Melatonin inhibits senescence-associated melanin pigmentation through the p53-TYR pathway in human primary melanocytes and the skin of C57BL/6J mice after UVB irradiation
Authors: Ma Li-Ping , Liu Meng-Meng , Liu Fang , Sun Bo , Wang Si-Nian , Chen Jie , Yu Hui-Jie , Yan Juan , Tian Mei , Gao Ling , Liu Qing-Jie 
Published: November 2022

Abstract
Purpose
UVB exposure accelerates skin aging and age-associated pigmentation, but their relationship remains unclear. UVB induces premature senescence and melanin production within melanocytes, along with the upregulation of p53 and cellular tyrosinase (TYR). As a tumor suppressor gene, p53 can keep the genome intact by modulating cell apoptosis and growth arrest during DNA injury. It is also associated with age-associated pigmentation, directly or indirectly regulating pigment-related gene expression. Melatonin effectively regulates tyrosinase activity and resists aging. In this study, we investigated the regulation of p53 on TYR to understand the association between premature senescence and senescence-associated pigmentation and determine the mechanism by which melatonin affects UVB-stimulated melanin production.
Methods
Primary melanocytes were extracted and identified from the male foreskin. The primary melanocytes were transduced using lentivirus pLKD-CMV-EGFP-2A-Puro-U6-TYR to knock down TYR expression. The melanin content was determined using the NaOH method, 3,4-Dihydroxy-L-phenylalanine (L-DOPA) was oxidized to dopachrome to determine TYR activity, and Western blotting was performed to detect the level of TYR protein. The primary melanocytes were pretreated with Nutlin-3 or PFT-α to upregulate or downregulate p53 levels or melatonin for 12 h and exposed to UVB irradiation at 80 mJ/cm2. The senescence-associated beta-galactosidase (SA-β-gal) kit was used to analyze premature senescence. The levels of p53, p-p53, and TYR protein were detected by performing the automated capillary electrophoresis Western blotting analysis in melanocytes 72 h after UVB irradiation. Wild-type and TYR(–/–) or TYR(+/–) knockout C57BL/6J mice were used to determine the regulatory role of TYR on melanin synthesis in vivo. Additionally, the effect of melatonin on skin erythema and pigmentation induced by UVB irradiation was analyzed in vivo.
Results
Primary melanocytes turned deep black after L-DOPA staining, indicating higher TYR protein and mRNA expression. Tyrosinase activity and melanin levels induced by UVB irradiation decreased significantly after the primary melanocytes were infected with pLKD-CMV-EGFP-2A-Puro-U6-TYR (P < 0.05). Premature senescence, tyrosinase activity, and melanin levels increased after exposure to UVB irradiation. There was a dramatic increase in primary melanocytes following Nutlin-3 treatment but significant inhibition after treatment with PFT-α (P < 0.05). Melatonin inhibited UVB-induced premature senescence, associated with decreased p53 level and phosphorylation at the serine-15 position, decreased UVB-induced tyrosinase activity and melanin levels, and reduced TYR expression.The TYR(–/–) knockout mice were recognized through white hair, whiskers, and paws, and loss of pigments in the eyes. The tyrosinase activity and melanin levels in the whisker follicles of TYR(–/–) knockout mice also decreased significantly (P < 0.05) relative to that in the wild-type (WT) mice. Skin erythema and melanin pigmentation induced by UVB irradiation decreased in the dorsal and ear skin of C57BL/6J mice topically pretreated with 2.5% melatonin.
Conclusion
Melanin synthesis induced by UVB irradiation is partly dependent on TYR in primary melanocytes and the C57BL/6J mice. Moreover, p53 links the UVB irradiation-induced premature senescence and senescence-associated pigmentation in primary melanocytes. It also directly regulates TYR in primary melanocytes after UVB irradiation. After UVB irradiation, melatonin partly inhibits senescence-associated pigmentation through the p53-TYR pathway in the primary melanocytes. Melatonin prevents skin erythema and melanin pigmentation induced by UVB irradiation in the dorsal and ear skin of C57BL/6J mice.