The Mechanisms Of P53 Signaling Pathway Activation In Regulating Arsenite-induced Autophagic Degradation Of IKKα And Transcriptional Inhibition Of IKKβ

Background and content Arsenite is a kind of heavy metal pollutant,which is widely distributed in the nature and has great harm to human health.In general,high-dose arsenite exposure mainly causes acute toxic effect,which is primarily characterized by “apoptosis”.However,long-term exposure to low-dose arsenite mainly triggers chronic toxic effect with “tumorigenesis” as the key feature.Therefore,the study on mechanism of health hazard effect of arsenite has important basic theoretical significance and applicable medical value.Our group has focused on elucidating the mechanisms involving in pro-apoptotic effect of arsenite for a long time.And a large number of valuable discoveries have been found in the previous studies,which have provided significant theoretical basis for the health risk assessment of arsenite and intervention targets for the control of arsenite toxicity.A novel finding in our recent work is that arsenite-induced cell apoptoticeffect reaction was accompanied with the down-regulation of two catalytic subunits of IKK kinase-IKKα and IKKβ,which served as a prerequisite event for the induction of apoptotic effect under arsenite exposure.In the attempt to figure out the molecular mechanisms involving in mediating IKKα and IKKβ down-regulation,we had found that p53 could be activated by arsenite and then induced autophagy leading to the specific degradation of IKKα.At the same time,p53 transactivated its target ETS-1 and cooperatively mediated transcriptional inhibition of IKKβ.Based on the above results,in this study,we investigated the signaling transduction mechanisms involving in mediating p53-dependent autophagic degradation of IKKα and transcriptional inhibition of IKKβ in HepG2 cells treated with arsenite.Results In the first part,we tried to identify the downstream targets of p53 in mediating arsenite-induced autophagic degradation of IKKα.We found that the strong induction of autophagy-related p53 targets,including DRAM1,ISG20L1,DAPK1,TIGAR and SESTRIN2,were observed in arsenite-treated HepG2 cells.DRAM1 played a major role in mediating the autophagic degradation of IKKα induced by arsenite;while TIGAR and SESTRIN2 were completely irrelevant with the above degradation reactions.Induction of ISG20L1 and DAPK1 also contributed to arsenite-induced autophagy,but was not involved in IKKαdegradation.Moreover,the induction of ISG20L1 was not controlled byp53 under the exposure of arsenite.The above experimental results indicated that DRAM1 is a p53 downstream target responsible for the autophagic degradation of IKKα induced by arsenite.In the second part,we further analyzed the upstream protein kinases responsible for mediating the activation of p53/DRAM1 signaling pathway in the arsenite response.The results showed that ATR,CHK1,LKB1,AMPKα and PERK were able to regulate the transcriptional activation of p53 under arsenite stimulation.However,only CHK1 and LKB1 had the ability to regulate DRAM1 induction and the subsequent IKKα degradation induced by arsenite.Therefore we concluded that CHK1 and LKB1 functioned as the upstream protein kinases of p53 in mediating DRAM1-dependent autophagic degradation of IKKα.In the third part,we investigated the signaling events for inducing IKKα into the autophagic degradation pathway.In fact,IKKα and IKKβare highly homologous proteins,but only IKKα was selectively degraded by the autophagic pathway.Therefore,we speculated that the structure of IKKα might be a primary determinant for initiating the selective degradation response.On the other hand,some unidentified autophagy receptors might be involved in the degradation of IKKα induced by arsenite.According to our results,IKKα was able to interact with LC3 through its kinase domain under arsenite exposure,and the potential LIR motif within the kinase domain of IKKα might be the critical structure formediating the IKKα/LC3 complex formation.Furthermore,CHK1 and LKB1 might also play a synergistic effect in triggering LC3 interaction and degradation of IKKα.Although we excluded the role of ISG20L1 and DAPK1 in mediating arsenite-induced IKKα degradation in the first part,we unexpectedly found that these two signaling proteins played critical roles in the transcriptional inhibition of IKKβ.Therefore,we analyzed the molecular mechanisms of ISG20L1 and DAPK1 in regulating the transcriptional inhibition of IKKβ induced by arsenite in the final part.It was found that DAPK1 and ISG20L1 synergistically transactivated p53 and then induced ETS-1 expression and IKKβ transcriptional inhibition followed by up-regulating MDM2-dependent GADD45α induction to mediate arsenite-induced pro-apoptotic effect.Based on these results,we have identified the upstream signaling events in mediating the p53-dependent transcriptional inhibition of IKKβ in the arsnite response.Conclusions In summary,data in this work have preliminarily disclosed the molecular mechanisms involving in selective degradation of IKKα by autophagy in response to arsenite.Moreover,we also figure out the upstream signal mechanisms in mediating p53-dependent transcriptional inhibition of IKKβ.These results thus have not only provided the novel findings for the negative regulation mechanisms of IKK kinase expression under stress conditions,but also extended our knowledge on the mechanisms of toxic effects of arsenite.