Effects of ultraviolet B on the expressions of nuclear factor-κB p65 and its inhibitory factor IκBα in keratinocytes

Abstract

Objective To investigate the regulatory effects of different doses of ultraviolet B (UVB) radiation on the expressions of nuclear factor (NF)-κB p65 and its inhibitory factor IκBα in human keratinocytes cultured in vitro.Methods A human keratinocyte cell line CRL-2310 and primary human keratinocytes were used in this study.Both CRL-2310 and primary human keratinocytes were irradiated with 4.5,10 and 50 mJ/cm2 UVB,with unirradiated cells serving as the control.Western blot was performed to determine the expression levels of total IκBα,NF-κB p65 and IKKβ1 proteins in the cells at 4 hours after the irradiation.Some primary human keratinocytes were irradiated with 50 mJ/cm2 UVB,and Western blot was conducted to measure the expression levels of total and phosphorylated IκBα,NF-κB p65 and IKKβ1 proteins in these cells at 2,4,8,and 12 hours after the irradiation respectively.The optical density of protein bands was analyzed by the Quantity One software (version 4.6.8),the product of the optical density and area of each band was calculated for IKKβ,IκBα,NF-κB p65,β-actin as well as phosphorylated IKKβ,IκBα,and NF-κB p65 separately,and the expression levels of target proteins were expressed as the ratio of the product for the target proteins to that for β-actin.All experiments were repeated for three times,and the average expression levels were calculated for these proteins.Results No significant changes were observed in the CRL-2310 keratinocytes or the primary keratinocytes for the protein expression levels of total IKKβ,IκBα and NF-κB p65 at 4 hours after UVB radiation at 4.5 and 10 mJ/cm2,or for those of total IKKβ at 4 hours after UVB radiation at 50 mJ/cm2 compared with the unirradiated controls (all P ＞ 0.05).However,there was a statistical decrease in the expression levels of IκBα and NF-κB p65 in primary keratinocytes (0.173 ± 0.055 vs.0.462 ± 0.142,t =5.64,P ＜ 0.05; 0.076 ± 0.030 vs.0.120 ± 0.034,t =5.40,P ＜ 0.05),as well as a slight reduction in the expression level of IκBα (0.160 ± 0.046 vs.0.398 ± 0.136,t =4.37,P ＜ 0.05) with no statistical changes in the expression level of NF-κB p65 (P ＞ 0.05) in CRL-2310 keratinocytes,at 4 hours after 50 mJ/cm2 UVB radiation when compared with the unirradiated controls.After irradiation with 50 mJ/cm2 UVB,the expression level of IκBα in primary keratinocytes began to decrease at 2 hours compared with the unirradiated keratinocytes (0.140 ± 0.034 vs.0.208 ± 0.031,t =17.55,P ＜ 0.05),and the degree of decrease increased over time; the expression level of NF-κB p65 showed no changes at 2 or 4 hours (both P ＞ 0.05),but a significant decrease at 8 and 12 hours (1.162 ± 0.345 vs.1.235 ± 0.349,t =36.67,P ＜ 0.05; 1.061 ± 0.246 vs.1.390 ± 0.226,t =28.71,P ＜ 0.05),but remained unchanged afterwards.No significant differences were noted in the protein expression levels of IKKβ,phosphorylated IKKα/β (ser176/180),phosphorylated IκBα (ser32) or phosphorylated NF-κB p65 (ser536) between the irradiated and unirradiated primary keratinocytes at the 4 time points (all P ＞ 0.05).Conclusions High dose of UVB radiation (50 mJ/cm2) can significantly suppress the protein expression of IκBα,but slightly decrease the protein expression of NF-κB p65 in human primary keratinocytes,suggesting that UVB radiation induces the activation of NF-κB p65 through another potential pathway. Key words: Ultraviolet rays; Keratinocytes; NF-kappa B