DNA-PKcs-SIN1 Complexation Mediates Low-dose X-ray Irradiation(LDI)-induced Akt Activation And Osteoblast Differentiation

Objective: To investigate the activity of AKT signaling pathway after LDI and elucidate the underlying mechanism of LDI-induced osteoblast differentiation.Methods:1. Osteoblasts were harvested from the calvariae of 2-or 3-day old mice and exposed to single dose of X- irradiation with 0.0 Gy、0.5 Gy、1.0 Gy、2.0 Gy and 4.0 Gy respectively. The cell viability was measured by the 3- [4,5-dim-ethylthylthiazol-2- yl]-2,5 diphenyltetrazolium bromide(MTT) assay. Alkaline Phosphate(ALP) activity was evaluated by ALP viability kit. The expression of type I collagen(Col I) and Runt-related transcription factor-2(Runx2) were measured by Real- time PCR.2. Osteoblasts were exposed to single dose of X- irradiation with 0.0 Gy and 1.0 Gy. The activity of AKT was measured by Western-blot. After using the kinase inhibitors against Akt or DNA-PKcs, as well as the sh RN A partial depletion of Akt1, Alkaline Phosphate(ALP) activity was evaluated by ALP viability kit. The expression of type I collagen(Col I)and Runt- related transcription factor 2(Runx2) were measured by Realtime PCR. The target of LDI- induced AKT activity was explored by immunoprecipitation(Co-IP) assay.Results : Results from MTT assay demonstrated that only 4 Gy X-ray irradiation reduced cell viability of mouse calvarial osteoblasts, no significant effect were showed on osteoblast survival at 0.0 Gy、0.5 Gy、1.0 Gy、2.0 Gy(*p < 0.05). X-ray irradiation from 0.5 Gy to 2 Gy significantly increased ALP activity in osteoblasts, while X-ray irradiation at a high dose(4 Gy) failed to do so(*p < 0.05). X-ray irradiation from 0.5 Gy to 2 Gy induced m RNA expression of Col I and Runx2(*p < 0.05). LDI at 1 Gy showed most significant effect on osteoblast differentiation. DNA-PKcs activation is required for LDI- induced ALP activity increase and Col I/Runx2 m RNA expression in osteoblasts(*p < 0.05). Akt activation is involved in LDI- induced osteoblast differentiation(*p < 0.05). Co-IP assay results showed that LDI induced DNA-PKcs-SIN1 complexation in mouse calvarial osteoblasts(*p < 0.05), which was inhibited by NU- 7441 or by SIN1 si RNA-knockdown.Conclusion: DNA-PKcs-SIN1 complexation is required for LDI- induced Akt activation and mouse calvarial osteoblast differentiation.