Junctional Epidermolysis Bullosa Keratinocytes in Culture Display Adhesive, Structural, and Functional Abnormalities

An unusual, elongated, refractile cell morphology was observed in keratinocytes cultured from three patients with non-lethalis forms of junctional epidermolysis bullosa (JEB). To determine whether these changes might be related to altered cell adhesion, keratinocyte strains established from one patient were examined for adhesive, structural, and functional characteristics. JEB keratinocytes expressed keratin tonofilaments, as determined by staining with AE1 monoclonal antibodies and direct observation of tonofilaments by electron microscopy. JEB keratinocytes showed diminished cell-substratum adhesions, judged by interference reflection microscopy. Areas of diminished cell-substratum adhesion corresponded to F-actin-rich cell adhesions (focal adhesions) and not to cellular areas that abundantly express hemidesmosomal antigens. Analysis of cell-substratum adhesion by electron microscopy revealed extensive areas of cell-substratum separation in JEB keratinocytes that were not present in normal keratinocytes maintained in serum-free medium. Normal keratinocytes displayed numerous regions of focal contact between the ventral plasma membrane and the culture substratum, but these structures were not seen in JEB keratinocytes. Bundled actin filaments (stress fibers) were greatly diminished in expected regions of cell-substratum adhesion in JEB keratinocytes and, instead, displayed disorganized individual filaments. The growth rate of JEB keratinocytes was quite slow in culture, with a population doubling time of 2.7 d versus 1.5 d for normal keratinocytes under identical conditions. JEB keratinocytes also displayed a reduced ability to aggregate into colonies upon exposure to medium with increased extracellular calcium. JEB keratinocytes thus display adhesive, structural, and functional abnormalities that suggest this cell type may be central to the pathogenesis of junctional epidermolysis bullosa. Study of affected keratinocytes could be important to characterize associated molecular pathologies