Heiko Büth

• In this work, the involvement of the lysosomal cysteine peptidase cathepsin B in regeneration after scratch-wounding and in spontaneous keratinocyte migration was investigated. As cellular model systems, the human keratinocyte cell line HaCaT and normal human epidermal keratinocytes were used. In a standardized scratch-wounding assay, it was shown that inhibition of extra- and intracellular cathepsin B using specific inhibitors led to an impairment of keratinocyte migration. This was true for the cell line HaCaT and also for NHEK, demonstrating that HaCaT cells represent a suitable model to study keratinocyte migration. Furthermore, we wanted to identify the binding-partner for cathepsin B at the plasma membrane of migrating keratinocytes. The possibility of annexin-II being the plasma membrane receptor for secreted cathepsin B was ruled out. As alternative candidate, we investigated the LRP1 as a possible binding partner. Using immunofluorescence, it was shown that cathepsin B and LRP1 co-localize at the plasma membrane. By incubating HaCaT keratinocytes with an endogenous inhibitor for LRP1, it was detected that blocking of the ligand binding domains of LRP1 after scratch-wounding resulted in increased amounts of soluble cathepsin B in the culture media. Likewise, the levels of plasma membrane-associated cathepsin B were decreased, suggesting that LRP1 is in fact the binding partner for cathepsin B. To enable the direct observation of cathepsin B sorting and trafficking in living keratinocytes, we designed a plasmid encoding for a chimeric protein of HaCaT cells derived cathepsin B and eGFP. Our results showed that the secreted lysosomal cysteine peptidase cathepsin B is important for the process of keratinocyte migration after scratch-wounding, and we additionally showed that LRP1 is most likely the binding-partner at the plasma membrane of migrating keratinocytes to localize cathepsin B.