The Effect And Mechanisms Of HIF-1? Mediated Osteoclast Differentiation Under Hypoxia Stress

Background:During normal growth and development,osteoblast-mediated bone formation is coupled with osteoclast-mediated bone resorption,with the participation and precise regulation of multiple cells,cytokines and stimulants.Keep bone tissue in a state of dynamic balance,maintaining the stability of normal bone structure and physiological function.Osteoclasts are formed by the fusion of mononuclear/macrophage lineages in the bone marrow and play an important role in the maintenance of skeletal homeostasis.It is the only cell with bone resorption function discovered so far,not only involved in participates in physiologic bone remodeling,but also plays an important role in pathological bone metabolism.Studies have shown that in bone metabolism-related diseases such as osteoporosis,tumor bone metastasis and rheumatoid arthritis,osteoclast differentiation and excessive bone resorption have an important impact on the progression and prognosis of these diseases.The normal physiological conditions of bone marrow is in a hypoxic microenvironment with an oxygen volume fraction of about 6.6%.Fractures,inflammation and tumors can cause blood flow or oxygen supply decrease in the bone,which makes the bone microenvironment in a more obvious pathological hypoxic state.Many studies have explored how hypoxia and hypoxia-inducible factor-1??HIF-1??affect osteoclast differentiation and activity,but there is no clear specific molecular mechanism at present.Objective:To study the regulation of hypoxia/HIF-1?pathway on the differentiation of RAW264.7cells into mature osteoclasts,and to explore its possible molecular mechanism.Methods:1.Real-time quantitative PCR and TRAP staining were used to detect the effect of CoCl₂-induced hypoxia on the differentiation of RAW264.7 cells into mature osteoclasts.2.Real-time quantitative PCR and Western Blotting were used to detect the effect of CoCl₂-induced hypoxia on the expression of HIF-1?during RAW264.7 cell differentiation from mRNA and protein levels respectively.3.HIF-1?-shRNA was used to silence the expression of HIF-1?in RAW264.7 cells.The differentiation of RAW264.7 cells was detected by real-time fluorescent quantitative PCR and TRAP staining.To determine whether HIF-1?mediates the regulation of CoCl₂-induced hypoxia on osteoclast differentiation.4.Real-time quantitative PCR and Western Blotting were used to detect the effect of CoCl₂-induced hypoxia on the expression of NRP-1 during RAW264.7 cell differentiation from mRNA and protein levels respectively.5.The expression of NRP-1 was detected by Western blot after HIF-1?down-regulation.To determine whether HIF-1?mediates the regulation of NRP-1 by CoCl₂-induced hypoxia.6.The expression of NRP-1 in RAW264.7 cells was down-regulation by NRP-1-shRNA.The differentiation of RAW264.7 cells was detected by real-time fluorescent quantitative PCR and TRAP staining.To clarify whether NRP-1 mediates the regulation of CoCl₂-induced hypoxia on osteoclast differentiation.Result:1.CoCl₂-induced hypoxia significantly decreased the mRNA expression of Osteoclast differentiation related genes?Cath K,MMP-9 and TRAP??P<0.05?,and decreased the number of TRAP-positive osteoclasts?P<0.05?.2.Hypoxia induced by CoCl₂ significantly up-regulated the expression of HIF-1?protein during the differentiation of RAW264.7 cells?P<0.05?,but had no effect on the expression level of HIF-1?mRNA.3.Under the condition of CoCl₂-induced hypoxia and down-regulation of HIF-1?protein in RAW264.7 cells,the mRNA expression of Osteoclast differentiation related genes?Cath K,MMP-9 and TRAP?were increased obviously?P<0.05?.as well as the number of TRAP-positive osteoclasts?P<0.05?.4.Hypoxia induced by CoCl₂ significantly up-regulated the expression of NRP-1 protein and mRNA during the differentiation of RAW264.7 cells?P<0.05?.5.Under the condition of CoCl₂-induced hypoxia,the silencing of HIF-1?expression in RAW264.7 cells significantly inhibited the expression of NRP-1 protein?P<0.05?.6.Under the condition of CoCl₂-induced hypoxia,Silenced the expression of NRP-1 in RAW264.7 cells up-regulated the expression levels of Cath K,MMP-9 and TRAP mRNA?P<0.05?and increased the number of TRAP-positive osteoclasts?P<0.05?.Conclusion:CoCl₂-induced hypoxia may inhibit the differentiation of RAW264.7 cells into mature osteoclasts via HIF-1?-mediated upregulation of NRP-1.