HaCaT keratinocytes secrete lysosomal cysteine proteinases during migration

Cathepsin B, a lysosomal cysteine proteinase, was detected within vesicles of cellular protrusions forming cell-cell contact sites between keratinocytes of the stratum spinosum of human skin. This observation suggested the possibility that secretion of the protease into the pericellular spaces could be involved in the dissociation of cell-cell contacts to enable intraepidermal keratinocyte migration. To determine whether Cathepsin B is indeed secreted from migrating keratinocytes, we first used subconfluent HaCaT cells as a culture model to study spontaneous keratinocyte migration. A Cathepsin B-specific fluorescent affinity label proved the association of mature Cathepsin B with the surfaces of HaCaT cells at the leading edges of growing cells. Second, we used scratch-wounds of confluent HaCaT monolayers as a model of induced keratinocyte migration. Cathepsin B was detected within lysosomes, i.e. vesicles within the perinuclear region of non-wounded cells. Expression of Cathepsin B was up-regulated and Cathepsin B-positive vesicles showed a redistribution from perinuclear to peripheral regions of keratinocytes at the wound margins within 4 h after wounding. Enzyme cytochemistry further showed that cell surface-associated Cathepsin B was proteolytically active at the leading fronts of migrating keratinocytes. In addition, increased amounts of mature forms of Cathepsin B were detected within the conditioned media of HaCaT cells during the first 4 h after scratch-wounding. In contrast, and as a control, the activity of the cytosolic enzyme Lactate Dehydrogenase was not significantly higher in media of wounded cells as compared with non-wounded controls, arguing for a specific induction of Cathepsin B secretion upon wounding and migration of the cells. This was further substantiated by applying various Cathepsin B-specific inhibitors after wounding. These experiments showed that the migration ability of keratinocytes was reduced due to the blockage of functional Cathepsin B. Thus, our results strongly suggest that cell surface-associated Cathepsin B is a protease that contributes to the remodelling of the extracellular matrix and thereby promotes keratinocyte migration during wound healing.