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

•An in vitro model for melanocyte senescence using UVB exposure is proposed.•The protocol relies on UVB exposure twice and subsequent 2-week cultivation.•Melanocyte senescence accompanies hyperpigmentation via prolonged p53 expression. 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. 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. Human epidermal melanocytes were exposed twice with 20 mJ/cm2 UVB over a 24-h interval and subsequently cultivated for 2 weeks. Senescent phenotypes were addressed morphologically, and by measuring the senescence-associated β-galactosidase (SA-β-Gal) activity, cell proliferation capacity with cell cycle analysis, and melanin content. 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-α at 2-weeks post-UVB irradiation, but not at 48 h, significantly reduced melanin content along with decreases in tyrosinase levels. 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.