Roles Of Ku80 In DNA-PK/JNK Signaling Pathway Mediates Silica-Induced DNA Double Strand Breaks Repair In Human Embryo Lung Fibroblasts

Background and objective:Silica is one of the most serious occupational hazards capable of inducing lung fibrosis and lung cancer after chronic exposure. Previous studies showed that silica exposure can induce cell cycle alternations and DNA double-strand breaks (DSBs).Our previous studies showed that silica exposure can induce cell cycle alternations, accompanied with the increased percentages of cells in S phase, and the marked activation of serine/threonine kinase protein kinase B (PKB/Akt), activator protein-1 (AP-1) as well as mitogen activated protein kinase (MAPK). Moreover, Akt/ERK, JNK pathway medicates silica-induced activator protein 1 (AP-1) transactivation, the expression of cyclin Dl and cyclin-dependent kinase 4 (CDK4) as well as cell cycle alternations.DNA-dependent protein kinase (DNA-PK), composing of a large catalytic subunit, DNA-PKcs, and a regulatory component, the Ku70-Ku80 heterodimer, is a molecular sensor for DNA damage, and is involved in the repair of DSBs by nonhomologous end-joining (NHEJ) pathway, in which Ku70-Ku80 plays a key role.It has been reported that DNA-PK phosphorylates a number of proteins, including Akt, JNK, p53, and many transcription factor such as c-Jun, c-Fos, which eventually contribute to damage-induced cell cycle arrest or cell apoptosis. Our previous studies showed that DNA-PKcs involves silica-induced cell cycle arrest and DSBs repair by DNA-PK/Akt/JNK/AP-1 signaling pathway.However, Roles of Ku80 in DNA-PK/JNK signaling pathway mediates silica-induced DSBs repair are not clear. Another, DNA-PK comprises same catalytic domain with Phosphatidylinositol 3 kinase (PI3K), which also involves silica-induced cell cycle alternations and activation of MAPK. Whether PI3K involves in repair of DSBs is neither known.Based on above studies, our current studies, which take DSBs and the repair as the breakthrough point, further focused on tracing the upstream sensor of our previous MAPKs pathway, the regulatory component (Ku80 mainly) of DNA-PK, and the biological endpoints of DSBs damage repair effect. In this study, RNAi, dominant negative mutants as well as chemical inhibitors, were used to investigate the roles of Ku80 in DNA-PK/JNK pathway mediates silica-induced DSBs repair as well as the potential effect of this pathway on silica-induced cell cycle and cell cycle regulatory proteins alternation in human embryonic lung fibroblast (HELF), and to detect the upstream or downstream relationship of signaling pathway.MethodsKu80 siRNA expression vectors was transfected into HELF by lipofectamine. Neutral comet assay and/or yH2AX recognition technology were applied to detect silica-induced DNA double strand breaks. According to the neutral comet experimental result, the DNA repair ability (DNA repair compentence, DRC) was calculated. According to the DRC value, the roles of Ku80 in the silica-induced DSBs repair were analyzed. The expression levels and activity of protein in HELF, such as DNA-PKcs, Akt, JNK, c-Jun, p53, p21, Cyclin D1, CDK4, Cyclin E, CDK2, E2F1, pRb were determined by WB. Cell cycle changes were identified by flow cytometry in HELF. The formation of yH2AX foci in HELF were analyzed by immunofluorescence microscopy.Result1. Stable transfectants, H-NC and H-Ku80 were established successfully.2. Both western blot and immunofluorescence assay analysis indicated that siRNA-mediated silencing of Ku80 strikingly downregulated the silica-induced expression of yH2AX in HELF. It indicates that H2AX phosphorylation is through Ku80 dependent pathway, and Ku80 effects the function of DNA-PK which acts as silica-induced DNA double strand breaks damage sensor.Silencing of Ku80 in HELF cells resulted in a decreased of silica-induced DNA damage repair competence compared with the negative control cell.After the expression of Ku80 was inhibited, the increase of phosphorylation of Akt at Ser473, JNK at Thr183/Tyr185, and c-Jun at ser63, which induced by silica exposure were potently blocked in HELF cells.3. When Ku80 expression was inhibited, the number of S phase cells was marked increased, and the overexpression of p21, p53, CyclinD1, CDK4, Cyclin E, CDK2, and the phosphorylation level of p53 at ser15 were potently blocked. however, the overexpression of E2F1 and the phosphorylation level of pRb-Ser780 were further increased.4. Blocking activation of PI3K by dominant negative mutant in DN-p85 cell, silica-induced DNA damage repair competence was decreased compared with the negative control cell induced by silica. And expression levels of Ku70 and Ku80, not DNA-PKcs, were also decreased.Conclusion1. Ku80 regulates the function of DNA-PK as silica-induced DSBs damage sensor and promotes DSBs repair by DNA-PK/Akt/JNK/AP-1 signaling pathway.2. Ku80 mediates silica-induced cell cycle change by DNA-PK/JNK and DNA-PK/p53 signaling pathway.3. PI3K promotes silica-induced DSBs damage repair by regulated expression levels of Ku70 and Ku80.In brief, this research had initially proven the roles of Ku80 in DNA-PK/JNK pathway in silica-induced DSBs repair. These findings will help us to understand the signal transduction mechanisms involved in the pathogenesis effects of silica at DNA damage reponse level.