Gla-rich protein is involved in the cross-talk between calcification and inflammation in osteoarthritis

Abstract

Osteoarthritis (OA) is a whole-joint disease characterized by articular cartilage loss, tissue inflammation, abnormal bone formation and extracellular matrix (ECM) mineralization. Disease-modifying treatments are not yet available and a better understanding of osteoarthritis pathophysiology should lead to the discovery of more effective treatments. Gla-rich protein (GRP) has been proposed to act as a mineralization inhibitor and was recently shown to be associated with OA in vivo. Here, we further investigated the association of GRP with OA mineralization-inflammation processes. Using a synoviocyte and chondrocyte OA cell system, we showed that GRP expression was up-regulated following cell differentiation throughout ECM calcification, and that inflammatory stimulation with IL-1β results in an increased expression of COX2 and MMP13 and up-regulation of GRP. Importantly, while treatment of articular cells with γ-carboxylated GRP inhibited ECM calcification, treatment with either GRP or GRP-coated basic calcium phosphate (BCP) crystals resulted in the down-regulation of inflammatory cytokines and mediators of inflammation, independently of its γ-carboxylation status. Our results strengthen the calcification inhibitory function of GRP and strongly suggest GRP as a novel anti-inflammatory agent, with potential beneficial effects on the main processes responsible for osteoarthritis progression. In conclusion, GRP is a strong candidate target to develop new therapeutic approaches.

Keywords: ECM mineralization; Gamma-carboxylated GRP; Gla-rich protein; Inflammation; Osteoarthritis.

Fig. 1

Characterization of control and osteoarthritic-derived (OA) chondrocyte and synoviocyte cell cultures through gene expression patterns determined by qPCR. a Expression of OA (OC, COMP and MMP13) and differentiation (Cola2a1, Cola10a1, CD68 and vimentin) gene markers in control (NHAC and HFLS) and OA-derived (OA-HAC and OA-HFLS) chondrocytes and synoviocytes. b Expression of VKDP-related genes (GRP, MGP, GGCX and VKOR) in control and OA-derived chondrocytes and synoviocytes. Values are relative to a reference sample (control cell culture) set to 1. All experiments were repeated at least three times. Multiple t tests were performed. Statistical significance was defined as P ≤ 0.05 (*), P ≤ 0.005 (**) and P ≤ 0.0005 (***)

Fig. 2

Accumulation patterns of GRP protein forms in both control (NHAC and HFLS) and OA (OA-HAC and OA-HFLS)-derived chondrocytes and synoviocytes. Immunofluorescence imaging was obtained for total GRP (CTerm-GRP), γ-carboxylated (cGRP) and undercarboxylated GRP (ucGRP) protein forms using specific antibodies. Cell nuclei were stained with DAPI; scale bar represents 100 µm. All experiments were repeated at least twice

Fig. 3

In vitro ECM mineralization of control and OA-derived chondrocytes and synoviocytes (NHAC and OA-HAC, and HFLS and OA-HFLS, respectively). a Representative von Kossa staining at week 3 (T3) in control or induced mineralizing conditions with 5.4 mM CaCl₂ (Ca). Scale bar represents 100 µm. b Mineralization rate was determined every week (T0–T3) through calcium quantification normalized to protein levels. Data are representative of three independent experiments. Two-way Anova and multiple comparisons were achieved with the Tukey’s test. Statistical significance was defined as P ≤ 0.05 (*) and P ≤ 0.005 (**)

Fig. 4

Association of GRP with calcification and cell differentiation in osteoarthritis. a Gene expression patterns of GRP, mineralization (MGP, OC and Osx) and differentiation (Col2a1 and Col10a1) markers during induced mineralization of chondrocytes (NHAC and OA-HAC). b Gene expression patterns of GRP, mineralization (MGP, OC and OPN) and differentiation (vimentin and CD68) markers during induced mineralization of synoviocytes (HFLS and OA-HFLS). Gene expression was determined every week during 3 weeks (T0–T3). Gene expression values are relative to the reference sample (control cell culture) and set to 1. Data are representative of three independent experiments. Two-way Anova and multiple comparisons were achieved with the Tukey’s test. Statistical significance was defined as P ≤ 0.05 (*) and P ≤ 0.005 (**)

Fig. 5

Effect of GRP in ECM mineral deposition in chondrocytes and synoviocytes. Mineralization rate was determined by calcium measurement (normalized to protein levels) after 3 weeks of NHAC and HFLS cells treatment with 5.4 mM CaCl₂ (Ca), or supplemented with 500 ng/mL of ucGRP (Ca+ucGRP) or cGRP (Ca+cGRP). Control corresponds to cells cultured in non-supplemented media. Data are representative of three independent experiments. Ordinary one-way ANOVA was performed. Statistical significance was defined as P ≤ 0.05 (*)

Fig. 6

In vitro and in vivo studies of GRP association with inflammatory events in osteoarthritis. a Gene expression of COX2 and MMP13 over 3 weeks of mineralizing treatment (5.4 mM calcium) in NHAC and HFLS (each week a set point, T0–T3). Values of gene expression are relative to the reference sample (T0) and set to 1. Data are representative of three independent experiments. Two-way Anova and multiple comparisons were achieved with the Tukey’s test. Statistical significance was defined as P ≤ 0.05 (*) and P ≤ 0.005 (**). b Gene expression levels of COX2, MMP13 and GRP in NHAC and HFLS cells, after 72-h inflammatory stimulation with 5 ng/mL IL-1β. Values are relative to the reference sample (0 h). Data are representative of three independent experiments. Two-way Anova and multiple comparisons were achieved with the Tukey’s test. Statistical significance was defined as P ≤ 0.05 (*). c–f Immunodetection of CD45, showing leucocyte infiltration sites (c and d) and total GRP (e and f, CTerm-GRP antibody) in consecutive sections of osteoarthritic synovial membrane samples. Counterstaining with HE; sale bar represents 100 µm

Fig. 7

Effect of GRP in the inflammatory process promoted by the addition of BCP crystals to articular cells. a Gene expression of COX2 and MMP13 in NHAC and HFLS cells supplemented for 72 h with BCP crystals (BCP) or BCPs coated with ucGRP (BCP + ucGRP) or cGRP (BCP + cGRP). Values are relative to the reference sample (untreated cells 0 h). Data are representative of three independent experiments. Two-way Anova and multiple comparisons were achieved with the Tukey’s test. Statistical significance was defined as P ≤ 0.05 (*), P ≤ 0.005 (**) and P ≤ 0.0005 (***). b PGE2 accumulation in NHAC and HFLS conditioned media of cells treated for 72 h as described in a. Control corresponds to culture media of non-treated cells. Ordinary one-way ANOVA was performed. Statistical significance was defined as P ≤ 0.05 (*), P ≤ 0.005 (**) and P ≤ 0.0005 (***)

Fig. 8

Effect of GRP in chondrocytes and synoviocytes under in vitro inflammatory conditions mimicking osteoarthritis. a Gene expression of COX2 and MMP13 in NHAC and HFLS cells pre-treated with 500 ng/mL of ucGRP or cGRP or 2 µM dexamethasone (DXM) followed by IL-1β stimulation (5 ng/mL) during 72 h. Cells untreated with GRP or DMX were also analyzed (IL-1β). Control corresponds to cells grown in advanced DMEM only. Values are relative to the reference sample (0 h). Data are representative of three independent experiments. Two-way Anova and multiple comparisons were achieved with the Tukey’s test. Statistical significance was defined as P ≤ 0.05 (*), P ≤ 0.005 (**) and P ≤ 0.0005 (***). b PGE2 accumulation in cell media of NHAC and HFLS treated for 24 h as described in (a). Control corresponds to culture media of non-treated cells. Ordinary one-way ANOVA was performed. Statistical significance was defined as P ≤ 0.05 (*), P ≤ 0.005 (**) and P ≤ 0.0005 (***)