Study On The Mechanism Of ANTXR1/integrin β1 Interaction In Response To Matrix Stiffness To Regulate ECM Synthesis In Chondrocytes

Cartilage defect is a complex and multi-factorial disease,the incidence of which is increasing year by year worldwide.After clinical treatment,there will be a large amount of fibrous tissue and fibrous cartilage in the repaired cartilage,called fibrosis of hyaline cartilage,which is mainly manifested by the extracellular matrix（ECM）component disorder and tissue stiffness decline,and ECM component disorder is an important reason for the decline in tissue stiffness.Therefore,improving the ECM component of the repaired cartilage is the key to the repair of cartilage defects.More and more evidence shows that mechanical microenvironment,especially stiffness,is the key factor affecting ECM homeostasis in cartilage tissue,but the related issue is still limited.ANTXR1 is a cell membrane protein that plays an important role in ECM regulation.Previous studies have shown that the expression of this molecule changes under different hydrostatic pressure,which is related to chondrogenic differentiation of stem cells,and interacts with the classical mechanosensor integrinβ1 in intracellular signal transduction.Therefore,ANTXR1 is likely to play an important role in the repair of cartilage defects.Starting with the participation of ANTXR1 in the repair of cartilage defects,this study explored the regulation of ANTXR1on cartilage tissue ECM in response to changes in matrix stiffness during cartilage repair,and further explored the interaction mode and mechanism of ANTXR1 and integrinβ1.The molecular mechanism of chondrocyte cell membrane protein"crosstalk"regulating ECM production was revealed.The successful implementation of this project is expected to provide new ideas and intervention targets for the repair of cartilage defects.【Objectives】（1）To clarify the correlation between ANTXR1 involvement in the process of cartilage defect repair and the generation of ECM in cartilage tissue.（2）To uncover the role of ANTXR1 in regulating the ECM synthesis pattern of chondrocyte in response to matrix stiffness.（3）To elucidate the specific molecular mechanisms of ANTXR1 regulates cartilage repair through interaction with integrinβ1 in response to matrix stiffness.【Methods】（1）Establish a rat cartilage defect animal model,and analyze the natural repair process of rat cartilage defects using Masson staining and Safranin O-Fast Green staining.Employ immunohistochemical staining to observe the spatiotemporal expression of ANTXR1 and ECM components during the cartilage repair process.Measure the stiffness of repaired cartilage using atomic force microscopy（AFM）.The correlation between matrix stiffness,ANTXR1 expression and ECM production was preliminarily established.（2）Construct mechanical environment using hydrogels in vitro.Utilize Western blotting and immunofluorescence to analyse the expression of ANTXR1 and major ECM components under different stiffness conditions.Lentivirus transfection adjusted the expression level of ANTXR1,and ANTXR1^-/-chondrocyte transcriptome analysis was used to clarify the role of ANTXR1 in regulating ECM production in chondrocytes.（3）The expression level and activation state of integrinβ1 were detected by Western blotting and flow cytometry.Co-IP and immunofluorescence co-localization were used to analyze the direct binding of ANTXR1 and integrinβ1 at different stiffness.MD,FRET and Western blotting were used to analyze and verify the specific mechanism of the interaction between ANTXR1 and integrinβ1.The expression level and activation state of ANTXR1and integrinβ1 were adjusted,the expression levels of SOX9 and RUNX2 were detected to explored the intracellular signaling pathway of ANTXR1 and integrinβ1 interacting on ECM generation in chondrocytes.【Results】（1）It was found in the rat knee cartilage defect animal model that the cartilage tissue showed"stratification"after the defect repair,the upper layer tended to be fibrous cartilage tissue,and the lower layer tended to be transparent cartilage tissue;ANTXR1,ECM components and tissue stiffness in the repair area showed significant changes during the repair process,in which the expression of ANTXR1 and collagen I and collagen VI components of fibrous cartilage tissue showed obvious spatiotemporal overlap,and the expression level was opposite to the stiffness of stratified tissue.（2）The results of in vitro stiffness microenvironment culture showed that ANTXR1and ECM components showed expression changes under different stiffness conditions,namely,ANTXR1,collagen I and collagen VI were highly expressed under low stiffness,while collagen II and aggrecan were highly expressed under high stiffness.By adjusting the expression level of ANTXR1 and analyzing the transcriptome of ANTXR1-/-chondrocytes,it was confirmed that ANTXR1 had a role in regulating ECM production in chondrocytes.（3）In vitro mechanism research found that ANTXR1 and integrinβ1 interact under soft conditions;Further studies showed that the main region of ANTXR1 and integrinβ1interaction occurred in the extracellular region,and this binding state caused the conformation of integrinβ1 to change from open to folded,thereby affecting its activation state and the expression levels of the intracellular transcription factors RUNX2 and SOX9.【Conclusion】This study reveals the mechanism of ECM disorder after cartilage defect repair from the perspective of membrane protein interaction firstly.It was found that in the process of cartilage defect repair,matrix stiffness can regulate the expression of ANTXR1 and integrinβ1 in chondrocytes and change their binding state,that is,low matrix stiffness in the cartilage surface of the repair area induces high expression of ANTXR1,resulting in binding to integrinβ1 extracellular region.This reduces the activation level of integrinβ1,which in turn affects the expression of transcription factors SOX9 and RUNX2,and ultimately alters ECM production in chondrocytes.This study identified the role of ANTXR1 and integrinβ1 interaction in the formation of ECM in cartilage defect repair area,providing new ideas and molecular targets for articular cartilage defect repair.