中波紫外线辐射角质形成细胞核因子kB和P53信号通路的交互作用

Cross-talk between nuclear factor-κB and P53 signal oathway in keratinocytes after ultraviolet B irradiation

目的研究中波紫外线(UVB)辐射对表皮角质形成细胞核因子 kB(NF-kB)和 P53信号通路的影响以及 NF-kB 和 P53信号通路的交互作用。方法正常人表皮角质形成细胞(NHEK)(含野生型 p53)和永生化人角质形成细胞株 HaCaT(含突变型 p53)各分2组于37℃、5%CO_2环境培养。当细胞融合达85%时进行 UVB(60 mJ/cm^2)辐射,其中1组于辐射前1 h 加入终浓度为5μmol/L 具有抗 NF-kB 活化作用的 BAY11-7082。分别提取辐射前后 NHEK 和 HaCaT 细胞蛋白和核蛋白,应用 Western 印迹检测 NF-kB、P53和 P21蛋白的表达水平,应用电泳迁移变更分析(EMSA)检测NF-kB 的转录情况。结果 UVB 辐射可激活 NHEK 和 HaCaT 的 NF-kB、P53和 P21的蛋白表达和NF-kB 的转录活性。BAY11-7082对 NHEK 和 HaCaT 的 NF-kB 的转录活性均具有显著的抑制作用。作为 NF-kB 的抑制剂,BAY11-7082还显著抑制了 NHEK 的 P53和 P21的蛋白表达(P53:0.08±0.07vs 0.78±0.32,P<0.01;P21:0.65±0.22 vs 1.58±0.77,P<0.05),但不影响 HaCaT 的 P53和 P21的蛋白表达。结论 UVB 辐射激活表皮角质形成细胞的 NF-kB 和 P53信号通路存在交互作用,这种交互作用与 P53功能状况相关。

Objective To investigate the cross-talk between nuclear factor κB （NF-κB） and P53 signal pathways in human epidermal keratinocytes （NHEKs） after ultraviolet B （UVB） irradiation. Methods Normal NHEKs harboring wild p53 gene and immortal human keratinocytes of the line HaCaT harboring mutant p53 gene were cultured at 37℃ in an atmosphere containing 5% CO2 and then underwent irradiation of UVB of the dose of 60 mJ/cm^2. Part of the NHEKs and HaCaT cells were pretreated with BAY11-7082, NF-κB inhibitor inhibiting IkB-a phosphorylation, of the final concentration of 5 μmol/L. Western blotting was used to detect the protein expression of NF-κB, P53, and P21. The transcriptional activity of NF-κB was analyzed by electrophoretic mobility shift assay （EMSA）. Results UVB irradiation increased the protein expression of NF-κB, P53, and P21 and triggered the transcriptional activity of NF-κB. BAY11-7082 significantly inhibited the NF-κB protein expression induced by UVB irradiation in the NHEKs and HaCaT cells. The P53 protein expression of the NHEKs undergoing pretreatment of BAY11-7082 and UVB irradiation was 0. 08 ± 0. 07, significantly lower than that of the NHEKs undergoing only UVB irradiation （0. 78 ±0. 32, P 〈 0. 01 ）. The P21 protein expression of the NHEKs undergoing pretreatment of BAY11- 7082 and UVB irradiation was 0. 65 ±0. 22, significantly lower than that of the NHEKs undergoing only UVB irradiation （ 1.58±0. 77,P 〈0. 05）. However, the P53 and the P21 protein expression of the HaCaT cells undergoing pretreatment of BAY11-7082 and UVB irradiation was not significantly different from that of the HaCaT cells undergoing only UVB irradiation. Conclusion There exists a UVB-induced cross-talk between NF-κB and P53 signal pathways in human epidermal keratinocytes. This cross-talk is correlated with P53 functional condition.