Modulation of OPG, RANK and RANKL by human chondrocytes and their implication during osteoarthritis

Abstract

Objectives: Earlier studies suggest the involvement of osteoprotegerin (OPG), RANK and RANK ligand (RANKL) in OA subchondral bone metabolism; however, few studies have looked at their functional consequences on chondrocytes. We compared the expression/production of OPG, RANK and RANKL on human normal and OA chondrocytes, and evaluated, on OA chondrocytes, their modulation by some catabolic factors. Furthermore, the role of OPG and RANKL on the production of catabolic/anabolic factors was assessed.

Methods: Expression was determined using real-time PCR, production of RANK and RANKL by flow cytometry and that of OPG by ELISA. Modulation of these factors was determined upon treatment with IL-1beta, TNF-alpha and PGE(2). The functional consequences were examined following treatment with soluble RANKL or OPG-Fc (OPG without the heparin-binding domain).

Results: OPG, RANK and RANKL were expressed and produced by human chondrocytes. Membranous RANK was produced only by an OA chondrocyte subpopulation (29%) localized throughout the cartilage. The OPG/RANKL ratio was significantly (P = 0.05) reduced on the OA chondrocytes, whereas the RANK/RANKL ratio was significantly (P < 0.03) increased. OPG and membranous RANKL levels were significantly enhanced by IL-1beta, TNF-alpha and PGE(2), whereas membranous RANK was significantly increased only with IL-1beta. Administration of soluble RANKL had no effect on the OA chondrocytes. However, addition of OPG-Fc significantly stimulated MMP-13 (P = 0.05) and protease-activated receptor-2 (PAR-2) (P < 0.04) production.

Conclusions: Our findings showed that human chondrocytes express and produce OPG, RANK and RANKL. OA chondrocyte treatment with catabolic factors pointed towards an increased biological effect of OPG. Interestingly, OPG appears to be involved in OA progression by increasing two catabolic factors involved in cartilage pathophysiology.

Fig. 1

Gene expression of (A) OPG, (B) RANKL, (C) RANK, and the ratio of (D) OPG/RANKL and (E) RANK/RANKL in human normal (n=8) and OA chondrocytes (n=8). Total RNA was extracted and processed for real-time PCR, and the data are expressed as the mean (S.E.M.) of arbitrary unit as described in ‘Materials and methods’ section. Of note, the RANKL arbitrary unit is represented as ×10⁻³ and RANK as ×10⁻⁵. Statistical significance was assessed by the Student’s t-test vs normal.

Fig. 2

Representative histograms of the membranous (A) RANKL (n=5) and (B) RANK (n=11) flow cytometry on human OA chondrocytes. A mouse IgG served as control (dotted line) for background fluorescence, whereas specific fluorescence (solid line) was obtained with an anti-human RANKL (anti-hRANKL) or anti-human RANK (anti-hRANK) antibody as described in ‘Materials and methods’ section. Of note, the gate M1 represents the number of OA chondrocytes expressing membranous RANK, and in this sample 28% of the cells were RANK positive.

Fig. 3

OPG protein production in human OA chondrocytes (n=9) incubated in the absence (Control) or presence of the catabolic factors IL-1β, TNF-α and PGE₂ at the indicated concentrations. Statistical analysis was assessed by the paired Student’s t-test vs control.

Fig. 4

Membranous RANKL level on human OA chondrocytes (n=8) incubated in the absence (Control) or presence of IL-1β, TNF-α and PGE₂. Data are expressed as arbitrary unit calculated from fluorescence intensity over control, which was attributed a value of 1. Statistical analysis was assessed by paired Student’s t-test vs control.

Fig. 5

(A) Percentage of cells positively labelled for membranous RANK on human OA chondrocytes (n=6) incubated in the absence (Control) or presence of IL-1β, TNF-α and PGE₂. (B) Representative histogram of the membranous RANK flow cytometry in the absence (Control, dotted line) or in the presence (solid line) of IL-1β. Of note, the gate M1 represents the number of OA chondrocytes expressing membranous RANK. (C) Membranous RANK level on OA chondrocytes (n=6) incubated in the absence (Control) or presence of the above factors. Data are expressed as arbitrary unit calculated from fluorescence intensity over control, which was attributed a value of 1. Statistical analysis was assessed by paired Student’s t-test vs control.

Fig. 6

Gene expression level (n=7) of (A) MMP-13 and (B) PAR-2, and protein production of (C) MMP-13 (n=5) and (D) PAR-2 (n=8) on human OA chondrocytes upon treatment in the absence (Control) or presence of OPG-Fc at concentrations of 50 and 100 ng/ml. The gene expression level is expressed as arbitrary units over control, which was attributed a value of 1. The protein production is expressed as nanograms per milligram of protein for MMP-13 and as arbitrary units for PAR-2. Statistical analysis was assessed by paired Student’s t-test vs control.