Effects of broadband ultraviolet B on non-canonical Wnt pathways in human epidermal melanocytes

Abstract

Objective To investigate the effects of broadband ultraviolet B (BB-UVB) on the proliferation of, tyrosinase activity and melanogenesis in melanocytes. Methods Melanocytes isolated from human foreskin were subjected to primary culture. Some cultured primary melanocytes were irradiated with BB-UVB at 10, 20, 30, 40, 50, 100, 200 and 300 mJ/cm2. Then, CCK-8 assay was performed to evaluate the proliferative activity of melanocytes, dopa oxidation assay to estimate the activity of tyrosinase, and sodium hydroxide (NaOH) -lysis method was used to determine melanin content. Real-time fluorescence-based quantitative PCR was conducted to measure the mRNA expressions of genes involved in non-canonical Wnt pathways in melanocytes after irradiation with BB-UVB at 30, 50 and 100 mJ/cm2. Western blot was carried out to determine the expressions of proteins involved in non-canonical Wnt pathways in melanocytes before and after irradiation with BB-UVB of 100 mJ/cm2. The melanocytes receiving no treatment served as the control group. Statistical analysis was carried out by one-way analysis of variance followed by least significant difference (LSD) -t test for multiple group comparisons and by the independent sample t test for two-group comparisons. Results After irradiation with BB-UVB at 10-300 mJ/cm2, the proliferative activity of melanocytes was gradually reduced compared with the control group (all P< 0.05) , and the survival rate of melanocytes was less than 50% when the irradiation dose of BB-UVB was higher than 100 mJ/cm2. Furthermore, tyrosinase activity gradually increased in melanocytes after irradiation with BB-UVB at 10-100 mJ/cm2 compared with the control group, and the increase was statistically significant at the radiation dose of 100 mJ/cm2 (P< 0.05). Compared with the control group, the WIF-1 mRNA expression level decreased, while c-Jun N-terminal kinase (JNK) , microphthalmia-associated transcription factor (MITF) , Ras-related C3 botulinum toxin substrate1 (RAC1) and tyrosinase (TYR) mRNA expression levels increased in melanocytes after irradiation with BB-UVB at 30, 50 and 100 mJ/cm2 (all P< 0.05) ; the WNT5A mRNA expression significantly decreased in melanocytes irradiated with 30 and 50 mJ/cm2 BB-UVB, but increased in those irradiated with 100 mJ/cm2 BB-UVB (all P< 0.05). The radiation with 100 mJ/cm2 BB-UVB significantly decreased the expression of WIF-1 protein, but enhanced the expressions of WNT5A, JNK, MITF, RAC1 and TYR proteins in melanocytes compared with the control group (all P< 0.05) . Conclusions BB-UVB can decelerate the proliferation of, elevate tyrosinase activity and melanin level in, melanocytes. The WIF-1 gene may inhibit melanogenesis, and the decrease in its expression may promote melanogenesis by activating the JNK/MITF/TYR pathway through the combined effects of proteins involved in non-canonical Wnt pathways. Key words: Melanocytes; Ultraviolet rays; Wnt signaling pathway; Cell proliferation; Melanin synthesis