Zyxin‐involved actin regulation is essential in the maintenance of vinculin focal adhesion and chondrocyte differentiation status

Abstract Objectives To investigate the role of zyxin‐involved actin regulation in expression level of vinculin focal adhesion and collagen production of chondrocyte and its possible underlying mechanism. Materials and methods Chondrocytes obtained from rabbit articular cartilage were used in this study. The expression of zyxin, actin and vinculin, as well as the extracellular matrix (ECM) protein collagen type I, II and X (COL I, II and X) of chondrocytes were compared between zyxin‐knockdown group and negative control group, and between transforming growth factor‐β1 (TGF‐β1) treatment group and non‐treatment group, respectively. Results 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 COL II as well as hypertrophy marker COL X. On the other hand, chondrocytes treated with TGF‐β1 showed an enhanced expression of F‐actin, and a lower expression of zyxin compared to non‐treatment group. In response to TGF‐β1‐induced actin polymerization, expression of vinculin and COL I was increased, while expression of COL II and aggrecan was decreased. Conclusions These results demonstrate supporting evidence 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 major role of zyxin‐actin interaction in the synthesis of collagen ECM and the remodelling of cytoskeleton‐ECM adhesion.


| INTRODUC TI ON
Articular cartilage is the highly specialized connective tissue of synovial joints. It is devoid of blood vessels and nerves and is kept healthy by cartilage producing cells called chondrocytes. 1 The chondrocytes maintain a balance between cartilage matrix synthesis and degradation in a biomechanically dynamic environment. 2 Once this balance is destroyed by an abnormal biological or mechanical stimulus to chondrocytes, the loss of cartilaginous tissue occurs, which finally leads to the degeneration of the whole joint. 3 Therefore, the biomechanics and mechanobiology of articular chondrocytes is of great significance to articular cartilage homeostasis and pathology and has been under extensive investigation. 4,5 It is acknowledged that the biomechanical properties of chondrocytes are largely dependent on the cytoskeleton, in particular the actin cytoskeleton, as actin has been shown to regulate a series of chondrocyte characteristics including phenotype, 6,7 viscoelasticity, 8,9 stiffness 10,11 and responses to chemical/mechanical stimuli. 12,13 Like other eukaryotic cells, the actin cytoskeleton of chondrocyte is connected with the extracellular matrix (ECM) 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. 14 Zyxin contains a number of dynamic structural features, including a proline-rich domain that interacts with Ena/vasodilator-stimulated phosphoprotein (VASP) family members, a nuclear export signal that allows translocation between the nucleus and cytoplasm, and three C-terminal LIM domains involved in protein-protein interactions. 15 Recent studies show that zyxin could reinforce the actin cytoskeleton of human airway smooth muscle cells 16 and could regulate endothelial von Willebrand factor secretion by reorganizing actin filaments around exocytic granules. 17 However, the modulating role of zyxin on actin in chondrocytes has not been elucidated.
Vinculin, an actin-binding protein considered to reinforce cellcell 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 ɑ-catenin, respectively. 18,19 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. 20 Moreover, studies have shown that vinculin and zyxin are colocalized at focal adhesions. 14,21 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 chondrocyte model. We performed immunofluorescence, qRT-PCR and Western blot to measure actin and vinculin expression as well as collagen production of chondrocyte. 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-β1 (TGF-β1), as TGF-β1 has been shown to mediate chondrocyte actin cytoskeleton through Ras homolog gene family, member A (RhoA) pathway. 22,23 2 | MATERIAL S AND ME THODS

| Chondrocytes isolation and culture
Primary chondrocytes were harvested according to a previous protocol. 24

| Quantitative real-time PCR
Total RNA was extracted from cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), and the RNA concentration was determined using a

| Immunofluorescence
The cells were washed with PBS and fixed with 4% formaldehyde for 10 minutes at room temperature (RT). Then, the cells were washed three times with cold PBS and treated with 0.2% v/v Triton X-100 in PBS for

| Alcian blue staining
The cells were washed with PBS and fixed with 4% formaldehyde for 10 minutes at RT. Then, the cells were washed three times with PBS and stained with Alcian blue (Cyagen, Santa Clara, CA, USA) for 30 minutes. The cells were washed again three times with PBS and imaged with microscopy. Staining was quantified by solubilizing the sample in 6 mol/L guanidine hydrochloride for 8 hours at RT according to a published method. 26 The OD value at 620 nm was measured by spectrophotometry.

| Western blot
The cells were lysed in RIPA with PMSF (Beyotime, Shanghai, China) on ice for 5 minutes and removed with a scraper. Then the lysate was

| Actin quantification
Quantification of F-actin to globular actin (G-actin) ratio was also performed by Western blot according to a previously published method. 27,28 The cells were lysed in 150 μL of extraction buffer

| Statistical analysis
Data were represented as mean ± standard deviation (SD) and obtained from three independent experiments. One-way analysis of variance (ANOVA) was performed for statistical analysis between control group (NC) and experiment groups (mostly #1009) using spss statistics 20 (IBM, Chicago, IL, USA) software. Mann-Whitney U test was performed for statistical analysis when the data didn't accord with the homogeneity of variance. P values less than 0.05 were considered as statistically significant.
Chondrocytes transfected with #1009 siRNA showed a similar morphology but a weaker zyxin staining compared to NC group ( Figure 1B). In line with the gene expression data, zyxin protein expression in chondrocytes transfected with #1009 siRNA was also much lower (63.98 ± 8.38%) than NC group ( Figure 1C).

| Zyxin knockdown reduces vinculin expression of chondrocyte
After observing the role zyxin plays in maintenance of actin cytoskeleton, we investigated the effect of zyxin knockdown on the expression of focal adhesion protein vinculin. Immunofluorescence staining showed that vinculin deposition in #1009 group was more concentrated at cell edge compared to that in NC group ( Figure 3A), and vinculin intensity in #1009 group was slightly lower ( Figure 3B).
PCR results showed that vinculin gene expression was decreased to 43.23 ± 1.48% in response to zyxin knockdown ( Figure 3C). Similarly, Western blot analysis demonstrated that vinculin deposition in chondrocytes reduced to 65.24 ± 5.94% after zyxin knockdown ( Figure 3D).

| Zyxin knockdown affects collagen production of chondrocyte
We next monitored the expression of chondrogenic marker COL II, de-differentiation marker COL I and hypertrophic marker COL X. Immunofluorescence staining showed that COL II and COL X deposition in chondrocytes of #1009 group was weaker than that of NC group ( Figure 4A,B). In line with the staining results, COL II and COL X gene expression decreased to 34.04 ± 11.61% and 84.32 ± 4.95%, respectively in response to zyxin knockdown ( Figure 4C). In line with the gene expression data, Western blot analysis also showed that COL II and COL X deposition in chondrocytes reduced to 44.92 ± 6.06% and 86.07 ± 4.47%, respectively after zyxin knockdown ( Figure 4D). There was no difference in COL I expression between the two groups at either gene or protein level.  Figure 5D), and Western blot analysis showed that vinculin deposition at protein level also increased by 35% after TGF-β1 treatment ( Figure 5E).

| TGF-β1 increases F-actin and vinculin expression but reduces zyxin expression, which in turn affects chondrocyte differentiation status
Since TGF-β1 treatment down-regulated the expression of zyxin, we therefore speculated that TGF-β1-treated chondrocytes had the same phenotypic change as zyxin knock-down chondrocytes. Alcian blue staining showed that the aggrecan (ACAN) deposition in chondrocytes decreased in response to TGF-β1 treatment ( Figure 6A,B). COL II gene expression showed approximately a 75% decrease, while COL I gene expression showed approximately an 80% increase in response to TGF-β1 treatment ( Figure 6C). Western blot analysis further confirmed that COL II deposition decreased, and COL I deposition increased in chondrocytes after TGF-β1 treatment ( Figure 6D).  38 This finding is contrary to our results here, which might be due to the different local activities of TGF-β1 within these two cell types.

| D ISCUSS I ON
Since TGF-β1 treatment down-regulated the expression of zyxin, we hypothesized that chondrocytes had the same phenotypic changes in response to TGF-β1 stimulation as to zyxin knockdown. Indeed, the GAG deposition and expression of COL II both decreased after TGF-β1 treatment ( Figure 6). The reason  (Figure 7). 39,40 It is noteworthy that the expression of COL I increased in response to TGF-β1 treatment. This might be owing to the nuclear localization of myocardin-related transcription factor (MRTF)-a G-actin-binding protein that has the ability to regulate COL I gene expression. 27,28 Here, the TGF-β1 treatment reinforced actin polymerization, and the polymerization F I G U R E 6 Transforming growth factor-β1 (tgf-β1) treatment affects ecm synthesis of chondrocyte. (a) alcian blue staining of chondrocytes treated with tgf-β1. (b) alcian blue staining quantification by od value at 620 nm; *P < 0.05. (C) mRNA expression of COL I, COL II and COL X of chondrocytes TREATED WITH TGF-Β1. DATA WERE normalized to control; *P < 0.05, **P < 0.01. (D) Western blot analysis of COL I, COL II and COL X of chondrocytes treated with TGF-β1; *P < 0.05, **P < 0.01 FIGURE 7 Schematic of zyxinbased regulation mechanism of actin cytoskeleton and differentiation status of chondrocyte. Zyxin expression and actin polymerization state have a bidirectional relationship and mutually adjust differentiation of chondrocyte. MRTF: myocardin-related transcription factor; PKA signalling: protein kinase A signalling; SRF: serum response factor state has been shown to regulate the actin-binding proteins into and out of the nucleus. 41 In particular, nuclear localization of MRTF allows it to associate and act as a transcriptional co-activator to serum response factor on promoter regions, 42 , therefore, enhancing COL I gene expression (Figure 7).
Overall, there are two limitations of this study. First, a zyxinknockdown model was used, which is inferior to zyxin-knockout model. Second, the chondrocytes were cultured in a 2D environment, which could induce de-differentiation and interfere with phenotype analysis. Nevertheless, our study demonstrated that the zyxin-involved actin regulation has an impact on vinculin focal adhesion and collagen matrix of chondrocytes. When zyxin level is lower than normal, the expression of COL II was mainly affected; when F-actin level is higher than normal, the expression of COL I was largely increased, and the expression of vinculin parallels with F-actin. In conclusion, our results revealed the important role of zyxin-involved actin regulation in vinculin focal adhesion and collagen matrix of chondrocytes.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.