Abstract (eng)
Plectin is a large (~500 kDa) and versatile member of a family of proteins called plakins or cytolinkers. It is able to interact with a multitude of cytoskeletal pro-teins including components of all major cytoskeletal filament systems of the cy-toskeleton, and is spliced into various isoforms. Plectin isoform 1a (P1a), which is predominantly expressed in skin, is an essential component of hemidesmosomes (HDs) where it binds to integrin β4 (ITGβ4). HDs are transmembrane adhesion complexes, which anchor basal keratinocytes of the epidermis to the underlying basal lamina, thus conferring mechanical stability to the skin. A single autosomal dominant missense mutation in the plectin gene causes the skin blistering disease Epidermolysis Bullosa Simplex-Ogna (EBS-Ogna), which manifests with dra-matically diminished P1a protein levels and reduced numbers of HDs. Calpains, cysteine proteases expressed in several tissues including the epidermis, have been demonstrated to degrade ITGβ4. Proteolytic degradation of HD components could be an important prerequisite for terminal differentiation of keratinocytes and could also be involved in the pathomechanism underlying EBS-Ogna. For these reasons, I assessed the role of calpains as possible candidates for P1a proteolysis at the onset of terminal differentiation and in the pathogenesis of EBS-Ogna.
Performing an enzymatic activity assay, I could demonstrate that calpains are ac-tive in immortalized mouse keratinocytes. I also found that in immortalized mouse keratinocytes a fraction of calpains is localized at the plasma membrane and asso-ciated with the cytoskeleton. Furthermore, hyperactivation of calpains in immorta-lized mouse keratinocytes led to P1a and ITGβ4 degradation, which could be blocked by treatment with a cell permeable peptide inhibitor of calpains. Results with the analysis of primary mouse keratinocytes undergoing differentiation sug-gested that a fraction of calpains must be active during terminal differentiation since treatment of the cells with a pharmacological inhibitor of cysteine proteases increased the protein levels of the inactive full length form of calpains. Further-more, using a mouse model mimicking EBS-Ogna I found that calpains are in-volved in the proteolysis of P1a in primary keratinocytes derived from mutant animals. Upon inhibition of calpains in primary EBS-Ogna keratinocytes, elevated P1a protein levels could be detected along with increased numbers of HD-like protein complexes (HPCs). Finally, I established clonal keratinocyte cell lines carrying the heterozygous EBS-Ogna mutation (Plecwt/Ogna). Compared to wild-type clones, Plecwt/Ogna keratinocytes showed signs of proliferation defects pointing towards a hitherto undetected role of HDs as regulators of cell proliferation.