IKKβ Reduction And Its Effect On Regulating Ubiquitination And Accumulation Of GADD45α In The Arsenite Responses

Background Arsenic is a kind of heavy metal toxicant, which is widely distributed in the earth crust, soil, water and air. Acute exposure to high dose of arsenite induces genotoxic and oxidative stress responses, leading to cell damage effect and apoptosis. However,the intracellular signaling events in mediating cell stress responses and the pro-apoptotic effects induced by arsenite have not been fully defined.We have been focusing on elucidating the signal transduction mechanism involving in pro-apoptotic effect of arsentie for many years. And one of our critical findings is that growth arrest and DNA damage inducible gene(GADD) 45α plays an important role in mediating arsenite cytotoxicity. In fact, GADD45α is also reactive to ultraviolet(UV), ionizing radiation(IR) and other stressors and exerting multiple functions in DNA damage repair, inhibiting cell cycle progression, inducing senescence and promoting cell apoptosis, etc. We found that GADD45α undergoes a constitutive ubiquitination-dependant degradation in both normal diploid cells and hepatocellular carcinoma, which contributes to trace level of this protein in the resting cells. Arsenite exposure blocks the degradation of GADD45α and therefore results to its accumulation and the subsequent activation of the downstream pro-apoptotic signal transduction pathways. During this process, GADD45α m RNA transcription keeps consistently, which is different from the transcriptional induction of GADD45α in response to UV irradiation. Based on these data, we have for the first time elucidated the critical contribution of regulation on GADD45α ubiquitination and protein stability to the cytotoxicity of arsenite. In the following studies, we further find that MDM2 acts as an E3 ubiquitin ligase for GADD45α and regulates its protein stability under both resting and arsenite exposure conditions. Arsentie treatment induces ribosomal stress responses mediated by the small subunit ribosomal protein S7, which blocks MDM2-dependent ubiquitination and degradation of GADD45α, leading to GADD45α accumulation in the cells.IKKβ(inhibitor kappa B kinase β) is a catalytic subunit of IKK complex, which plays critical role in mediating IKK/NF-κB pathway activation. The best known function of IKKβ is to induce the phosphorylation and degradation of Iκ-Bα, thereby releasing NF-κB into the nucleus to regulate the expression of downstream target genes. With the discovery of more and more NF-κB-independent new substrates for IKK complex, the functions of IKK have gone far beyond NF-κB pathway. Our group has focused on elucidating NF-κB-independent functions of IKK in the cellular stress responses for a long time. And a series of NF-κB-independent new roles of IKKα or IKKβ have been disclosed under stress conditions. In the research to reveal the signal transduction mechanisms in mediating arsenite-induced pro-apoptotic effects, we found that up-regulation of GADD45α was accompanied with down-regulation of both IKKα and IKKβ. Most importantly, reduction of both IKK subunits was also critical for mediating arsenite cytotoxicity. Therefore we raised the hypothesis that there might be some functional link between the signal events of GADD45αup-regulation and down-regulation of IKKα and IKKβ. So we carried out the following study to answer this question.Objectives1. To explore the signaling pathway downstream of IKKβ in regulating the ubiquitination and protein stability of GADD45α under both resting and arsenite exposure conditions. Based on these results, we hope to clarify the new NF-κB-independent mechanism of IKKβ in regulating pro-apoptotic effects induced by arsenite.2. To explore the mechanism regarding IKKβ down-regulation in the arsenite-induced responses, and to clarify the contributions of IKKβ expression levels changes to executing its NF-κB-independent function.Methods In this study, all of the experiments were performed in the arsenite-sensitive human hepatocellular carcinoma cell line Hep G2. Hep G2 cells were treated with arsenite for different time periods and then the expression and activation of the signal proteins,like GADD45α, IKKα, IKKβ, MDM2, p53 and so on, were detected by western blot;The m RNA levels of IKKβ, MDM2 and p53 were detected by reverse transcription PCR; Co-immunoprecipitation were used to detect the protein-protein interactions between IKKβ, MDM2, S7, p53, Ets-1, etc; Dual luciferase reporter assay was performed to detect the transcriptional activity of IKKβ promotor; chromatin immunoprecipitation assay was used to detect the recruitment of p53 and Ets-1 to IKKβ promotor in the arsenite responses.Results In the first part, we focused on elucidating the signal transduction mechanisms downstream of IKKβ in regulating the ubiquitination and degradation of GADD45αunder both resting and arsenite exposure conditions. We found that up-regulation of GADD45α was accompanied with down-regulation of IKKα and IKKβ in the arsenite-treated Hep G2 cells. Knockdown IKKβ significantly up-regulated GADD45αinduction by As2O3; while increasing of GADD45α induction was observed after overexpression of IKKβ. However, the expression levels of GADD45α did not show obvious difference after knockdown or overexpression of IKKα in the arsenite-treated Hep G2 cells. These results suggest that IKKβ is involved in GADD45α accumulation in the arsenite responses. We further found that overexpression of IKKβ significantly inhibited pro-apoptotic responses in the Hep G2 cells, while knockdown IKKβincreases the percentage of apoptotic cells. Based on these data, we believe that IKKβplays a negative role in regulating the induction of GADD45α and therefore functions as a protector in the pro-apoptotic responses induced by arsenite.In the following study, we found that IKKβ was involved in constitutive ubiquitination of degradation of GADD45α, thereby reducing the stability of this protein. This event contributes largely to keep trace level of GADD45α in resting cells.Furthermore, IKKβ down-regulated GADD45α expression levels by interacting and inhibiting ubiquitination and degradation of MDM2 and increasing its protein stability.During this process, no interaction of IKKβ and S7 was observed, and the expression levels of S7 did not change under conditions of overexpression or knockdown IKKβ.Therefore, we conclude that IKKβ acts as a co-activator of MDM2, and then negatively regulates the stability of GADD45α under both resting and arsenite exposure conditions. Arsenite-induced IKKβ reduction terminates its negative effects on GADD45α accumulation and therefore promotes the cells undergoing apoptosis.IKKβ kinase activity was required for mediating the above functions, which provides us new discoveries regarding the NF-κB-independent functions of IKK under stress conditions.In the second part, we investigated the signaling pathways involving in IKKβreduction in the arsenite responses. We found that the down-regulation of IKKβ was due to the inhibition of IKKβ transcription. After analyzing IKKβ promoter sequences,we found three p53-binding sites and two Ets-1-binding sites within the promotor.Cooperation of p53 and Ets-1 in mediating transcriptional inhibition of IKKα has been reported in the previous literature. Therefore, we speculated that they might also contribute to transcriptional inhibition of IKKβ in the arsenite responses. Subsequent experimental results showed that arsenite exposure induced p53 activation and up-regualtion of Ets-1 expression. After knockdown p53 or Ets-1, arsenite-induced down-regulation of IKKβ expression was almost completely blocked, indicating that p53 and Ets-1 were indeed involved in of IKKβ reduction in response to arsenite.Then, we found the inducible interaction between p53 and Ets-1 after arsenite stimulation, suggesting that these two transcription factors may exert a synergistic effect in the regulation of IKKβ expression. The results from chromatin immunoprecipitation and luciferase reporter analysis further confirmed that p53 and Ets-1 could bind to the specific functional sites within IKKβ promoter after arsenite stimulation. Mutations of these sites resulted to almost complete block of IKKβreduction mediated by p53 and Ets-1. Based on the above results, we had disclosed the mechanism of transcriptional inhibition of IKKβ mediated by p53 and Ets-1induced by arsenite, which had provided a new discovery for the mechanism of the regulation on IKK expression.Conclusions Data in this work reveal that IKKβ is a novel co-stabilizor of the E3 ubiquitin ligase MDM2. IKKβ could negatively regulate GADD45α expression through enhancing the stability of MDM2. By these mechanism, IKKβ suppresses the accumulation of GADD45α and the subsequent pro-apoptotic effect under arsenite exposure. Moreover,arsenite induces p53 activation and Ets-1 expression, which synergistically mediate the transcription inhibition of IKKβ. IKKβ reduction removes its inhibitory effect on GADD45α induction and thus promotes the cells undergoing apoptosis. In conclusion,the induction of p53-Ets-1-IKKβ-MDM2-GADD45α signaling pathway plays an important role in mediating the pro-apoptotic effect induced by arsenite.