Regulatory effects of different doses of ultraviolet B on the expressions of p62, Beclin-1, Atg12 and Atg3 in HaCaT cells

Abstract

Objective To evaluate the regulatory effects of different doses of ultraviolet B (UVB) radiation on the expressions of p62,Beclin-1,Atg12 and Atg3 proteins in the human keratinocyte cell line HaCaT.Methods Cultured HaCaT cells were divided into several groups to be irradiated with three doses of UVB (4.5,10 and 50 mJ/cm2) immediately followed by additional culture with or without the lysosomal protease inhibitors E64D of 10 μg/L and pepstatin of 10 μg/L,both of which were used to block p62 degradation,for 4 and 12 hours respectively.Those HaCaT cells receiving the same treatment but no irradiation served as the control.Subsequently,Western blot was performed to determine the expression levels of p62,Beclin-1,Atgl2 and Atg3 proteins in these cells.The protein expression level was represented as the ratio of the product of absorbance value and area of the electrophoretic bands of target proteins to that of β-tubulin.Paired t test was performed to compare the expression levels of these proteins among different groups.Results After irradiation with UVB of 50 mJ/cm2,the expression level of p62 was significantly upregulated at 4 hours in unblocked HaCaT cells compared with unblocked unirradiated cells (0.473 ± 0.022 vs.0.246 ± 0.038,t =15.27,P ＜ 0.05),and in blocked HaCaT cells compared with blocked unirradiated cells (0.445 ± 0.035 vs.0.244 ± 0.016,t =7.62,P ＜ 0.05).Increased p62 expression was also observed in unblocked and blocked HaCaT cells at 12 hours after irradiation with UVB of 4.5 mJ/cm2 compared with the corresponding unirradiated cells (unblocked cells:0.497 ± 0.047 vs.0.254 ± 0.035,t =22.89,P ＜ 0.05; blocked cells:0.548 ± 0.051 vs.0.257 ± 0.025,t =17.42,P ＜ 0.05).No significant differences were noted in the protein expression levels of Beclin-1,Atgl2 or Atg3 between unblocked or blocked HaCaT cells and corresponding unirradiated cells at 4 or 12 hours after irradiation with UVB at 4.5 or 50 mJ/cm2 (all P ＞ 0.05).Conclusions The expression level of p62 is different at different time points after different doses of UVB radiation,and there seems no biological association between UVB radiation and autophagosome formation. Key words: Ultraviolet rays; Keratinocytes; Proto-oncogene proteins; Beclin-1; Atg12; Atg3