Zyxin-invovled Actin Regulation Is Essential In The Maintenance Of Chondrocyte Differentiation Status

Articular cartilage,the highly specialized connective tissue of synovial joints that devoid of nerves and blood vessels,is kept healthy by the only resident cartilage producing cells called chondrocytes.Studies have proved that the chondrocytes maintain a balance between synthesis and degradation of cartilage matrix in a biomechanically dynamic environment.The cartilaginous tissue loss,which finally leads to the whole joint degeneration,often occurs once this balance is destroyed by an abnormal mechanical or biological stimulus to chondrocytes.Therefore,the biomechanics and mechanobiology of articular chondrocytes play a critical role in homeostasis and pathology of articular cartilage and has been under extensive investigation.It has been acknowledged that the biomechanical properties of chondrocytes are mainly dependent on the cytoskeleton,especially the actin cytoskeleton,as actin has been shown to regulate a series of chondrocyte characteristics including phenotype,stiffness,viscoelasticity,and responses to mechanical/chemical stimuli.Like other eukaryotic cells,the actin cytoskeleton and extracellular matrix(ECM)of chondrocyte are connected through focal adhesion.Zyxin is a LIM protein that concentrates at focal adhesions,and is known to function as a mechanosensitive protein due to its ability to mobilize and relocate from focal adhesions to actin stress fibres in response to mechanical cues.Zyxin contains a number of dynamic structural features,including three C-terminal LIM domains involved in protein-protein interactions,a proline-rich domain that interacts with Ena/vasodilator-stimulated phosphoprotein(VASP)family members,anda nuclear export signal that allows translocation between the nucleus and cytoplasm.Zyxin has been reported to drive the actin cytoskeleton remodeling in fibroblasts and reinforce the actin cytoskeleton of human airway smooth muscle cells.It has also been shown that zyxin could reorganize actin filaments around exocytic granules to regulate endothelial von Willebrand factor secretion.However,the role that zyxin plays in modulating actin cytoskeleton in chondrocytes has not been elucidated.Vinculin,an actin-binding protein considered to reinforce cell-cell and cell-matrix adhesions,is a component of both integrin- and cadherin-based adhesion complexes and is recruited to both types of adhesions rapidly in response to mechanical cues through its interactions with talin and ?-catenin,respectively.Vinculin plays an important role in maintaining tissue integrity and is essential for persistent directional cell migration,indicating a role in forming a polarized connection between the actin cytoskeleton and adhesions.Studies have showed that vinculin and zyxin are colocalized at focal adhesions.Therefore,vinculin might be involved in the interaction between actin and zyxin.In this study,we investigated the modulating effects of zyxin on actin cytoskeleton and vinculin focal adhesion by constructing zyxin-knockdown rabbit articular cartilage chondrocyte model.We performed immunofluorescence,q RT-PCR and Western blot to measure actin and vinculin expression as well as collagen production of chondrocyte.Knockdown of zyxin increased the ratio of globular actin(G-actin)to filamentous actin(F-actin)of chondrocyte,which further inhibited expression of vinculin and chondrogenic marker collagen type II(COL II)as well as hypertrophy marker collagen type X(COL X).Moreover,to study the influence of actin on zyxin and vinculin,we tested the changes of zyxin and vinculin when chondrocytes were treated with transforming growth factor-?1(TGF-?1),as TGF-?1 has been shown to mediate chondrocyte actin cytoskeleton through Ras homolog gene family,member A(Rho A)pathway.Chondrocytes treated with TGF-?1 showed an enhanced expression of F-actin,and a lower expression of zyxin compared to non-treatment group.In response to TGF-?1-induced actin polymerization,expression of vinculin andde-differentiation marker collagen type I(COL I)was increased,while expression of COL II and aggrecan was decreased.These results demonstrate that in chondrocytes the level of zyxin is closely associated with the state of actin polymerization.In particular,the change of zyxin and F-actin parallels with the change of COL II and vinculin,respectively,indicating a critical role of zyxin-actin interaction in the synthesis of collagen ECM and the remodelling of cytoskeleton-ECM adhesion.