bromochloroacetic-acid and 4-phenylbutyric-acid

Missense mutations in keratin 5 and 14 genes cause the severe skin fragility disorder epidermolysis bullosa simplex (EBS) by collapsing of the keratin cytoskeleton into cytoplasmic protein aggregates. Despite intense efforts, no molecular therapies are available, mostly due to the complex phenotype of EBS, comprising cell fragility, diminished adhesion, skin inflammation and itch.. We extensively characterized KRT5 and KRT14 mutant keratinocytes from patients with severe generalized EBS following exposure to the chemical chaperone 4-phenylbutyrate (4-PBA).. 4-PBA diminished keratin aggregates within EBS cells and ameliorated their inflammatory phenotype. Chemoproteomics of 4-PBA-treated and untreated EBS cells revealed reduced IL1β expression- but also showed activation of Wnt/β-catenin and NF-kB pathways. The abundance of extracellular matrix and cytoskeletal proteins was significantly altered, coinciding with diminished keratinocyte adhesion and migration in a 4-PBA dose-dependent manner.. Together, our study reveals a complex interplay of benefits and disadvantages that challenge the use of 4-PBA in skin fragility disorders.

Topics: Animals; Apoptosis; Biomarkers; Biopsy; Cell Adhesion; Cell Communication; Cell Line; Cytoskeleton; Disease Models, Animal; Epidermolysis Bullosa; Extracellular Matrix; Humans; Immunohistochemistry; Keratinocytes; Keratins; Mice; Phenotype; Phenylbutyrates; Protein Transport; Proteome; Proteomics; Signal Transduction; Skin

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.. To generate immortalized keratinocyte lines from patients with EI for studies of EI cell pathology and the effects of chemical chaperones as putative therapies.. 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.. 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.. These immortalized cell lines represent a useful model for studying EI biology and novel therapies.

Topics: Adolescent; Adult; Cell Line; Cell Transformation, Viral; Epidermis; Hot Temperature; HSP70 Heat-Shock Proteins; Humans; Hyperkeratosis, Epidermolytic; Keratinocytes; Keratins; Male; Methylamines; Models, Biological; Phenotype; Phenylbutyrates; Stress, Physiological; Young Adult