Extracellular vesicles are transferred from melanocytes to keratinocytes after UVA irradiation

Wäster, Petra, Ida Eriksson, Linda Vainikka, Inger Rosdahl, and Karin Öllinger. Scientific reports 6 (2016): 27890.

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.

Cells release extracellular vesicles (EVs) from at least two different origins, namely exosomes and microvesicles. Exosomes are intraluminal vesicles that are <100 nm in size and are derived from multivesicular endosomes that fuse with the plasma membrane1,2. Microvesicles, also called ectosomes, are 100 to 1,000 nm in diameter and are formed in a more rapid process than exosomes through the outward budding of the plasma membrane, which leads to the shedding of the vesicles3,4. The vesicles comprise components that are typical to their cell of origin. EVs show a preference for certain target cells, although the mechanism of recognition remains undefined1.

Exposure to ultra violet (UV) irradiation is the major risk factor for the development of skin cancer, in particular malignant melanoma, although the etiology of this condition involves a complex interplay between genetics, host characteristics and environmental factors5. The UVB component of sunlight (280–320 nm) induces DNA oxidation directly, while UVA (320–400 nm) alters the redox balance of the cell and induces oxidative stress, eventually leading to indirect DNA damage6,7. Recent studies of keratinocytes, melanocytes and melanoma cell lines have shown that UVA irradiation causes plasma membrane damage that is repaired through lysosomal exocytosis8,9,10.

Although lysosomes are the central degradation unit of the cell, their function goes far beyond its degradative mission and includes regulation of cell death, maintenance of cholesterol homeostasis and repair of plasma membrane damage through exocytosis11,12. Upon plasma membrane damage, Ca2+ influx from the extracellular compartment triggers lysosomal exocytosis and fusion with the membrane, forming a resealing patch to rescue the cell13,14,15. Previous studies have shown that lysosomes promote resealing through the secretion of acid sphingomyelinase (aSMase), an enzyme that generates ceramide through the cleavage of the abundant membrane lipid sphingomyelin16. In addition to conventional lysosomes, melanocytes harbor melanosomes, which are specialized lysosome-related organelles that contain the photoprotective pigment melanin. Melanosomes share some proteins with lysosomes but also contain unique membrane proteins, such as premelanosome protein (PMEL), tyrosinase, and tyrosinase-related protein 1, which are important for melanogenesis17,18. Furthermore, lysosomal marker proteins such as cathepsin D and lysosomal-associated membrane protein-1 (LAMP-1) are present in low levels in mature melanosomes19. Mature melanosomes bind microtubules and undergo bi-directional actin-dependent transport from the perinuclear area towards dendrites20,21. This transfer is stimulated and regulated through keratinocyte-derived factors22,23, although the delivery mechanism has not been fully characterized. Different mechanisms have been suggested including the heterophagocytosis of melanocyte dendrites by keratinocytes, the release of melanosome-loaded vesicles, the exocytosis of the melanin core with subsequent endocytosis by keratinocytes, transfer by nanotubes or via melanocyte filopodia, and direct fusion with the keratinocyte membrane24,25,26.

In previous reports, we have demonstrated the novel finding of UVA induced plasma membrane damages that is followed by repair through lysosomal exocytosis and accompanied by release of lysosomal constituents8,9. The aim of the present study was to examine UV-induced signaling between melanocytes and keratinocytes in order to determine the relationship between exocytosis of lysosomes and the transfer of melanosomes from melanocytes to keratinocytes. Interestingly, we found melanosome transfer to be mechanistically unrelated to lysosomal exocytosis. Moreover, the lysosomal exocytosis was followed by EV shedding from the melanocyte plasma membrane and uptake of EVs by keratinocytes through endocytosis.

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