Protective role of frizzled-related protein B on matrix metalloproteinase induction in mouse chondrocytes

Abstract

Introduction: Our objective was to investigate whether a lack of frizzled-related protein B (FrzB), an extracellular antagonist of the Wnt signaling pathways, could enhance cartilage degradation by facilitating the expression, release and activation of matrix metalloproteinases (MMPs) by chondrocytes in response to tissue-damaging stimuli.

Methods: Cartilage explants from FrzB-/- and wild-type mice were challenged by excessive dynamic compression (0.5 Hz and 1 MPa for 6 hours). Load-induced glycosaminoglycan (GAG) release and MMP enzymatic activity were assessed. Interleukin-1β (IL-1β) (10, 100 and 1000 pg/mL for 24 hours) was used to stimulate primary cultures of articular chondrocytes from FrzB-/- and wild-type mice. The expression and release of MMP-3 and -13 were determined by RT-PCR, western blot and ELISA. The accumulation of β-catenin was assessed by RT-PCR and western blot.

Results: Cartilage degradation, as revealed by a significant increase in GAG release (2.8-fold, P = 0.014) and MMP activity (4.5-fold, P = 0.014) by explants, was induced by an excessive load. Load-induced MMP activity appeared to be enhanced in FrzB-/- cartilage explants compared to wild-type (P = 0.17). IL-1β dose-dependently induced Mmp-13 and -3 gene expression and protein release by cultured chondrocytes. IL-1β-mediated increase in MMP-13 and -3 was slightly enhanced in FrzB-/- chondrocytes compared to wild-type (P = 0.05 and P = 0.10 at gene level, P = 0.17 and P = 0.10 at protein level, respectively). Analysis of Ctnn1b and Lef1 gene expression and β-catenin accumulation at protein level suggests that the enhanced catabolic response of FrzB-/- chondrocytes to IL-1β and load may be associated with an over-stimulation of the canonical Wnt/β-catenin pathway.

Conclusions: Our results suggest that FrzB may have a protective role on cartilage degradation and MMP induction in mouse chondrocytes by attenuating deleterious effects of the activation of the canonical Wnt/β-catenin pathway.

Figure 1

Load-induced glycosaminoglycan release and matrix metalloproteinase activity in mouse cartilage explants from FrzB^−/− and wild-type mice. Explants were subjected to dynamic compression for 6 hours (0.5 Hz, 1 MPa). FrzB^−/− cartilage explants (black bars, n = 4) or wild-type (WT) explants (gray bars, n = 4) were loaded. Results from the loaded cartilage explants were normalized to those from the corresponding nonloaded explants (Ctrl), so that the graphs represent the fold-induction in response to compression. (A) Amount of glycosaminoglycan (GAG) released from cartilage explants into culture supernatant. (B) Matrix metalloproteinase (MMP) enzymatic activity was measured in the culture supernatant of cartilage explants. Load-induced MMP activity tends to be enhanced in FrzB^−/− explants compared with WT (P = 0.17). Bars represent the mean ± standard error of the mean. *P ≤ 0.05 versus Ctrl. FrzB, frizzled-related protein B; KO, knockout.

Figure 2

IL-1β-mediated increase in MMP-13 in cultured articular chondrocytes is enhanced in absence of FrzB. Primary chondrocytes were treated for 24 hours with interleukin (IL)-1β (10, 100 or 1,000 pg/ml). Results from the treated chondrocytes were normalized to those from nontreated samples (Ctrl), so that the graphs represent the fold-induction in response to IL-1β. (A) Real-time polymerase chain reaction analysis of Mmp-13 gene expression. IL-1β-induced Mmp-13 gene expression tends to be enhanced in FrzB^−/− chondrocytes compared with wild-type (WT) chondrocytes, especially with 1,000 pg/ml (P = 0.05, n = 3). (B) Culture media were analyzed for MMP-13 by western blotting. Quantification of the MMP-13 blot is shown above the blot. IL-1β-induced MMP-13 protein release tends to be enhanced in FrzB^−/− chondrocytes compared with WT chondrocytes, especially with 1,000 pg/ml (P = 0.17, n = 4). Bars represent the mean ± standard error of the mean. *P ≤ 0.05 versus Ctrl, #P ≤ 0.10 between FrzB^−/− and WT. FrzB, frizzled-related protein B; KO, knockout; MMP, matrix metalloproteinase; ND, not detected.

Figure 3

IL-1β-mediated increase in MMP-3 in cultured articular chondrocytes is enhanced in absence of FrzB. Primary chondrocytes were treated for 24 hours with interleukin (IL)-1β (10, 100 or 1,000 pg/ml). (A) Real-time polymerase chain reaction analysis of Mmp-3 gene expression. Results from the treated chondrocytes were normalized to those from nontreated samples (Ctrl), so that the graphs represent the fold-induction in response to IL-1β. The IL-1β-induced Mmp-3 gene expression tends to be enhanced in FrzB^−/− chondrocytes compared with wild-type (WT) chondrocytes, especially with 1,000 pg/ml (P = 0.10, n = 3). (B) Culture media were analyzed for total MMP-3 by enzyme-linked immunosorbent assay. MMP-3 protein release in response to 100 pg/ml IL-1β tends to be enhanced in FrzB^−/− chondrocytes compared with WT chondrocytes (P = 0.10, n = 3). Bars represent the mean ± standard error of the mean. *P ≤ 0.05 versus Ctrl, #P ≤ 0.10 between FrzB^−/− and WT. FrzB, frizzled-related protein B; KO, knockout; MMP, matrix metalloproteinase; ND, not detected.

Figure 4

IL-1β-mediated and load-mediated regulation of Wnt/β-catenin signaling in chondrocytes from FrzB^−/− and wild-type mice. Cultured articular chondrocytes from FrzB^−/− mice or wild-type (WT) mice were treated for 24 hours with interleukin (IL)-1β (1 ng/ml). Results from the IL-1β-treated samples were normalized to the control ones (Ctrl), so that the graphs represent the fold-induction in response to IL-1β. (A)Ctnnb1 gene expression (coding β-catenin) and Lef1 gene expression (a Wnt/β-catenin target gene) were analyzed by real-time polymerase chain reaction (PCR; n = 4 and n = 3, respectively). Ctnnb1 gene expression was not modulated by IL-1β in WT chondrocytes and Lef1 gene expression was decreased (P = 0.05). In contrast, the treatment induced Ctnnb1 and Lef1 gene expression in FrzB^−/− chondrocytes (P = 0.014 and P = 0.05, respectively). IL-1β-mediated regulation of β-catenin expression was thus different between FrzB^−/− and WT (P = 0.057 for Ctnnb1, P = 0.05 for Lef1). (B) Intracellular extracts were analyzed for total β-catenin by western blotting. Quantification of β-catenin blot suggested that IL-1β-mediated β-catenin accumulation was different between FrzB^−/− and WT (P = 0.10, n = 4). (C) FrzB^−/− cartilage explants or WT explants were subjected to dynamic compression for 6 hours (0.5 Hz, 1 MPa). Ctnnb1 gene expression was analyzed by real-time PCR (n = 3). Results from the loaded cartilage explants were normalized to those from the corresponding nonloaded explants (Ctrl), so that the graphs represent the fold-induction in response to compression. Ctnnb1 gene expression was not affected by load in WT explants but was increased 2.7 fold in compressed FrzB^−/− samples (P = 0.05). The load-induced increase in Ctnnb1 mRNA tends to be enhanced in FrzB^−/− explants compared with WT explants (P = 0.20). Bars represent the mean ± standard error of the mean, *P ≤ 0.05 versus Ctrl, #P ≤ 0.10 between FrzB^−/− and WT. FrzB, frizzled-related protein B; KO, knockout.