Mechanistic Study Of Direct And Indirect Pathways Of CXCR4 Contribute To Fibrocyte Proliferation And Ossification In Ankylosing Spondylitis

Objective:Ankylosing spondylitis(AS)is a chronic autoimmune disease characterized by inflammation and abnormal osteogenesis.The incidence of AS in China is about 0.3%,and the number of patients is about 4 million.Lumbosacral pain is accompanied by progressive destruction of joint structures in the spine and limbs,leading to joint stiffness and eventual disability.At present,the clinical treatment mainly focuses on the control of chronic inflammation,and there is no effective treatment for abnormal ossification.The mechanism is unclear.Fibroblasts are the most common cells in the periarticular connective tissues and have osteogenic ability under pathological conditions.Studies have shown that the inducing inflammation in vitro can promote the proliferation of fibroblasts and increase collagen synthesis,and enentuallylead to bone formation and connective tissue fibrosis,mechanism of which is still unclear.CXCR4 is a member of the CXC chemokine family,is a specific receptor for SDF1(stromal cell-derived factor-1).The SDF1/CXCR4 signaling pathway plays an important role in biological activities.It is involved in the activation,migration and homing of various cells,especially tumor cells and stem cells.It has been found that the CXCR4 signaling pathway has the dual effects of promoting osteogenesis and destroying bone.Our previous studies found that CXCR4 is highly expressed in ligaments and synovium of AS,and CXCR4 was positively correlated with the WNT/β-catenin pathway in cell models.It was also found that CXCR4 was positively correlated with IL6,and exogenous IL-6 significantly up-regulates Annexin A2 expression and ALP activity.Thus in AS,does the CXCR4 signaling pathway play the role in osteogenesis or osteoclast in bone metabolism in AS? Therefore,this study aims to investlgate the regulatory mechanism of CXCR4 on fibroblasts ossification:firstly,the regulatory ability of CXCR4 on osteogenesis of fibroblasts derived from AS ligaments was clarified.;Secondly,the molecular mechanism of osteogenesis was studied.In our study,it was found that CXCR4 activation can also promote THE secretion of IL6,and IL-6 can stimulate the ossification of fibroblasts themselves.Therefore,we also revealed another mechanism of CXCR4 regulating ossification through an indirect way.Materials and Methods:1.The pathological ligament tissues of the hip joint of AS patients with ossification or ossification tendency in the middle and late stage of the disease and normal ligament tissues were collected for primary fibroblast culture.2.A viral vector targeting CXCR4 expression regulation was designed and constructed to transfect AS primary fibroblasts,and the transfection efficiency was evaluated and optimized.3.The expression level of CXCR4 in fibroblasts was changed,and the proliferation,osteoblast differentiation indexes and expression changes of major proteins in Wnt pathway of fibroblasts were detected;4.To evaluate the regulation effect of CXCR4 agonist SDF-1 and inhibitor AMD3100 on fibroblast osteogenesis;5.Western blot and ELISA verifiecd that CXCR4 could promote the expression and secretion of IL6 through the PI3K-Akt pathway;6.To study the effect and mechanism of IL-6 on fibroblast ossification.Results:1.Compared with the control group,the expression of CXCR4 in the ligament tissues of AS patients was significantlyincresased,and the expression of CXCR4 in the primary cultured fibroblasts of the ligament tissue was also higher than that of the control group.The proliferation ability of fibroblasts composed of AS was higher than that of control group.2.The proliferation ability,bone formation and mineralization ability of fibroblasts were significantly improved by increasing the expression level of CXCR4;Otherwise,it weakens;3.CXCR4 expression was increased,and the activity of Wnt /β-catenin pathway was also increased;Moreover,this pathway can be activated by SDF-1 and inhibited by AMD3100.4.After the activation of CXCR4 pathway,the expression of cyclin-D1 and c-myc,which are related to proliferation,were increased;The expression of alkaline phosphatase(ALP)and osteocalcin(OCN),a marker protein associated with osteogenesis,were also increased.After blocking CXCR4,proteins related to proliferation and ossification were also inhibited.5.Endogenic knockdown of CXCR4 inhibited PI3K-Akt pathway activity and reduce the expression of IL-6;SDF-1 activates CXCR4,further activates PI3K-Akt pathway,and enhances IL-6 expression.6.After treatment of fibroblasts with different concentrations of il-6(0,10,20 ng/ml)for 24 h,it was found that Annexin A2 gene transcription and protein expression were promoted,and the protein expression increased significantly with the increase of il-6concentration.7.There was a positive correlation between serum IL-6 and Annexin A2 m RNA level in the corresponding ligament tissue samples(r=0.8727),and the Sperman analysis of correlation coefficient(P =0.0233)indicated that there was a positive correlation between the two.8.Annexin A2 plays a role in promoting fibroblast ossification through activation of ERK pathway,with significantly increased expression levels of ossification-related proteins such as BMP-2,OCN and OSX.The ratio of OPG to RANK as an osteogenic marker increased significantly.Knockdown of Annexin A2 expression or blocking of the ERK pathway weakens the ossification effect of fibroblasts and reduces the expression of corresponding marker proteins.Conclusion:CXCR4 increased in fibroblasts,which directly activated the proliferation and osteogenic ability of fibroblasts through the Wnt/β-catenin signaling pathway(direct effect).In addition,the pi3K-Akt pathway can stimulate the secretion of IL-6 by fibroblasts,and high level of IL-6 can play an indirect role in the ossification of fibroblasts through Annexin A2-MAPK/ ERK pathway.