0.5 Gy X-ray radiation promotes osteoblast differentiation by Wnt/β-Catenin signaling

Abstract

Objective: To investigate the important roles of Wnt signaling in the processes of 0.5Gy X-ray promoting osteoblast differentiation, and make clear the molecular mechanisms involved. Methods: Flow cytometry was employed to detect the apoptosis after osteoblast exposure to 0.5 Gy X-ray radiation.The protein level of osteoblast differentiation markers, such as collagen Iα (Col1α), alkaline phosphatase (ALP), osteocalcin (OCN), were detected by Western-blot and ALP activity staining was performed. Real-time PCR and Western-blot were utilized to evaluate the variations of key factors in Wnt signaling pathways, while specific inhibitor of Wnt/β-Catenin, XAV939 was used to block the Wnt signaling. Results: Low-dose (0.5 Gy) X-ray induced significant decline in MC3T3-E1 osteoblast apoptosis at three days after radiation.The dynamic variations in the expression of osteoblast differentiation markers, including Col1α, ALP, OCN, were observed after 0.5 Gy X-ray irradiation by Western blot analysis.The protein levels of Col1α have a reduction temporarily at 4 days of radiation (34.5%±5.8%, t=9.912, P<0.001), then a significant increase is detected at 10 day after radiation (162.5%±6.5%, t=2.673, P<0.05). OCN levels dropped by 83% (t=3.968, P<0.01) at 4 day after 0.5 Gy X-ray radiation, and raised at 10 day (39.5%±4.1%, t=3.219, P<0.05) and 14 day (79.4%±7.5%, t=6.708, P<0.001), respectively. ALP levels increased at 7 day (79.7%±22.3%, t=6.257, P<0.001) and 10 day(128.3%±6.1%, t=4.340, P<0.01)after radiation. At the same time, 0.5 Gy X-ray radiation can activate Wnt/GSK-3/β-Catenin signaling.The mRNA levels of Wnt3a、LPR5 and TCF-4 increased by 1.7 fold (t=6.573, P<0.001), 1.1 fold (t=5.323, P<0.05) and 1.4 fold (t=3.054, P<0.05) at 7 day after radiation.In addition, p-GSK-3β level reduced by 42.1% (t=4.460, P<0.01), and active β-Catenin increased by 1.9 fold (t=3.528, P<0.05). However, the specific inhibitor of Wnt/β-Catenin, XAV939 completely abrogated Wnt/β-Catenin signaling and the increase in ALP expression and activity induced by 0.5 Gy X-ray radiation. Conclusion: These results demonstrated that low dose X-ray radiation promoted osteoblast survival at early differentiation, and promoted differentiation at middle and late stage, in which Wnt signaling participated the regulation processes.