TEMPERATURE SENSITIVITY OF THE KERATIN CYTOSKELETON AND DELAYED SPREADING OF KERATINOCYTE LINES DERIVED FROM EBS PATIENTS

Point mutations in the keratin intermediate filament genes for keratin 5 or keratin 14 are known to cause hereditary skin blistering disorde rs such as epidermolysis bullosa simplex, in which epidermal keratinoc ytes are extremely fragile and the skin blisters on mild trauma. We sh ow that in 2 phenotypically diverse cases of epidermolysis bullosa sim plex, the keratin mutations result in a thermoinstability of the inter mediate filament cytoskeleton which can be reproducibly demonstrated e ven in the presence of tissue culture-induced keratins and in conditio ns where filament fragility is not otherwise obvious. SV40-T antigen a nd HPV16 (E6^E7) immortalised keratinocyte cell lines were examined, e stablished from control and epidermolysis bullosa simplex-affected ind ividuals with either severe (Dowling-Meara) or mild (Weber-Cockayne) f orms of the disease. In standard tissue culture conditions no signific ant and consistent abnormality of the keratin cytoskeleton could be de monstrated. However after thermal stress a reduced stability of the ke ratin filaments was demonstrable in the epidermolysis bullosa simplex cell lines, with filaments breaking into aggregates similar to those s een in skin from EBS patients. These aggregates were maximal at 15 min utes after heat shock and the filament network structure was substanti ally reversed by 60 minutes. Differences were also seen in the cells d uring respreading after replating: cells containing mutant keratins we re slower to respread than controls and fine aggregates were seen at t he cell margins in the Dowling-Meara derived cell line. Such delays in restoring the normal intermediate filament network after physiologica l processes involving cytoskeleton remodelling may render the cells vu lnerable to cytolysis in vivo if physically challenged during this tim e window. The steady reduction in the mitotic index of the epidermis d uring the first few years of life could then explain the clinical impr ovement which is frequently observed in growing children.