Effects of ultraviolet B on the MAPK signaling pathway in cultured HaCaT cells in vitro

Abstract

Objective To explore the regulatory effects of ultraviolet B (UVB) radiation on the mitogenactivated protein kinase (MAPK) signaling pathway in cultured HaCaT cells in vitro at different post-radiation time points. Methods Some cultured HaCaT cells were classified into several groups to be exposed to UVB of 1.5, 4.5, 7.5, 10, 20, 30 and 50 mJ/cm2 for 1, 3, 5, 7, 15, 20 and 34 seconds respectively, or UVB of 50 mJ/cm2 for 34 seconds, or remain untreated (control group). Western blot was performed to determine the total and phosphorylation levels of ERK1/2, JNK and p38 in HaCaT cells at 8 hours after exposure to 1.5, 4.5, 7.5,10, 20, 30 and 50 mJ/cm2 UVB, as well as at 2, 4, 8 and 12 hours after exposure to 50 mJ/cm2 UVB. Quantity One software was used to measure the absorbance value of protein bands, and protein levels were expressed as the absorbance ratio of target bands to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Statistical analysis was carried out by using one- way analysis of variance, least significant difference (LSD) - t test and multivariate analysis of variance. Results Compared with the control group, there was a significant increase at 8 hours in the phosphorylation levels of ERK 1/2 and JNK in HaCaT cells after exposure to 7.5, 10, 20, 30 and 50 mJ/cm2 UVB (all P< 0.05), and in the phosphorylation level of p38 after exposure to 20, 30 and 50 mJ/cm2 UVB (all P < 0.05), with the strongest increase in phosphorylation levels of ERK 1/2, JNK and p38 all observed in HaCaT cells exposed to 50 mJ/cm2 UVB (all P< 0.05). After exposure to 50 mJ/cm2 UVB, the phosphorylation level of ERK1/2 began to increase in HaCaT cells at 8 hours, and significantly increased at 12 hours(44.844±2.023 vs. 10.277±0.320, P< 0.05) compared with the control group; however, the phosphorylation levels of p38 and JNK increased in HaCaT cells as early as 2 hours after exposure (both P< 0.05), and remained significantly higher compared with the control group at 4, 8 and 12 hours (all P< 0.05), while the increasing trend in phosphorylated JNK level weakened over time (P< 0.05). Conclusions The ERK1/2, JNK and p38 MAPK signaling pathways were activated in HaCaT cells after exposure to 50 mJ/cm2 UVB, and the degree of their activation varied over time. Key words: Ultraviolet rays; Cells, cultured; Keratinocytes; Signal transduction; MAP kinase kinase kinases