Effect of Ultraviolet B Irradiation on Melanin Content Accompanied by the Activation of p62/GATA4-Mediated Premature Senescence in HaCaT Cells

Abstract

Objective: To explore the effect and mechanism of ultraviolet B (UVB) on melanin synthesis and premature senescence in human immortalized keratinocytes (HaCaT) cells. Methods: HaCaT cells were irradiated with 0, 20, 50, 80, 100, 150, and 200 mJ/cm2 of UVB. NaOH method was used for melanin content assay, cellular tyrosinase (TYR) activity was determined by 3,4-Dihydroxy-L-phenylalanine (L-DOPA) oxidation to dopachrome, premature senescence was analyzed by senescence-associated beta-galactosidase (SA-β-gal) staining kit, and the levels of p21, p16, p62, and GATA4 proteins were detected by Western blotting. Premature senescence was inhibited by the inhibitors of ataxia telangiectasia mutated (ATM) or ataxia telangiectasia and Rad3–related (ATR), and the p53 signaling pathway was activated by Nutlin-3. The mRNA levels of senescence-associated secretory phenotype (SASP) factors including tumor necrosis factor alpha (TNF-α), vascular endothelial growth factor A (VEGF-A), and interleukin-8 (IL-8) were measured by real-time quantitative polymerase chain reaction in HaCaT cells after 80 mJ/cm2 of UVB irradiation. Results: The melanin level increased significantly with the elevation of irradiation dose (F = 28.19, 43.82, 143.60, P