Extracellular localization of galectin-3 has a deleterious role in joint tissues

Abstract

In this study we examine the extracellular role of galectin-3 (gal-3) in joint tissues. Following intra-articular injection of gal-3 or vehicle in knee joints of mice, histological evaluation of articular cartilage and subchondral bone was performed. Further studies were then performed using human osteoarthritic (OA) chondrocytes and subchondral bone osteoblasts, in which the effect of gal-3 (0 to 10 microg/ml) was analyzed. Osteoblasts were incubated in the presence of vitamin D3 (50 nM), which is an inducer of osteocalcin, encoded by an osteoblast terminal differentiation gene. Genes of interest mainly expressed in either chondrocytes or osteoblasts were analyzed with real-time RT-PCR and enzyme immunoassays. Signalling pathways regulating osteocalcin were analyzed in the presence of gal-3. Intra-articular injection of gal-3 induced knee swelling and lesions in both cartilage and subchondral bone. On human OA chondrocytes, gal-3 at 1 microg/ml stimulated ADAMTS-5 expression in chondrocytes and, at higher concentrations (5 and 10 microg/ml), matrix metalloproteinase-3 expression. Experiments performed with osteoblasts showed a weak but bipolar effect on alkaline phosphatase expression: stimulation at 1 microg/ml or inhibition at 10 microg/ml. In the absence of vitamin D3, type I collagen alpha 1 chain expression was inhibited by 10 microg/ml of gal-3. The vitamin D3 induced osteocalcin was strongly inhibited in a dose-dependent manner in the presence of gal-3, at both the mRNA and protein levels. This inhibition was mainly mediated by phosphatidylinositol-3-kinase. These findings indicate that high levels of extracellular gal-3, which could be encountered locally during the inflammatory process, have deleterious effects in both cartilage and subchondral bone tissues.

Figure 1

Mouse knee swelling measurement. Galectin-3 (gal-3) was injected in both knees. Animals were examined daily and knee diameter measured using a digital calliper as described in Materials and methods. The swelling corresponded to the difference between joint diameter measured every day and joint diameter prior to the injection (D0). D0 was given the value of 0. Control (Ctl): injection of PBS. Each group contained four animals. *p versus same conditions as D0; ^#p versus control of the corresponding day.

Figure 2

Histological score for mice four days after intra-articular galectin-3 (gal-3) injection. (a) Total score, (b) cartilage score and (c) bone histomorphometric score. Data are expressed as median and (range) and are presented in box plot, where the boxes represent the 1st and 3rd quartiles, the line within the box represents the median, and the lines outside the box represent the spread of the values. Control (Ctl): mice injected with PBS. P versus control group; n = four animals per group.

Figure 3

Representative histological sections of specimens from mice stained with (a-d) safranin O or (e-h) toluidine blue (magnification ×100). Control group (a,e). Mice were injected with 0.1 μg (b,f), 1 μg (c,g), or 10 μg (d,h) galectin-3.

Figure 4

Effects of exogenous galectin-3 (gal-3) on human osteoarthritis chondrocytes. Chondrocytes were treated with increasing concentrations of recombinant human gal-3. Both ADAMTS-5 and matrix metalloproteinase (MMP)-3 expression were analyzed by real time RT-PCR. P versus control (Ctl); n = 5.

Figure 5

Effects of exogenous galectin-3 (gal-3) on markers of subchondral bone osteoblasts. Osteoblasts were treated with 1 or 10 μg/ml of recombinant human gal-3 in the presence or absence of vitamin D₃. Both (a) alkaline phosphatase and (b) osteocalcin expression were analyzed by real time RT-PCR. Insert illustrates the protein level of osteocalcin. N = 4.

Figure 6

Signalling pathways of inhibition by galectin-3 (gal-3) of vitamin D₃-stimulated osteocalcin production. Osteoblasts were treated with 5 μg/ml of recombinant human gal-3 in the presence of vitamin D₃ and osteocalcin was determined. Inhibitor concentrations were: KT5720, 2 μM; KT5823, 2 μM; Genistein (Genist.), 1 μM; Taxifollin, 1 μM; wortmannin (Wortma.), 250 nM; PD98059, 10 μM; and SB202190, 2 μM. *P versus the autologous control; n = 5.

Figure 7

Effects of exogenous galectin-3 (gal-3) on type I collagen expression in osteoblasts. Osteoblasts were treated with 1 or 10 μg/ml of recombinant human gal-3 in the presence or not of vitamin D₃. Collagen type I α1 chain expression was analyzed by real time RT-PCR. *P versus control (Ctl; without vitamin D3 or gal-3); **p versus 1 μg gal-3 alone; n = 4.