- •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.
Abbreviations:UVR (ultraviolet radiation), UVsen (UVB-induced senescence), SAP (senescence-associated pigmentation), SA-β-Gal (senescence-associated β-galactosidase), TPA (tetradecanoyl phorbol acetate), CT (cholera toxin), pTpT (thymine dinucleotides), HEMn (human epidermal melanocyte neonatal), CDKN1A (cyclin dependent kinase inhibitor 1A), PFTα (pifithrin-α), TYR (tyrosinase), TYRP1 (tyrosinase-related protein 1), POMC (pro-opiomelanocortin), α-MSH (α-melanocyte-stimulating hormone), KITLG (c-KIT ligand), HGF (hepatocyte growth factor)
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Published online: February 26, 2018
Accepted: February 22, 2018
Received in revised form: December 29, 2017
Received: September 15, 2017
© 2018 Japanese Society for Investigative Dermatology. Published by Elsevier B.V. All rights reserved.