DNA-PKcs/JNK/p53 Signaling Pathway Mediates Silica-Induced DNA Double Strand Breaks Repair In Human Embryo Lung Fibroblasts

Background and objective: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 DNA double strand breaks (DSBs) by non-homologous end-joining (NHEJ) pathway.Silica is one of the most serious occupational hazards capable of inducing lung fibrosis and lung cancer after chronic exposure. 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 D1 and cyclin-dependent kinase 4 (CDK4) as well as cell cycle alternations. However, the sensor molecules of silica-induced DNA double strand breaks are not clear.Based on above studies, our current studies, which take DNA double strand breaks and the repair as the breakthrough point, further focused on tracing the upstream sensor of our previous MAPKs pathway, the catalytic subunit of DNA-PK, and the biological endpoints of DNA double strand breaks repair effect. In this study, RNAi, dominant negative mutants as well as chemical inhibitors, were used to investigate the role of DNA-PKcs/JNK/p53 pathway in silica-induced DNA double strand breaks repair as well as the potential effect of this pathway on silica-induced cell cycle and cell cycle regulatory proteins alternations in human embryonic lung fibroblast (HELF), and to detect the upstream or downstream relationship of signaling pathway.Methods1. DNA-PKcs siRNA expression vectors together with AP-1 luciferase reporter plasmid were transfected into HELF by lipofectamine. Hygromycin was used to select the transfected cell lines, which were consequently identified by western blot (WB).2. Neutral comet assay and/orγH2AX 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.3. The expression levels and activity of protein in HELF, such as DNA-PKcs, Akt, JNK, p53, p21, cyclin D1, CDK4, E2F1, pRb, were determined by WB. Cell cycle changes were identified by flow cytometry in HELF. The formation ofγH2AX foci in HELF were analyzed by immunofluorescence microscopy. AP-1 luciferase activity was determined by the luciferase reporter gene assay using a luminometer.Results1. Stable transfectants were established successfully.2. Silica induces the expression ofγH2AX in HELF in a dose- and time-dependent manner.After treatment with different doses of silica for 12 h, 25μg/ml silica can dramatically induce the expression ofγH2AX. Along with the concentration increasing, theγH2AX level was increased gradually and reached a peak at 200μg/ml. After treatment with 200μg/ml silica for different times, the levels ofγH2AX increased in a time-dependent manner. The expression ofγH2AX was significantly increased at 1 h, and reaching maximum at 12 h and then decreasing at 24 h.3. Analysis of DNA double strand breaks and repairAfter treatment with different doses of silica for 12 h, Olive tail moment increased in concentration-dependent manner. After treatment with 200μg/ml silica for different times, Olive tail moment increased significantly at 6 h, and reaching maximum at 12 h and then decreasing at 24 h.4. DNA-PKcs is silica-induced DNA double strand breaks damage sensor Both western blot and immunofluorescence assay analysis indicated thatsiRNA-mediated silencing of DNA-PKcs strikingly downregulated silica-induced the expression ofγH2AX in HELF. It indicates that H2AX phosphorylation is through DNA-PKcs dependent pathway, and DNA-PKcs is silica-induced DNA double strand breaks damage sensor.5. The role of DNA-PKcs, Akt, INK, AP-1 and p53 in silica induced DNA double strand breaks repair by neutral comet assay.Silencing of DNA-PKcs in HELF cells resulted in a decreased of silica-induced DNA damage repair competence (DRC=22.47 %), compared with the negative control cell induced by silica (DRC=59.67 %). Silica-induced DNA damage repair competence was markedly decreased in dominant negative mutants of Akt or JNK. Inhibition of the activation of AP-1 by curcumin significantly inhibited DNA double strand breaks repair in response to silica treatment. After p53 expression was inhibited, silica-induced DNA damage repair competence was markedly increased. These results indicate that DNA-PKcs, Akt, JNK and AP-1 can promote silica-induced DNA damage repair, however, the expression of p53 inhibit silica-induced DNA damage repair.6. Effect of DNA-PKcs and p53 on cell cycle and cell cycle regulatory proteins.Silica increased the percentage of S phase as compared to the controls. Silica markedly increases in the expression of cyclin D1, E2F1 and p21 as well as the phosphorylation level of pRb-Ser780. There were not marked changes of pRb and CDK4 expression. When DNA-PKcs and p53 expression was inhibited, the number of S phase cells was marked increased, and the overexpression of p21 was inhibited, however, the overexpression of E2F1 and the phosphorylation level of pRb-Ser780 were further increased. Expression of cyclin D1 were not changed after the inhibiton of DNA-PKcs and p53 expression. These results suggest that DNA-PKcs and p53 are involved in silica-induced cell cycle change through positively regulating the expression of p21, negatively regulating the overexpression of E2F1 and the phosphorylation level of pRb-Ser780.7. Relationship among DNA-PKcs, Akt, JNK, AP-1 and p53 in silica-treated HELFExposure of HELF cells to silica markedly increased the phosphorylation of Akt at Ser473 and Thr308, JNK at Thrl83/Tyrl85,and p53 at Ser15 as well as the expression of p53 and transactivation of AP-1. After the expression of DNA-PKcs was inhibited, silica-induced phosphorylation level of Akt-Ser473, JNK and p53 were potently blocked, indicating that DNA-PKcs is upstream kinase of Akt, JNK, AP-1, p53. After the expression of Akt,JNK and AP-1 transactivation were inhibited , silica-induced the phosphorylation of p53 and the expression was not changed, suggesting that the phosphorylation of p53 is through Akt,JNK and AP-1 independent pathway. In our previous study, Akt/JNK/AP-1 signaling pathway has been tested. These results indicate that DNA-PKcs activate two pathways in silica-induced DNA double strand breaks repair, including DNA-PKcs/Akt/JNK/AP-1 and DNA-PKcs/p53 pathway, which is different in the role of silica-induced DNA double strand breaks repair.Conclusion 1. DNA-PK is silica-induced DNA double strand breaks damage sensor.2. DNA-PKcs/Akt/JNK/AP-1 signaling pathway promote silica-induced DNA double strand breaks repair, however, the expression of p53 inhibited silica-induced DNA double strand breaks repair.3. DNA-PKcs/p53 pathway mediates silica-induced cell cycle change through positively regulating the expression of p21, negatively regulating the overexpression of E2F1 and the phosphorylation level of pRb-Ser780.In brief, this research had initially proven the role of the DNA-PKcs/JNK/p53 pathway in silica-induced DNA double strand breaks repair. These findings will help us to understand the signal transduction mechanisms involved in the pathogenesis effects of silica at DNA damage reponse level.