IL-23 Induces Osteoclasts To Express Osteogenic Factors

Introduction Ankylosing spondylitis(AS)is a rheumatic disease that inflammation back pain is the main symptoms,combined with sacroiliac joint stiffness and spinal rigidity.The pathological features of AS is enthesitis,bone erosion,and syndesmophyte(new bone)formation.The pathogenesis of new bone formation in AS is still unknown.At present,there is a view that IL-23 may play an important role in AS new bone formation.Recently,a study showed that IL-23 caused opposite changes in enthesitis,osteoblast-mediated bone formation and osteoclast-driven bone erosion.IL-23 could regulate the differentiation of osteoclasts by RANKL signal,but it has no direct effect on osteoblasts.Under normal conditions,bone metabolism(bone remodeling)is always in the dynamic balance.Osteoblasts regulate the differentiation of osteoclasts by expressing M-CSF,RANKL and OPG.Osteoclasts express Ephrin B2,SPHK1,BMP2,BMP6,Wnt10 b to induce osteoblasts recruitment and promote osteoblast differentiation and mature cell survival.Osteoclasts increase semaphorin 4D and Htr A1 expression to inhibit osteoblast differentiation.Therefore,this study aims to explore the osteoclasts expression level of Ephrin B2,SPHK1,BMP2,BMP6,Wnt10 b,Htr A1 and semaphorin 4D after stimulated by IL-23.Methods Two methods were used to induce osteoclasts.The first method was to induce osteoclasts by mouse bone marrow mononuclear cells.The second method was to use RAW264.7 cells to induce osteoclasts.TRAP staining was used to identify osteoclasts.After comparing the efficiency of two methods,one method was chosen for subsequent experiments.The expression of TRAP and NFATc1 in osteoclasts was also tested by real-time PCR.After osteoclasts stimulated by IL-23,m RNA expression levels of Ephrin B2,SPHK1,BMP2,BMP6,Wnt10 b,semaphorin 4D and Htr A1 were evaluated by real-time PCR.Ephrin B2,SPHK1 expression levels were detected by western-blot.Results1.In experimental group of the two methods,some cells were TRAP-positive,containing more than three nuclei,while cells were small and TRAP-negative in control group.It was found that the numbers of osteoclasts induced by mouse bone marrow mononuclear cells were significantly higher than those induced by RAW264.7 cells.So we used mouse bone marrow mononuclear cells to induce osteoclasts in subsequent expriments.The m RNA expression levels of TRAP and NFATc1 in the experimental group were significantly higher than those in the control group.It further confirmed that this method can induce osteoclasts and can be used for subsequent experiments.2.The expression of Ephrin B2,SPHK1 and BMP2 were increased at m RNA level.At 3hours,the expression level of Ephrin B2 was 2.10 times higher than that in control group.Three hours later,SPHK1 expression level was 2.30 times higher than that of control group.Six hours later,SPHK1 was 2.46 times higher than control.Compared with control group,the expression level of BMP2 at 3 hours was 1.46 times higher.Western blot was used to detect the expression of Ephrin B2 and SPHK1.The results showed that Ephrin B2 and SPHK1 expression was significantly increased after IL-23 stimulation.3.Compared with the control group,the expression of BMP6、Wnt10b、Semaphorin 4D and Htr A1 at m RNA level did not change significantly.Conclusions1.IL-23 induces osteoclasts to express Ephrin B2,SPHK1 and BMP2.It is indicated that IL-23 may promote the differentiation of osteoblasts by osteoclasts indirectly.2.IL-23 does not induce osteoclasts to express BMP6,semaphorin 4D and Wnt10 b.