| Introduction Ultraviolet B (UVB) mainly targets keratinocytes of epidermis, resulting in protein damage in keratinocytes. Effectively degenerating damaged proteins performs a fundermental role of maintaining cell homeostasis. The polyubiquitin-binding protein p62,with a variety of biological functions, can recognize ubiquitination sites of misfolded protein via the C-terminal UBA domain.And p62can directly bind LC3through the LC3-interacting region (LIR). Poly-ubiquitinated proteins with p62and LC3colocalized are degraded by autophagy.It’s suggesting that p62acts as an adaptor between ubiquitinated proteins and autophagy.Autophagy,as a physiological phenomenon, extensively exists in eukaryotic cells. Autophagy performs a fundermental role of degeneration of denatured protains and injured organelles. Moreover, it is be suggested that autophagy exsits in keratinocytes and alleviates inflammation in keratinocytes.Whereas, it remains unclear that whether UVB-induced damage can induce p62protein expression changes in keratinocyte cell. And the difference of p62expression level is uncertain having biological association between autophagosome formation. In this study, we make a preliminary exploration of these issues. Our study contributes to clear that whether UVB-induced damage have an impact on protein degradation pathway of p62as the core in keratinocyte.Objective To evaluate the regulatory effects of different doses of ultraviolet B (UVB) irradiation on the expressions of p62, Beclin-1, Atg12and Atg3proteins in the human keratinocyte cell line HaCaT.Methods At4hours after irradiation with UVB(4.5,10and50mJ/cm2), ultrastructural changes of irradiated HaCaT cells were detected through transmission electron microscopy.Cultured HaCaT cells were divided into several groups to be irradiated with three doses of UVB (4.5,10and50mJ/cm2) immediately followed by additional culture with or without the lysosomal protease inhibitors E64D of10μg/L and pepstatin of10μg/L, both of which were used to block p62degradation, for4and12hours 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, Atg12and Atg3proteins 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 P-tubulin. Paired t test was performed to compare the expression levels of these proteins among different groups.Results After irradiation with UVB of4.5and10mJ/cm, autophagosome appeared in HaCaT cells, especially in4.5mJ/cm group.However,the autophagosome was rarely found in50mJ/cm group.After irradiation with UVB of50mJ/cm, the expression level of p62was significantly upregulated at4hours in unblocked HaCaT cells compared with unblocked control cells (0.473±0.022vs.0.246±0.038, t=15.27, P<0.05), and in blocked HaCaT cells compared with blocked control cells (0.445±0.035vs.0.244±0.016, t=7.62, P<0.05). Increased p62expression was also observed in unblocked and blocked HaCaT cells at12hours after irradiation with UVB of4.5mJ/cm2compared with the corresponding control cells (unblocked cells:0.497±0.047vs.0.254±0.035, t=22.89, P<0.05; blocked cells:0.548±0.051vs.0.257±0.025, t=17.42, P<0.05). No significant differences were noted in the protein expression levels of Beclin-1, Atgl2or Atg3between unblocked or blocked HaCaT cells and control cells at4or12hours after irradiation with UVB at4.5and50mJ/cm2(all P>0.05).Conclusions Low doses of UVB irradiation can induce formation of autophagosome in keratinocytes; while there is no significantly formation of autophagosome in cells after high doses of UVB irradiation.The expression level of p62is different at different time points after different doses of UVB irradiation, and there seems no biological association between UVB irradiation and autophagosome formation. |
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