Abstract
Activation of the Wnt/beta-catenin and retinoid signaling pathways is known to tilt cartilage matrix homeostasis toward catabolism. Here, we investigated possible interactions between these pathways. We found that all-trans-retinoic acid (RA) treatment of mouse epiphyseal chondrocytes in culture did increase Wnt/beta-catenin signaling in the absence or presence of exogenous Wnt3a, as revealed by lymphoid enhancer factor/T-cell factor/beta-catenin reporter activity and beta-catenin nuclear accumulation. This stimulation was accompanied by increased gene expression of Wnt proteins and receptors and was inhibited by co-treatment with Dickkopf-related protein-1, an extracellular inhibitor of Wnt/beta-catenin signaling, suggesting that RA modulates Wnt signaling at Wnt cell surface receptor level. RA also enhanced matrix loss triggered by Wnt/beta-catenin signaling, whereas treatment with a retinoid antagonist reduced it. Interestingly, overexpression of retinoic acid receptor gamma (RARgamma) strongly inhibited Wnt/beta-catenin signaling in retinoid-free cultures, whereas small interfering RNA-mediated silencing of endogenous RARgamma expression strongly increased it. Small interfering RNA-mediated silencing of RARalpha or RARbeta had minimal effects. Co-immunoprecipitation and two-hybrid assays indicated that RARgamma interacts with beta-catenin and induces dissociation of beta-catenin from lymphoid enhancer factor in retinoid-free cultures. The N-terminal domain (AF-1) of RARgamma but not the C-terminal domain (AF-2) was required for association with beta-catenin, whereas both AF-1 and AF-2 were necessary for inhibition of beta-catenin transcriptional activity. Taken together, our data indicate that the Wnt and retinoid signaling pathways do interact in chondrocytes, and their cross-talks and cross-regulation play important roles in the regulation of cartilage matrix homeostasis.
Highlights
In recent studies, we found that Wnt/-catenin signaling can strongly affect matrix anabolic and catabolic metabolism in chondrocytes [6]
We show that the Wnt/-catenin and retinoid signaling pathways interact and affect matrix homeostasis and phenotypic expression in chondrocytes
We find that retinoic acid (RA) stimulates Wnt/-catenin signaling, increases gene expression of Wnt proteins and receptors, and enhances the inhibitory effects of Wnt/-catenin signaling on matrix accumulation (Fig. 7A)
Summary
Chondrocyte and Limb Bud Cell Cultures—Mouse primary epiphyseal chondrocytes were isolated from neonatal C57BL/6 mice as previously described [6]. Isolated limb mesenchymal cells were inoculated at 2.5 ϫ 105 cells/20 l and cultured in Dulbecco’s modified Eagle’s medium/Ham’s nutrient mixture F-12 containing 2% fetal bovine serum and 100 ng/ml recombinant BMP2 for 7 days to induce chondrogenic differentiation. Isolated chondrocytes or limb bud cells were plated at an initial density of 4 ϫ 104/well or 2 ϫ 104/well, respectively, into 96-well plates that had been coated with a Wnt/-catenin reporter plasmid (Super 8x TOPFlash, Addgene Inc., Cambridge, MA) in the presence of Lipofectamine 2000. SiRNA for each RAR (1 pmol) and control siRNA (1 pmol) (to exclude potential off-target effects caused by siRNA) were co-transfected with Super 8x TOPFlash reporter plasmid and pRL-TK-luc into freshly isolated mouse chondrocytes seeded at an initial density of 4 ϫ 104/well in a 96-well plate by reverse transfection using Lipofectamine 2000. Statistical Analysis—One-way analysis of variance followed by Boneferroni/Dunn post hoc multiple comparison tests (Prism 5, GraphPad Software Inc., La Jolla, CA) was used to determine statistical significance between groups. p values less than 0.01 were considered significant (*, p Ͻ 0.01 as indicated by brackets)
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