Wnt3a knockdown promotes collagen type II expression in rat chondrocytes

Abstract

Osteoarthritis (OA) is a chronic condition caused by cartilage degradation, and there are currently no effective methods for preventing the progression of this disease; gene therapy is a relatively novel method for treating arthritis. Decreased collagen type II (Col2) expression within the cartilage matrix is an important factor for the development of OA, and Wnt3a serves a significant role in cartilage homeostasis. The present study assessed whether Wnt3a knockdown promoted Col2 expression in chondrocytes. Lentivirus-introduced small interfering RNA was used to knock down the expression of Wnt3a in primary rat chondrocytes, and then IL-1β treatment was used to establish an OA chondrocyte model. The expression of target genes (Wnt3a, Col2, MMP-13 and β-catenin) was analyzed using reverse transcription-quantitative PCR, western blotting and immunocytochemistry. There was significantly less MMP-13 and β-catenin expression in the Wnt3a knockdown cells compared with the other controls. Col2 expression was significantly higher in the Wnt3a-knockdown cells compared with the control cells, indicating that knockdown of Wnt3a may promote Col2 expression. Consequently, Wnt3a was indicated to be an important factor in cartilage homeostasis, and Wnt3a knockdown may serve as a novel method for OA therapy.

Keywords: Wnt3a; chondrocyte; collagen type II; knockdown.

Figure 1

Putative effects of LV-Wnt3a-RNAi. IL-1β upregulates Wnt3a expression in chondrocytes, which activates the β-catenin-dependent canonical pathway to induce expression of MMPs and reduce Col2 expression. A LV-Wnt3a-RNAi was designed to knock down Wnt3a expression, which is hypothesized to result in an increase in Col2 expression. COL2, collagen type II; IL-1β, interleukin-1β; LV-Wnt3a-RNAi, lentivirus vector-mediated Wnt3a-specific RNA interference; MMPs, matrix metalloproteinases.

Figure 2

Characteristics of the cultured chondrocytes. (A) Cultured chondrocyte cells were stained with toluidine blue. Cultured cells were also incubated with anti-collagen II antibody, and signal was detected using green fluorescence (magnification, x200). (B) Determination of transfection. OA-like chondrocytes were observed by optical microscopy after LV-Wnt3a-RNAi transfection. Transfected cells were confirmed using fluorescence microscopy, which indicated the optimum multiplicity of infection to be 60 (magnification, x100). (C) Live-Dead staining was used to assess the viability of transfected chondrocytes. Transfected chondrocytes treated with calcein AM (green) and ethidium homodimer-1 (red) demonstrated viable chondrocytes with only a minority of dead cells. The merged image indicates that viable chondrocytes accounted for 96% of the total cells (magnification, x100). (D) Chondrocytes were cultured in DMEM with 10 ng/ml IL-1β for 1, 2 or 6 h. MMP-13 gene expression and protein abundance at 6 h were significantly higher compared with that in other groups. (E) Quantitative PCR was used to assess transfection efficiency. The experiment was divided into three groups: control group, empty vector group and LV-wnt3a group. Relative Wnt3a mRNA expression was detected. The experiments were performed on five samples per group. ^****P<0.0001. IL-1β, interleukin-1β; LV-Wnt3a-RNAi, lentivirus vector-mediated Wnt3a-specific RNA interference; MMP, matrix metalloproteinase; OA, osteoarthritis.

Figure 3

Wnt3a knockdown increases Col2 expression and decreases MMP-13 and β-catenin expression. (A) Quantitative PCR analysis: Quantitative PCR was performed to detect expression of Wnt3a, Col2, MMP-13 and β-catenin following Wnt3a knockdown. Wnt3a, MMP-13 and β-catenin expression in LV-wnt3a group was significantly lower than that in control group and empty vector group, while expression of Col2 in LV-wnt3a group was significantly higher than that in other groups. (B) Western blotting analysis. Western blot was performed to detect expression of Wnt3a, Col2, MMP-13 and β-catenin following Wnt3a knockdown. Protein expression data were normalized to the β-actin loading control. Wnt3a, MMP-13 and β-catenin expression in LV-wnt3a group was significantly lower than that in control group and empty vector group, while expression of Col2 in LV-wnt3a group was significantly higher than that in other groups. (C) Immunocytochemistry analysis: Representative images of immunocytochemical staining in all groups(magnification, x200). IOD analysis of the immunocytochemical images indicated that Col2 expression was significantly higher in LV-wnt3a group compared with control group and empty vector group. The experiments were performed on five samples per group. ^****P<0.0001. COL2, collagen type II; IL-1β, interleukin-1β; IOD, integrated optical density; MMP, matrix metalloproteinase.