The expression of Vangl2, Wnt5a, and MMPs increased after IL-1β stimulation:
Exogenous IL-1β (10ng/mL) enhanced the mRNA expression of Vangl2, Wnt5a, MMPs, and related proinflammatory factors in a time-dependent manner. The time-course study showed that the expression of Vangl2, MMP3, and MMP13 increased at early stages. Vangl2 reached its peak at 4 h and remained at a high level for the rest of period (Fig.1 a). The expression of Wnt5a gradually elevated after IL-1β treatment and then decreased after 6 h (Fig.1 b). Although differences of MMP9 expression between adjacent time points were not statistically significant, all mRNA levels of MMP3, MMP9, MMP13, IL-6, IL-8 and TNF-α increased in IL-1β-treated chondrocyte (Fig.1 c). The expression of MMP3 changed most significantly after IL-1β stimulation and almost reached at 35 times of the initial level at 12 h. MMP13 expression also increased around 12 times of the initial value at 4 h.
Induction of Vangl2 protein by IL-1β was further verified in ATDC5 by Western blotting and IF analysis (Fig.2 a, b). A significant increase of Vangl2 immunostaining was observed after stimulation with IL-1β for 4 h, showing the consistent effects of both the mRNA and protein levels. Moreover, the increased level of Vangl2 in OA chondrocytes is also confirmed by IF staining (Fig.2 b).
Fig1. The expression of Vangl2, Wnt5a, MMPs and proinflammatory cytokines were up-regulated in OA chondrocyte: (a, b) Vangl2 and Wnt5a expression in ATDC5 were detected by RT-qPCR after stimulation with 10 ng/mL IL-1β for 0, 2, 4, 6, 8 and 12 h. (c) Expression trend of MMP3, MMP9, MMP13, IL-6, IL-8, and TNF-α after IL-1β stimulation were determined by RT-qPCR. *P <0.05.
Fig2. Vangl2 expression was upregulated in OA chondrocytes. (a) The protein levels of Vangl2 in normal and OA chondrocytes were detected by Western blotting and then quantified. **P <0.01 compared with the control group. (b) Immunofluorescence for Vangl2 (green) and DAPI (blue) in control and IL-1β treated ATDC5, Scale bar, 50μm.
Knockdown of Vangl2 alleviated IL-1β-induced MMP 3, MMP9, MMP13, and IL-6 expression at both the gene and protein levels
To further evaluate the effects of Vangl2 on the inflammatory response of OA chondrocyte, we analyzed whether suppression of endogenous Vangl2 modulates the expression of MMPs and IL-6. Western blotting and RT-qPCR showed that the knockdown efficiency of siRNA targeting Vangl2 was around fifty percent (Fig.3 a, b). The chondrocytes were divided into three groups: Group I (nc), chondrocytes were only transfected by siRNA of nc; Group II (IL-1β+nc), cells were further stimulated with IL-1β; Group III (IL-1β+si-Vangl2), OA chondrocytes were transfected with siRNA targeting Vangl2. The RT-qPCR and Western blotting analysis showed that gene expressions of MMP3, MMP9, MMP13, and IL-6 were significantly increased in IL-1β stimulation compared with nc group (Fig. 3c, d). Then, we found that Vangl2 silencing notably alleviated the IL-1β-induced gene expression of MMPs and IL-6 (Fig. 3c). Western blotting also revealed that knockdown of Vangl2 reduced the protein expression of MMP3, MMP9, MMP13, and IL-6 significantly (Fig.3d).
Fig.3 Knockdown of Vangl2 attenuated IL-1β-induced inflammatory activation in ATDC5. (a, b) The knockdown efficiency of siRNA targeting Vangl2 was detected by Western blotting (a) and RT-qPCR (b). (c) Effects of Vangl2 inhibition on MMP3, MMP9, MMP13, and IL-6 mRNA levels were measured by RT-qPCR. (d) Western blotting for MMP3, MMP9, MMP13, and IL-6 in three groups. Densitometric quantification, values were normalized for GAPDH. **P <0.01, *** P <0.001.
Vangl2 silencing reduces the loss of cartilage ECM in vitro
To explore the relationship between Vangl2 and chondrocyte anabolism in OA, we analyzed the expression of the major structural proteins in cartilage ECM, Col-2 and aggrecan (Fig.4). As shown in the fig.4, the protein and gene levels of Col-2 declined to approximately 50% after stimulated with IL-1β (Fig.4 a, b). It is of note that Vangl2 inhibition almost eliminates the effects of IL-1β on Col-2 and aggrecan. Both Western blotting and RT-qPCR showed that the expression levels of Col-2 in group III returned to normal (Fig.4 a, b). The results of ICC staining further confirmed our findings (Fig.4 c, d). Similarly, Vangl2 silencing also notably suppressed the degradation of aggrecan in OA chondrocytes (Fig.4 e).
Fig.4 Silencing of Vangl2 prevents Col-2 and aggrecan degradation in OA. (a) Western blotting of Col-2 in three groups. The images were quantitatively analyzed and normalized to GAPDH. (b) The change of Col-2 mRNA was quantitated by RT-qPCR. (c, d) Representative ICC images of Col-2 (c), and the IOD analysis (d). Scale bar, 50μm. (e) The aggrecan expression was measured by Western blotting and quantified for three groups. ns: Not Statistically Significant, *P <0.05, **P <0.01. IOD, integrated option density.
Inhibition of Vangl2 attenuates the proinflammatory functions of Wnt5a and the underlying mechanism.
We constructed Wnt5a-overexpressing chondrocytes by plasmid transfection and investigated the role of Wnt5a in the IL-1β induced inflammatory response of chondrocytes (Fig.5 a). Wnt5a promoted the reduction of Sox9, Col-2 and the upregulation of MMP3, MMP9, MMP13 in OA (Fig.5 b, c). As expected, Vangl2 silencing almost eliminated the proinflammatory effects of Wnt5a in OA chondrocyte (Fig.5 b, c). Specifically, the expression of Sox9 and Col-2 was up-regulated and the expression of MMP3, MMP9, and MMP13 was reduced in Vangl2-silencing chondrocyte.
To further investigate the mechanisms by which Vangl2 silencing protects chondrocytes, we examined the phosphorylation levels of P38 MAPK, extracellular signal-regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK), and P65 by Western blotting (Fig.6). The results showed that Wnt5a further upregulated the expression of phosphorylated-P38, JNK, ERK, and P65, which had been induced by IL-1β. (Fig.6 a, c). Knockdown of Vangl2 significantly inhibited the expression of P-P38, P-JNK, and P-ERK induced by Wnt5a (Fig. 6 a, b). Simultaneously, Vangl2 silencing also notably decreased the phosphorylation level of P65 enhanced by Wnt5a (Fig. 6 c).
Fig.5 Vangl2 silencing can eliminate the effect of Wnt5a on inflammatory activation. (a) RT-qPCR analysis shows Wnt5a-overexpression efficiency of Wnt5a plasmid in ATDC5. (b) Effects of Vangl2 and Wnt5a on the expression of Sox9 and Col-2 were measured by Western blotting. (c) Protein expression of MMP3, MMP9, MMP13 were also measured by Western blotting. GAPDH was used as a loading control. ns: Not Statistically Significant, *P <0.05, **P <0.01; nc/NC, negative control; +, overexpression; -, silencing.
Fig.6 Chondrocyte signaling in response to Wnt5a and Vangl2. (a) The influence of Wnt5a and Vangl2 on MAPK signaling was detected by Western blotting. (b) MAPK signaling activation is quantified by the ratio of phosphorylated protein to total protein. (c) The effect of Wnt5a and Vangl2 on NF-κB signaling was also detected by Western blotting. *P <0.05, **P <0.01.