关键词: 白癜风, 黑素细胞, 黑素合成, 细胞周期蛋白依赖性激酶5, 细胞分裂周期蛋白42, 小眼畸形相关转录因子

Abstract: 【Abstract】 Objective To investigate the role of cyclin-dependent kinase 5 （CDK5） in regulating the cell division cycle protein 42 （CDC42）-microphthalmia-associated transcription factor （MITF） signaling pathway in the mechanism underlying melanin synthesis in melanocytes. Methods CDK5 was overexpressed or knocked down in the immortalized human melanocyte cell line （PIG1）, and the cells were divided into the following groups: （1） control group treated with transfection reagents alone; （2） empty vector group transfected with empty plasmids; （3） CDK5 group transfected with CDK5 overexpression plasmids; （4） si-CDK5 group transfected with CDK5-specific siRNA; （5） si-NC group transfected with negative control siRNA. RT-qPCR and Western blot analysis were performed to determine the mRNA and protein expression of CDK5, CDC42, and MITF in PIG1 cells, respectively, while the DOPA oxidation method and the sodium hydroxide solubilization method were used to evaluate the tyrosinase （TYR） activity and to determine the melanin content, respectively. In experiments with CDC42 knockdown in PIG1 cells, the cells were divided into a si-CDC42 group and a si-NC group transfected with CDC42 siRNA and negative control siRNA, respectively; the mRNA and protein expression of CDK5 and MITF were determined using the aforementioned methods, so were the TYR activity and melanin content. Skin lesion tissues from 10 patients with vitiligo were collected as the vitiligo group, and normal skin tissues adjacent to melanocytic nevi or sebaceous cysts from 10 individuals were collected as the control group; immunofluorescence staining and Western blot analysis were employed to determine the localization and expression of CDK5 and CDC42 in the skin tissues. Comparison of measurement data among multiple groups was performed using one-way analysis of variance, followed by least significant difference-t test for multiple comparisons, and the two-independent-sample t test was used for comparisons between two independent samples. Results RT-qPCR and Western blot analysis revealed that overexpression of CDK5 in PIG1 cells led to significantly increased mRNA and protein expression levels of CDK5, CDC42, and MITF in the CDK5 group compared with the control group and empty vector group （all P < 0.001）. Compared with the si-NC group, the si-CDK5 group showed significantly decreased protein expression levels of CDC42 （0.40 ± 0.05 vs. 1.00 ± 0.31, t = 3.35, P = 0.029） and MITF （0.42 ± 0.02 vs. 1.00 ± 0.29）, as well as significantly decreased TYR activity （0.67 ± 0.03 vs. 1.00 ± 0.20, t = 2.90, P = 0.044） and melanin content （0.60 ± 0.03 vs. 1.02 ± 0.25, t = 2.85, P = 0.047）. There were no significant differences in the CDK5 mRNA or protein expression level between the si-CDC42 group and the si-NC group （both P > 0.05）; compared with the si-NC group, the si-CDC42 group showed significantly decreased MITF mRNA and protein expression levels （P = 0.003, < 0.001, respectively）, as well as significantly decreased TYR activity and melanin content （P = 0.034, 0.037）. Immunofluorescence staining revealed that CDK5 and CDC42 were highly expressed in melanocytes in normal human skin tissues. Western blot analysis showed that the protein expression levels of CDK5 and CDC42 were significantly higher in the control group than in the vitiligo group （both P < 0.05）. Conclusions CDK5 may enhance the TYR activity and promote melanin production in melanocytes through the CDC42-MITF signaling pathway. The expression of CDK5 and CDC42 was reduced in melanocytes from vitiligo lesions.

Key words: Vitiligo, Melanocytes, Melanin synthesis, CDK5, CDC42, MITF