Keratins are intermediate filament proteins expressed in all epithelial cell types. Keratin gene mutations cause a number of severe hereditary skin fragility disorders. Among these is the simplex subtype of epidermolysis bullosa (EBS), linked to mutations in keratins 5 and 14. Mutated keratins form a defective intermediate filament cytoskeleton and place EBS keratinocytes in a state of stress. This causes a continuous activation of MAP kinase signaling pathawys and alters gene expression of hundreds of other proteins, many of which have a function in cell-cell and cell-surface adhesion. Patients with the severe EBS-DM phenotype have an increased cumulative risk for basal cell carcinoma that correlates with sun exposure, but may not entirely correlate with the regions of frequent skin blistering.
To study the effects of keratin mutations, we first used patient-derived cell lines in cell based assays and demonstrate that regardless of the mutation their different genetic background may affect the way cells respond to experimental treatment in cell-based assays, so the accurate definition of cell culture conditions (the composition of medium, number of cells plated and number of days in the culture) is essential for the comparison.
We compared patient-derived EBS keratinocytes with control cell lines and tested how they behave in the face of two different types of stressors that are able to induce cell death: ionizing radiation and cytokines TNF-a and TRAIL. The data point out to a substantial difference between how normal and keratin mutant keratinocytes deal with such stresses. Within the complex picture of events following exposure to ionizing radiation we established that the expression of a number of proteins involved in DNA damage response, such as pChk2, pH2AX, pBRCA1 and pATM, was changed in mutant keratinocytes. The most striking decrease was observed for pChk2, which appears constitutively down regulated in severe EBS mutants, and so may even modulate the G2/M cell cycle checkpoint in these cells. Keratin mutants also appear less sensitive than normal cells to treatment with TNF-a and TRAIL, and this may be linked to the up-regulation of two pro-survival proteins, Bcl-2 and FLIP. These changes may be a protective mechanism that helps promote survival of EBS keratinocytes, but may also allow severely damaged cells to escape checkpoint control and apoptosis, and in theory this also may be a contributing factor to the cell transformation.
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