WNT16 Regulation of the Articular Chondrocyte Phenotype in Mice

Abstract

The anabolic effects of WNT16 on osteoblasts are well established, however, little is known regarding the role of WNT16 in chondrocytes. In this study, we evaluated Wnt16 expression and its biological effects on mouse articular chondrocytes (ACs), since these cells are key to the development of osteoarthritis. While ACs derived from the long bone epiphysis of 7-day old C57BL/6J mice express multiple Wnts, Wnt5b and Wnt16 represent the two most highly expressed Wnts (expressed at several-fold higher levels than other Wnts). Treatment of serum-free AC cultures, with 100 ng/mL of recombinant human (rh) WNT16 for 24 h (hrs), increased proliferation (20%, p < 0.05) and expression levels of makers (Sox9 and Col2) of immature chondrocytes at both 24 h and 72 h, while Acan increased at 72 h. Expression of Mmp9, a marker of mature chondrocytes was decreased at 24 h. Additionally, WNT16 treatment regulated expression levels of Wnt ligands in a biphasic manner, inhibiting its expression at 24 h, while stimulating expression at 72 h. To determine whether WNT16 exerted anabolic effects on the AC phenotype, ex vivo cultures of tibial epiphyses were treated with rhWNT16 or vehicle for 9 days, and the articular cartilage phenotype was evaluated by safranin O cartilage staining and expression of articular cartilage marker genes. Both articular cartilage area and expression levels of AC markers were increased after rhWNT16 treatment. Our data suggest that Wnt16 expressed in ACs may play a role in regulating joint cartilage homeostasis via its direct effect, as well as through modulating the expression of other Wnt ligands.

Keywords: WNT16; cell culture; chondrocytes; chondrogenesis; mice; osteoarthritis.

Figure 1

Expression patterns of Wnts and chondrocyte marker genes in primary articular chondrocyte cultures in vitro. (A) Expression of Wnts in cultured articular chondrocytes isolated from 1-week-old C57BL/6J mice (fold-change data vs. Wnt5b is expressed in the log scale). (B) Quantitation of expression of genes 24 (n = 5) and 72 h post rhWNT16 treatment (n = 5) in chondrocytes derived from epiphyses of 1-week-old C57BL/6J mice. (C) Quantitation of expression of Wnt ligands 24 and 72 h post rhWNT16 treatment in chondrocytes isolated from the epiphyses of 1-week-old C57BL/6J mice (n = 3). Values are the mean ± SEM. ^A p < 0.05 and ^B p = 0.06 vs. vehicle, * p = 0.08 vs. vehicle. The x-axis represents the genes studied and y-axis reflects fold-change in expression.

Figure 2

Illustration of the femoral head safranin O staining and expression patterns of chondrocyte marker genes in ex vivo cultures of tibia epiphysis regions which contain articular cartilage. (A) Histology image of safranin O stained areas and (B) quantitation of safranin-O-stained areas in 6-month-old male femoral heads cultured ex vivo and treated with rhWNT16 vs. vehicle. (C) Quantitation of expression levels of genes from epiphyseal regions of the tibia with articular cartilage, isolated from 6- and 10-month-old male C57BL/6J mice and cultured ex vivo with 100 ng rhWNT16 vs. vehicle for 7 days in vitro. n = 5/group, values are the mean ± SEM, ^A p < 0.05 vs. vehicle and ^B p = 0.08 vs. vehicle.