Signaling Events Involving In Mediating IKKα-Dependent P53 Transactivation In The Cellualr UVB Responses

BackgroundUV(ultraviolet radiation) is a typical environmental stressor, which can be divided into UVA(320-400nm), UVB(280-320nm) and UVC(200-280nm) due to their different wavelengths. Among them, Ultraviolet B irradiation is the key environmental factor for skin damage response. UVB irradiation not only induces the inflammatory response via up-regulating the expressions of the inflammatory cytokines, such as TNFα and VEGF, but also triggered the DNA damage response featured by p53 transactivation. We have disclosed the molecular mechanism invovling in mediating UVB-induced inflammatory response in our previous studies, so we focused on elucidating the molecular mechanism involving in mediating the UVB-induced DNA damage response featured by p53 transactivation in the current study.With the further research on IKK/NF-κB signaling pathway, some IKK kinase substrate that are unrelated to NF-κB and IκB have been found, suggesting that IKK kinase may have new function independent of NF-κB. IKKα, as an important regulatory subunit of IKK kinase can activate a series of signaling molecules unrelated of the activation IKK/NF-κB signaling pathway, and participates in various biological reactions. Our team has engaged in investigating the new IKKβ and NF-κB-independent function of IKKα for a long term. We found that the induction of VEGF upon UVB exposure is IKKα-dependent while NF-κB–independent signaling event, which is critical for mediating the inflammatory response. Therefore, we focused on explore the potential role of IKKα involving in mediating UVB-induced p53 transactivation and the DNA damage response. ObjectiveTo figure out the signal transduction mechanism of IKKα, one of the catalytic subunit of IKK complex, involving in mediating the UVB-induced DNA damage response featured by p53 transactivation. MethodsThe transactivation of p53 was determined by dual-luciferase reporter gene analysis system; p53ser15(h)/ser18(m)、ATR、ATM、CHK1、LKB1 and PERK expression was analyzed by Western Blot in Hacat and MEFs cell lines. Whether IKKα with ATR、ATM、CHK1、LKB1、PERK could interact in vivo, even in a more physiological context was exzamined by co-immunoprecipitation. We investigated the sensitivity anaslysis of p53 and IKKα on apoptosis reaction by flow cytometry assay. ResultsFirst, we found that UVB exposure significantly induced p53 transactivation and therefore mediated the pro-apoptotic effect in Hacat and MEFs cells. Furthermore, IKKα played a key role in mediating UVB-induce p53 activation and the pro-apoptotic effect.Second, we found that IKKα was involved in the activation of the ATR-ATR/CHK1 pathway, the well-known upstream signaling event in mediating p53 activation in the DNA damage responses. ATR, ATM, CHK1 and p53 formed a protein complex and the binding of IKKα with CHK1 is critical for the formation and p53 transactivaion. In the absence of IKKα, ATR and ATM lost the binding ability to CHK1, which resulted to the inhibition of CHK1 and p53 activation.Third, we found that IKKα also exerted a role in activating the energy metabolic stress factor LKB1, which functioned as an upstream activator for p53 in the UVB response. The presence of IKKα is critical for the formation of LKB1/p53 protein complex. Without IKKα expression, LKB1 failed to bind with and activate p53.Fourth, we found that UVB could significantly induce the activation PERK, one of the key regulators for ER stresses. Moreover, IKKα was able to activate p53 via PERK-dependent manner in Hacat and MEFs cells. Similarly, UVB exposure induced the binding of IKKα to PERK/p53 protein complex. But surprisingly, the presence of IKKα is not required for the formation of PERK/p53 protein complex. ConclusionFirst, IKKα is involved in p53 transactivation by activaing ATR-ATM/CHK1 pathway in the cellular UVB response.Second, LKB1 is another mediator for IKKα–dependent p53 transactivation in the UVB response.Third, IKKα is involved in p53 transactivation by activaing in regulatin PERK/p53 pathway in the cellular UVB response.