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SummaryUltraviolet (UV) irradiation is a major environmental carcinogen involved in the development of skin cancer. To elucidate the initial signaling during UV-induced damage in human keratinocytes, we investigated lysosomal exocytosis and apoptosis induction. UVA, but not UVB, induced plasma membrane damage, which was repaired by Ca 2+ -dependent lysosomal exocytosis. The lysosomal exocytosis resulted in extracellular release of cathepsin D and acid sphingomyelinase (aSMase). Two hours after UVA irradiation, we detected activation of caspase-8, which was reduced by addition of anti-aSMAse. Furthermore, caspase-8 activation and apoptosis was reduced by prevention of endocytosis and by the use of cathepsin inhibitors. We conclude that lysosomal exocytosis is part of the keratinocyte response to UVA and is followed by cathepsin-dependent activation of caspase-8. The findings have implications for the understanding of UV-induced skin damage and emphasize that UVA and UVB initiate apoptosis through different signaling pathways in keratinocytes.

Ultraviolet (UV) irradiation induces skin pigmentation, which relies on the intercellular crosstalk of melanin between melanocytes to keratinocytes. However, studying the separate effects of UVA and UVB irradiation reveals differences in cellular response. Herein, we show an immediate shedding of extracellular vesicles (EVs) from the plasma membrane when exposing human melanocytes to UVA, but not UVB. The EV-shedding is preceded by UVA-induced plasma membrane damage, which is rapidly repaired by Ca2+-dependent lysosomal exocytosis. Using co-cultures of melanocytes and keratinocytes, we show that EVs are preferably endocytosed by keratinocytes. Importantly, EV-formation is prevented by the inhibition of exocytosis and increased lysosomal pH but is not affected by actin and microtubule inhibitors. Melanosome transfer from melanocytes to keratinocytes is equally stimulated by UVA and UVB and depends on a functional cytoskeleton. In conclusion, we show a novel cell response after UVA irradiation, resulting in transfer of lysosome-derived EVs from melanocytes to keratinocytes.

Lysosomes are central organelles for cellular degradation and energy homeostasis. In addition, lysosomal membrane permeabilization (LMP) and subsequent release of lysosomal content to the cytosol can initiate programmed cell death. The extent of LMP and available repair mechanisms determine the cell fate after lysosomal damage. In this study, we aimed to investigate the premises for lysosomal membrane repair after LMP and found that lysosomal membrane damage initiated by L-leucyl-L-leucine methyl ester (LLOMe) caused caspase-dependent apoptosis in almost 50% of the cells, while the rest recovered. Immediately after LLOMe addition, lysosomal proteases were detected in the cytosol and the ESCRT-components ALIX and CHMP4B were recruited to the lysosomal membrane. Next, lysophagic clearance of damaged lysosomes was evident and a concentration-dependent translocation of several lysosomal membrane proteins, including LAMP2, to the cytosol was found. LAMP2 was present in small vesicles with the N-terminal protein chain facing the lumen of the vesicle. We conclude that lysophagic clearance of damaged lysosomes results in generation of lysosomal membrane protein complexes, which constitute small membrane enclosed units, possibly for recycling of lysosomal membrane proteins. These lysosomal membrane complexes enable an efficient regeneration of lysosomes to regain cell functionality.

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