Roles Of Erythropoietin In Bone Regeneration Through EphrinB2/EphB4Signal Pathway

Bone repair or bone regeneration has become an important strategy in thetreatment of bone deformity and bone defects caused by trauma, tumor, andinflammation. Bone regeneration is a complex process of balance betweenbone-forming activity of osteoblasts and bone-resorbing activity of osteoclasts. Manymolecules are involved in the communication between osteoblasts and osteoclasts. Itwas believed that some proteins and signal pathways could affect both osteoclast andosteoblast at the same time to balance bone regeneration, such as ephrinB2/EphB4signal pathway. It was found that ephrinB2, which is synthesized in osteoclasts andbecome a transmembrane ligand in osteoclasts, and EphB4, a tyrosine kinase receptorin osteoblasts, are involved in the control of bone regeneration. Erythropoietin (EPO)is not only involved in red blood cell production, but recently was also found to beinvolved in many other biological functions, such as inducing bone regeneration.However, little is known about how EPO regulates bone regeneration. In this study,we hypothesized that EPO might be the double-edged protein that plays interestingroles by ephrinB2/EphB4in bone regeneration.Objective:(1) The role of EPO in the osteoblast/osteoclast differentiation.(2) In order to observe the roles of erythropoietin in bone regeneration throughephrinB2/EphB4signal pathway. We used ephrinB2-Fc to partially mimic theinteractions of osteoclasts and osteoblasts and gene knockdown assays.(3) The effects of EPO resulted in promoting new bone formation and inhibitingbone resorption in the animal model of jaw bone destruction.Methods:(1) To further understand mechanisms of effects of EPO on bone formation,murine bone marrow stromal cell line ST2cellas the osteoblast precursor werecultured with osteogenic medium. We will study the effect of EPO on the expressionof EphB4and their down stream molecules in progress of osteoblast differention byReal time PCR, cytochemical staining (ALP) and calcium deposition. In order to prove that the role of EPO on bone remodeling is mediated by bi-directional signalingof ephrinB2/EphB4, human EPO protein was used in osteoclast precursor (RAW264.7murine cell line) in vitro. The effect of EPO on the osteoclast differention by Realtime PCR, cytochemical staining (TRAP), and bone resorption assays in vitro.(2) In order to prove that the role of EPO on bone remodeling is mediated bybi-directional signaling of ephrinB2/EphB4, we will study the effect of EPO on theexpression of ephrinB2/EphB4and their downstream molecules in osteoclast andosteoblast co-culture system by Real time PCR and cytochemical staining, etc.Toobserve the effect of EPO on osteoclast/osteoblast differention by blockEphB4/ephrinB2signaling pathway by EphB4shRNA and ephrinB2siRNAin vitro.(3) Identify the role and mechanism of EPO on bone remodeling mediated byephrinB2/EphB4axis, and observe the effect of EPO on the regeneration of residualalveolar bone following tooth extraction in rats by soft X ray, bone mineral density(BMD) and histology, which will settle a foundation for treatment of jaw bonedestruction by EPO.Results:(1) The process of osteoblast differention showed that EPO increased expressionof EphB4in osteoblast. In addition, EPO increased the expressions of Runx2, Sp7,and ColⅠ in osteoblast at various time points. And EPO also enhance the activity ofALP and calcium deposition. The process of osteoclast differention EPO increasedexpression of ephrinB2in osteoclast and the expressions of c-fos, NFATc1, MMP9and Ctsk at early time points in osteoclast, but later decreased expressions of MMP9and Ctsk. The data from TRAP staining and bone resorption assays showed that EPOdecreased bone resorption. These results suggest that EPO can increase theproliferation of osteoclast, but not increase the bone resorption function.(2) EphrinB2-Fc interaction model was used as a stimulator to mimic theinteractions between osteoclast and osteoblast.EPO also increased expression ofosteogenicgenes and ALP positive osteoblasts and calcium deposition. The effects ofEPO in the osteoblast/osteoclast differention were attenuated by blockingephrinB2/EphB4gene transcription, suggesting that the regulation of bone remodelingby EPO might be through EphB4/ephrinB2signal pathway.(3) In vivo animal assays, BMD analysis and measurement of relative height ofresidual alveolar ridgeand histological findings (H&E staining) clearly demonstrate that EPO can efficiently induce new bone formation in the alveolar bone regenerationmodel.Conclusions:(1) EPO can promote osteoblast differentiation and the ability of bone formation;(2) EPO can increase the number of osteoclasts to inhibite resorption ability;(3) EPO directly stimulates ephrinB2expression in osteoclasts and EphB4expression inosteoblasts leading to the enhancement of bone formation while blacking the bone resorptionactivity of osteoclasts;(4) EPO promotes bone formation in an alveolar bone regeneration model.