent in severe keratin-defective cell lines and less so in normal cells. The proportion of cells containing aggregates were dramatically reduced by TMAO and 4-PBA pretreatment. Furthermore, heat stress greatly induced MAP kinase activation (p38 and JNK) and increased Hsp70/Hsc70 expression, and TMAO was able to transiently modulate these effects. The results suggest that TMAO rescue may involve components of the endogenous chaperone and MAPK machineries, which may represent novel targets for the development of more effective treatments for EBS and other keratin-related genetic disorders.

Keyword [en]
EBS, keratinocytes, keratin, MAPK, immunostaining, Hsp, TMAO, 4-PBA
National Category
Medical and Health Sciences
Research subject
Dermatology and Venerology
URN: urn:nbn:se:uu:diva-123064OAI: oai:DiVA.org:uu-123064DiVA: diva2:311762
Available from: 2010-04-23 Created: 2010-04-23 Last updated: 2010-05-18Bibliographically 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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Chamcheu, Jean ChristopherTörmä, Hans
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