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Disease-causing Keratin Mutations and Cytoskeletal Dysfunction in Human Skin: In vitro Models and new Pharmacologic Strategies for Treating Epidermolytic Genodermatoses
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Dermatology and Venereology. (Dermatology and Venereology)
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. , p. 85
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 564
Keywords [en]
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: urn:nbn:se:uu:diva-123071ISBN: 978-91-554-7816-2 (print)OAI: oai:DiVA.org:uu-123071DiVA, id: diva2:311783
Public defence
2010-06-04, Rosénsalen, AS Akademiska sjukhuset, ingång 95/96, Uppsala, 13:15 (English)
Opponent
Supervisors
Available from: 2010-05-12 Created: 2010-04-23 Last updated: 2011-03-15Bibliographically approved
List of papers
1. 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
Open this publication in new window or tab >>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
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2010 (English)In: British Journal of Dermatology, ISSN 0007-0963, E-ISSN 1365-2133, Vol. 162, no 5, p. 980-989Article 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
Keywords
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:nbn:se:uu:diva-114415 (URN)10.1111/j.1365-2133.2009.09615.x (DOI)000276853600006 ()20128788 (PubMedID)
Available from: 2010-05-03 Created: 2010-02-15 Last updated: 2017-12-12Bibliographically approved
2. Characterization of immortalized human epidermolysis bullosa simplex (KRT5) cell lines: trimethylamine N-oxide protects the keratin cytoskeleton against disruptive stress condition
Open this publication in new window or tab >>Characterization of immortalized human epidermolysis bullosa simplex (KRT5) cell lines: trimethylamine N-oxide protects the keratin cytoskeleton against disruptive stress condition
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2009 (English)In: Journal of dermatological science (Amsterdam), ISSN 0923-1811, E-ISSN 1873-569X, Vol. 53, no 3, p. 198-206Article 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
Keywords
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
Identifiers
urn:nbn:se:uu:diva-88245 (URN)10.1016/j.jdermsci.2008.11.003 (DOI)000263766300005 ()19157792 (PubMedID)
Available from: 2010-05-03 Created: 2009-01-27 Last updated: 2017-12-14Bibliographically approved
3. Chemical chaperones protect epidermolysis bullosa simplex keratinocytes from heat stress-induced keratin aggregation::  Involvement of heat shock proteins and MAP kinases
Open this publication in new window or tab >>Chemical chaperones protect epidermolysis bullosa simplex keratinocytes from heat stress-induced keratin aggregation::  Involvement of heat shock proteins and MAP kinases
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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.

Keywords
EBS, keratinocytes, keratin, MAPK, immunostaining, Hsp, TMAO, 4-PBA
National Category
Medical and Health Sciences
Research subject
Dermatology and Venerology
Identifiers
urn:nbn:se:uu:diva-123064 (URN)
Available from: 2010-04-23 Created: 2010-04-23 Last updated: 2017-12-12Bibliographically approved
4. Immortalized keratinocytes derived from patients with epidermolytic ichthyosis reproduce the disease phenotype: A useful in vitro model for testing new treatments
Open this publication in new window or tab >>Immortalized keratinocytes derived from patients with epidermolytic ichthyosis reproduce the disease phenotype: A useful in vitro model for testing new treatments
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2011 (English)In: British Journal of Dermatology, ISSN 0007-0963, E-ISSN 1365-2133, Vol. 164, no 2, p. 263-272Article 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
National Category
Medical and Health Sciences
Research subject
Dermatology and Venerology
Identifiers
urn:nbn:se:uu:diva-123067 (URN)10.1111/j.1365-2133.2010.10092.x (DOI)000286668300009 ()20977447 (PubMedID)
Available from: 2010-04-23 Created: 2010-04-23 Last updated: 2017-12-12Bibliographically approved

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Citation style
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