Exposure of human melanocytes to UVB twice and subsequent incubation leads to cellular senescence and senescence-associated pigmentation through the prolonged p53 expression

Background: Ultraviolet radiation (UVR) is a well-known factor in skin aging and pigmentation, and daily exposure to subcytotoxic doses of UVR might accelerate senescence and senescence-associated phenomena in human melanocytes. Objective: To establish an in vitro melanocyte model to mimic the conditions of repeated exposure to subcytotoxic doses of UVB irradiation and to investigate key factor(s) for melanocyte senescence and senescence-associated phenomena. Methods: Human epidermal melanocytes were exposed twice with 20 mJ/cm(2) UVB over a 24-h interval and subsequently cultivated for 2 weeks. Senescent phenotypes were addressed morphologically, and by measuring the senescence-associated beta-galactosidase (SA-beta-Gal) activity, cell proliferation capacity with cell cycle analysis, and melanin content. Results: The established protocol successfully induced melanocyte senescence, and senescent melanocytes accompanied hyperpigmentation. Prolonged expression of p53 was responsible for melanocyte senescence and hyperpigmentation, and treatment with the p53-inhibitor pifithrin-alpha at 2-weeks post-UVB irradiation, but not at 48 h, significantly reduced melanin content along with decreases in tyrosinase levels. Conclusion: Melanocyte senescence model will be useful for studying the long-term effects of UVB irradiation and pigmentation relevant to physiological photoaging, and screening compounds effective for senescence-associated p53-mediated pigmentation. (C) 2018 Japanese Society for Investigative Dermatology. Published by Elsevier B.V. All rights reserved.