| 1. |
- Chamcheu, Jean Christopher, et al.
(författare)
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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
- 2011
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Ingår i: Journal of Investigative Dermatology. - : Elsevier BV. - 0022-202X .- 1523-1747. ; 131:8, s. 1684-1691
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Tidskriftsartikel (refereegranskat)abstract
- Epidermolysis bullosa simplex (EBS) is a blistering skin disease caused by 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 other protein-folding disorders, involvement of molecular chaperones and the ubiquitin-proteasome system may modify disease severity. In this study, the effects of heat stress on keratin aggregation in immortalized cells from two patients with EBS (KRT5) and a healthy control were examined with and without addition of various test compounds. Heat-induced (43 °C, 30 minutes) aggregates were observed in all cell lines, the amount of which correlated with the donor phenotype. In EBS cells pre-exposed to proteasome inhibitor, MG132, and p38-mitogen-activated protein kinase (MAPK) inhibitor, SB203580, the proportion of aggregate-positive cells increased, suggesting a role of proteasomes and phosphorylation in removing mutated keratin. In contrast, aggregates were reduced by pretreatment with two chemical chaperones, trimethylamine N-oxide (TMAO) and 4-phenylbutyrate (4-PBA). TMAO also modulated stress-induced p38/c-jun N-terminal kinase (JNK) activation and expression of heat shock protein (HSPA1A), the latter of which colocalized with phosphorylated keratin 5 in EBS cells. Taken together, our findings suggest therapeutic targets for EBS and other keratinopathies.
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| 2. |
- Chamcheu, Jean Christopher, et al.
(författare)
-
Chemical chaperones protect epidermolysis bullosa simplex keratinocytes from heat stress-induced keratin aggregation: : Involvement of heat shock proteins and MAP kinases
-
Ingår i: Journal of Investigative Dermatology. - 0022-202X .- 1523-1747.
-
Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 3. |
- Kolundzic, Nikola, et al.
(författare)
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Induced pluripotent stem cell (iPSC) line from an epidermolysis bullosa simplex patient heterozygous for keratin 5 E475G mutation and with the Dowling Meara phenotype
- 2019
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Ingår i: Stem Cell Research. - : ELSEVIER SCIENCE BV. - 1873-5061 .- 1876-7753. ; 37
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Tidskriftsartikel (refereegranskat)abstract
- We have generated MLi002-A, a new induced pluripotent stem cell (iPSC) line derived from keratinocytes of a skin punch biopsy of a female patient with the severe epidermolysis bullosa simplex Dowling-Meara phenotype and the keratin K5 E475G mutation. Keratinocytes were reprogrammed using non-integrating Sendai virus vectors, and xeno-free culture conditions were used throughout. The characterization of MLi002-A cell line consisted of molecular karyotyping, mutation screening using restriction enzyme digestion and Sanger sequencing, and testing of the pluripotency and differentiation potentials by immunofluorescence of associated markers both in vitro and in vivo. This is the first iPSC model of EB Simplex.
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| 4. |
- Zupancic, Tina, et al.
(författare)
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Keratin gene mutations influence the keratinocyte response to DNA damage and cytokine induced apoptosis
- 2017
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Ingår i: Archives of Dermatological Research. - : Springer Science and Business Media LLC. - 0340-3696 .- 1432-069X. ; 309:7, s. 587-593
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Tidskriftsartikel (refereegranskat)abstract
- The keratin filament cytoskeleton is vital to the normal function of epithelial cells. It provides structural support and regulates different aspects of cell metabolism. Mutations in keratins 5 and 14 cause a skin fragility disorder, epidermolysis bullosa simplex (EBS). Patients with severe EBS have an increased cumulative risk for basal cell carcinoma. In this study, we tested how keratin 5 and 14 mutant EBS patient-derived keratinocytes behave in the face of two different types of stressors that are able to induce cell death: ionizing radiation and cytokines TNF-alpha and TRAIL. The data point out to a substantial difference between how normal and keratin mutant keratinocytes deal with such stresses. When case of DNA damage, the ATM/Chk2-pathway is one of the two main tracks that can prevent the progression of mitosis and so allow repair. This was altered in all investigated keratin mutants with a particular down-regulation of the activated form of checkpoint kinase 2 (pChk2). Keratin mutants also appear less sensitive than normal cells to treatment with TNF-alpha or TRAIL, and this may be linked to the up-regulation of two pro-survival proteins, Bcl-2 and FLIP. Such changes are likely to have a profound effect on mutant keratinocytes ability to survive and withstand stress, and in theory this may be also a contributing factor to cell transformation.
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| 5. |
- Zupancic, Tina, et al.
(författare)
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Keratinocyte-based cell assays : their potential pitfalls
- 2012
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Ingår i: Archives of Dermatological Research. - : Springer Science and Business Media LLC. - 0340-3696 .- 1432-069X. ; 304:9, s. 765-768
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Tidskriftsartikel (refereegranskat)abstract
- As an in vitro model system, patient-derived epidermolysis bullosa simplex keratinocytes have had an immense impact on what we know today about keratin filament function and their role in disease development. In the absence of gene therapy, screening compound libraries for new or better drugs is another approach to improve existing treatments for genodermatoses. However in this study, we report of the potential pitfalls when using this type of cell lines as a "reporter" system. When cell lines with different genetic backgrounds are being used in cell-based assays, the greatest obstacle is to determine the most appropriate culture conditions (i.e., the composition of medium, number of cells plated and number of days in culture). We demonstrate how culture conditions can greatly interfere with the cellular response in cell-based assays (cell proliferation, metabolic activity and migration), potentially also giving rise to misleading data.
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