Human Osteoarthritic Subchondral Bone Osteoblasts Research Articles

The ephrin-B2/EphB4 system is required in musculoskeletal development and protects the articulation during osteoarthritis

Ephrin ligands and their Eph receptors have been implicated in the control of extracellular matrix of some tissues. Although ephrin-B2 and its specific receptor EphB4 were found to be involved in postembryonic control of bone homeostasis, their roles were unclear in musculoskeletal growth and development as well as in osteoarthritis pathology. The role of this ephrin system in musculoskeletal growth and development was delineated in vivo using a cartilage-specific ephrin-B2 knockout mouse model. Its role in osteoarthritis in vivo was explored in mice using a bone-specific overexpression of EphB4 in which osteoarthritis was induced, and in vitro in human osteoarthritic subchondral bone osteoblasts and chondrocytes. In vivo , ephrin-B2 demonstrated to be essential for normal long bone growth and development and its absence in cartilage led to knee and hip osteoarthritis features in aged mice. In vitro data showed that the ephrin-B2-induced EphB4 receptor positively impacted the abnormal metabolism of both osteoarthritic subchondral bone osteoblasts and chondrocytes. The bone‑specific EphB4 overexpression in mice validated the in vitro data in that it had beneficial effects not only on the osteoarthritic subchondral bone but also on the cartilage and synovial membrane, and further substantiated the hypothesis that by prophylactically protecting the subchondral bone, the genesis of osteoarthritis could be, at least in part, inhibited. In the context of identifying new candidates targeting osteoarthritis progression, this ephrin system is extremely attractive as a potential novel therapeutic avenue, as therapies having a more global articular approach may prove to be the most successful to arrest or slow the progression of this disease.

Hypoxia and vitamin D differently contribute to leptin and dickkopf-related protein 2 production in human osteoarthritic subchondral bone osteoblasts.

IntroductionBone remodelling and increased subchondral densification are important in osteoarthritis (OA). Modifications of bone vascularization parameters, which lead to ischemic episodes associated with hypoxic conditions, have been suspected in OA. Among several factors potentially involved, leptin and dickkopf-related protein 2 (DKK2) are good candidates because they are upregulated in OA osteoblasts (Obs). Therefore, in the present study, we investigated the hypothesis that hypoxia may drive the expression of leptin and DKK2 in OA Obs.MethodsObs from the sclerotic portion of OA tibial plateaus were cultured under either 20% or 2% oxygen tension in the presence or not of 50 nM 1,25-dihydroxyvitamin D3 (VitD3). The expression of leptin, osteocalcin, DKK2, hypoxia-inducible factor 1α (Hif-1α) and Hif-2α was measured by real-time polymerase chain reaction and leptin production was measured by enzyme-linked immunosorbent assay (ELISA). The expression of Hif-1α, Hif-2α, leptin and DKK2 was reduced using silencing RNAs (siRNAs). The signalling pathway of hypoxia-induced leptin was investigated by Western blot analysis and with mitogen-activated protein kinase (MAPK) inhibitors.ResultsThe expression of leptin and DKK2 in Obs was stimulated 7-fold and 1.8-fold, respectively (P <0.05) under hypoxia. Interestingly, whereas VitD3 stimulated leptin and DKK2 expression 2- and 4.2-fold, respectively, under normoxia, it stimulated their expression by 28- and 6.2-fold, respectively, under hypoxia (P <0.05). The hypoxia-induced leptin production was confirmed by ELISA, particularly in the presence of VitD3 (P <0.02). Compared to Obs incubated in the presence of scramble siRNAs, siHif-2α inhibited VitD3-stimulated leptin mRNA and protein levels by 70% (P =0.004) and 60% (P <0.02), respectively, whereas it failed to significantly alter the expression of DKK2. siHif-1α has no effect on these genes. Immunoblot analysis showed that VitD3 greatly stabilized Hif-2α under hypoxic conditions. The increase in leptin expression under hypoxia was also regulated, by p38 MAPK (P <0.03) and phosphoinositide 3-kinase (P <0.05). We found that the expression of leptin and DKK2 were not related to each other under hypoxia.ConclusionsHypoxic conditions via Hif-2 regulation trigger Obs to produce leptin, particularly under VitD3 stimulation, whereas DKK2 is regulated mainly by VitD3 rather than hypoxia.

Strontium ranelate inhibits key factors affecting bone remodeling in human osteoarthritic subchondral bone osteoblasts

In osteoarthritis (OA) the progression of cartilage degeneration has been associated with remodeling of the subchondral bone. Human OA subchondral bone osteoblasts were shown to have an abnormal phenotype and altered metabolism leading to an abnormal resorptive process. Bone resorption is suggested to occur, at least in part, through the increased levels of two proteolytic enzymes, MMP-2 and MMP-9, and RANKL, which are mainly produced by osteoblasts. In this study, we investigated in human OA subchondral bone osteoblasts the modulatory effect of strontium ranelate on the above key factors. Human subchondral bone osteoblasts were cultured in a medium containing 0.1, 1 and 2 mM of strontium ranelate for 18 h for mRNA and 72 h for protein determination. The effect of strontium ranelate was evaluated on the expression (qPCR) of MMP-2, MMP-9, OPG, RANKL (total), RANKL-1, and RANKL-3, on the production of OPG (ELISA), membranous RANKL (flow cytometry), and MT1-MMP, ADAM17, and ADAM19 (Western blot). After incubation of osteoblasts with pre-osteoclasts (i.e., differentiated human peripheral blood mononuclear cells), the resorbed surface was measured using a sub-micron synthetic calcium phosphate thin film. Firstly, the expression levels of MMP-2, MMP-9, OPG, and RANKL were determined in normal and OA subchondral bone osteoblasts. As expected, the gene expression of MMP-9 and RANKL were not detectable in normal cells, whereas MMP-2 was very low but detectable and OPG demonstrated high gene expression. Further experiments looking at the effect of strontium ranelate on expression levels, except for OPG, were performed only on the OA subchondral bone osteoblasts. In OA cells, the expression levels of MMP-2 and MMP-9 were significantly decreased by strontium ranelate at 1mM (p≤0.005, p≤0.02, respectively) and 2 mM (p≤0.003, p≤0.007), and for MMP-9 only at 0.1 mM (p≤0.05). In normal cells, the expression of OPG was increased with strontium ranelate at 2 mM, and in OA both the expression (p≤0.02) and synthesis (p≤0.002) of OPG were significantly increased with strontium ranelate at 1 and 2 mM. RANKL (total) as well as the isoforms RANKL-1 and RANKL-3 were significantly increased by strontium ranelate at 1 and 2 mM. Of note, it is known that the different RANKL isoforms differentially regulate RANKL membranous localization: RANKL-3, in contrast to RANKL-1, prevents such membranous localization. This is reflected by the significant (p≤0.02) reduction in the level of membranous RANKL by strontium ranelate at 2 mM. This latter finding was not likely to be related to a proteolytic cleavage of membranous RANKL, as the enzymes known to cleave it, MT1-MMP, ADAM17 and ADAM19, were unaffected by strontium ranelate. In addition, OA osteoblasts treated with strontium ranelate induced a significant (p≤0.002) decrease in resorbed surface at the three tested concentrations. This study provides new insights into the mode of action of strontium ranelate on the metabolism of human OA subchondral bone osteoblasts. These data suggest that strontium ranelate may exert a positive effect on OA pathophysiology by inhibiting, in these cells, the synthesis of key factors leading to bone resorption, a feature associated with the OA process.

Intra‐articular injection of hyaluronan restores the aberrant expression of matrix metalloproteinase‐13 in osteoarthritic subchondral bone

Subchondral bone is a candidate for treatment of osteoarthritis (OA). We investigated the effects of intra-articular injection of hyaluronan (IAI-HA) on subchondral bone in rabbit OA model. OA was induced by anterior cruciate ligament transection, with some rabbits receiving IAI-HA. OA was graded morphologically, and expression of mRNA was assessed by real-time RT-PCR. Tissue sections were stained with hyaluronan-binding protein, and penetration of fluorescent hyaluronan was assessed. The in vitro inhibitory effect of hyaluronan on MMP-13 was analyzed in human osteoarthritic subchondral bone osteoblasts (OA Ob) by real-time RT-PCR and ELISA. Binding of hyaluronan to OA Ob via CD44 was assessed by immunofluorescence cytochemistry. Expression of MMP-13 and IL-6 mRNA in cartilage and subchondral bone, and morphological OA grade, increased over time. IAI-HA ameliorated the OA grade and selectively suppressed MMP-13 mRNA in subchondral bone. IAI-HA enhanced the hyaluronan staining of subchondral bone marrow cells and osteocyte lacunae. Fluorescence was observed in the subchondral bone marrow space. In OA Ob, hyaluronan reduced the expression and production of MMP-13, and anti-CD44 antibody blocked hyaluronan binding to OA Ob. These findings indicate that regulation of MMP-13 in subchondral bone may be a critical mechanism during IAI-HA.

Proteinase-activated receptor (PAR)-2 activation impacts bone resorptive properties of human osteoarthritic subchondral bone osteoblasts

Introduction In osteoarthritis (OA), the subchondral bone undergoes a remodelling process involving several factors synthesized by osteoblasts. In this study, we investigated the expression, production, modulation, and role of PAR-2 in human OA subchondral bone osteoblasts. Materials and methods PAR-2 expression and production were determined by real-time PCR and flow cytometry, respectively. PAR-2 modulation was investigated in OA subchondral bone osteoblasts treated with IL-1β (100 pg/ml), TNF-α (5 ng/ml), TGF-β1 (10 ng/ml), PGE 2 (500 nM), IL-6 (10 ng/ml) and IL-17 (10 ng/ml). Membranous RANKL protein was assessed by flow cytometry, and OPG, MMP-1, MMP-9, MMP-13, IL-6 and intracellular signalling pathways by specific ELISAs. Bone resorptive activity was measured by using a co-culture model of human PBMC and OA subchondral bone osteoblasts. Results PAR-2 expression and production ( p < 0.05) were markedly increased when human OA subchondral bone osteoblasts were compared to normal. On OA osteoblasts, PAR-2 production was significantly increased by IL-1β, TNF-α and PGE 2. Activation of PAR-2 with a specific agonist, SLIGKV-NH 2, induced a significant up-regulation of MMP-1, MMP-9, IL-6, and membranous RANKL, but had no effect on MMP-13 or OPG production. Interestingly, bone resorptive activity was also significantly enhanced following PAR-2 activation. The PAR-2 effect was mediated by activation of the MAP kinases Erk1/2 and JNK. Conclusion This study is the first to demonstrate that PAR-2 activation plays a role in OA subchondral bone resorption via an up-regulation of major bone remodelling factors. These results shed new light on the potential of PAR-2 as a therapeutic target in OA.

New Perspective in Osteoarthritis: The OPG and RANKL System as a Potential Therapeutic Target?

Bone remodelling is tightly regulated by a molecular triad composed of OPG/RANK/RANKL. The receptor activator of NF-kappaB ligand (RANKL) (localized on osteoblasts) enhances osteoclastogenesis via interaction with its receptor RANK (localized on osteoclasts), whereas osteoprotegerin (OPG) (produced by osteoblasts) inhibits this osteoclastogenesis by binding to RANKL. The equilibrium between OPG and RANKL plays a crucial role in the pathophysiology of bone. Although some studies have shown the efficacy of OPG as a therapeutic agent against bone resorption, its bioavailability and mechanism of action after binding to RANKL have only recently been studied. A mechanistic investigation based on what becomes of OPG after binding to cells expressing membranous RANKL demonstrated an internalization process of OPG through the clathrin pathway prior to proteasomal and/or lysosomal degradation. Interestingly, the OPG internalization process reduced the half-life of RANKL. Recent evidence has shown that subchondral bone alterations in osteoarthritis (OA) are intimately involved in cartilage degradation, and that OPG/RANKL may be implicated. Data show that human OA subchondral bone osteoblasts have abnormal OPG and RANKL levels and consequently an altered OPG and RANKL ratio. Further data also reveal the involvement of some osteotropic factors in these altered levels and that some of these factors generally target RANKL with a differential modulation of the RANKL isoforms. Altogether, data suggest that this system could be targeted as a new strategy for the treatment of OA.

Activation of the receptor EphB4 by its specific ligand ephrin B2 in human osteoarthritic subchondral bone osteoblasts

Abnormal subchondral bone metabolism is involved in osteoarthritis (OA). It has been suggested that ephrin B2 and its specific receptor EphB4 participate in bone homeostasis. We previously reported that human OA subchondral bone osteoblasts could be classified into 2 subpopulations: low (L), having proresorption properties, and high (H), having proformation properties. The purpose of this study was to investigate the importance of the ephrin system in OA subchondral bone osteoblasts. The presence of the EphB4 receptor was determined by immunohistochemistry, and its expression level, modulation upon treatment, and consequences of activation by ephrin B2 were determined by quantitative polymerase chain reaction. The effects of ephrin B2 activation of the EphB4 receptor on bone resorption activity were also determined. EphB4 receptor activation signaling pathways were investigated by specific enzyme-linked immunosorbent assay. EphB4 receptors were present in subchondral bone osteoblasts and osteocytes. Compared with normal and H-OA osteoblasts, EphB4 receptor expression levels were significantly increased in L-OA osteoblasts, with no difference between normal and H-OA osteoblasts. EphB4 receptor levels in L-OA osteoblasts were significantly up-regulated by prostaglandin E2 (PGE2) and interleukin-17 (IL-17). Ephrin B2, PGE2, and IL-17 significantly inhibited bone resorption activity in these cells. EphB4 activation by ephrin B2 significantly inhibited the expression of IL-1beta, IL-6, matrix metalloproteinase 1 (MMP-1), MMP-9, MMP-13, and RANKL, but not MMP-2 and osteoprotegerin. EphB4 receptor activation significantly inhibited the phosphatidylinositol 3-kinase/Akt pathway. This study is the first to provide evidence that EphB4 receptor activation by ephrin B2 in OA subchondral bone could affect abnormal metabolism in this tissue by inhibiting resorption factors and their activities. Ephrin B2 could be targeted as a specific therapeutic approach in the development of a disease-modifying OA drug.

Differential modulation of RANKL isoforms by human osteoarthritic subchondral bone osteoblasts: Influence of osteotropic factors

Background Osteoarthritis (OA) is the most common human joint disease. Recent studies suggest that an abnormal subchondral bone metabolism is intimately involved in the genesis of this disease. Bone remodelling is tightly regulated by a molecular triad composed of OPG/RANK/RANKL. RANKL exists as 3 isoforms: RANKL1, 2, and 3. RANKL1 and 2 enhance osteoclastogenesis whereas RANKL3 inhibits this phenomenon. We previously reported that human OA subchondral bone osteoblasts can be discriminated into two subgroups according to their level of PGE 2 [low (L) or high (H)]. Moreover, we also showed that L-OA osteoblasts express higher levels of total RANKL compared to H-OA osteoblasts. In this study, we investigated the level of membranous RANKL, comparing L- and H-OA subchondral bone osteoblasts, as well as its modulation by osteotropic factors. The impact of the modulation of RANKL1 and 3 on the membranous RANKL level was also studied. Methods Gene expression was determined using real-time PCR for RANKL1 and semi-quantitative PCR for RANKL3. Membranous RANKL was measured by flow cytometry. The modulation of membranous RANKL and RANKL isoforms was monitored on the L- and H-OA osteoblasts and also following treatment with osteotropic factors, including vitamin D 3 (50 nM), IL-1β (100 pg/ml), TNF-α (5 ng/ml), PGE 2 (500 nM), PTH (100 nM), IL-6 (10 ng/ml) and IL-17 (10 ng/ml). Results Membranous RANKL levels were significantly increased in L-OA osteoblasts compared to normal ( p < 0.01) and H-OA ( p < 0.05). The gene expression level of the RANKL1 profile was reminiscent of the membranous RANKL level. Although RANKL3 gene expression was lower on the H-OA osteoblasts than on normal and L-OA osteoblasts ( p < 0.03), the overall outcome favoured RANKL1. Treatment with the tested factors showed a significant increase in membranous RANKL on the L-OA osteoblasts, with the exception of PTH and IL-17. Interestingly in this subpopulation, the RANKL3 gene expression level was significantly increased upon PTH and IL-17 treatment. No effect of the tested osteotropic factors was found on the H-OA. Conclusion Our findings showed that the normal, L- and H-OA subchondral bone osteoblasts differentially express membranous RANKL and RANKL isoforms, and that treatment with osteotropic factors generally favours increased membranous localization of RANKL on L-OA compared to H-OA osteoblasts. This phenomenon appears to take place through differential modulation of each RANKL isoform.

Chondroitin and glucosamine sulfate in combination decrease the pro-resorptive properties of human osteoarthritis subchondral bone osteoblasts: a basic science study.

Early in the pathological process of osteoarthritis (OA), subchondral bone remodelling, which is related to altered osteoblast metabolism, takes place. In the present study, we explored in human OA subchondral bone whether chondroitin sulfate (CS), glucosamine sulfate (GS), or both together affect the major bone biomarkers, osteoprotegerin (OPG), receptor activator of nuclear factor-kappa B ligand (RANKL), and the pro-resorptive activity of OA osteoblasts. The effect of CS (200 μg/mL), GS (50 and 200 μg/mL), or both together on human OA subchondral bone osteoblasts, in the presence or absence of 1,25(OH)2D3 (vitamin D3) (50 nM), was determined on the bone biomarkers alkaline phosphatase and osteocalcin, on the expression (mRNA) and production (enzyme-linked immunosorbent assay) of bone remodelling factors OPG and RANKL, and on the pro-resorptive activity of these cells. For the latter experiments, human OA osteoblasts were incubated with differentiated peripheral blood mononuclear cells on a sub-micron synthetic calcium phosphate thin film. Data showed that CS and GS affected neither basal nor vitamin D3-induced alkaline phosphatase or osteocalcin release. Interestingly, OPG expression and production under basal conditions or vitamin D3 treatment were upregulated by CS and by both CS and GS incubated together. Under basal conditions, RANKL expression was significantly reduced by CS and by both drugs incubated together. Under vitamin D3, these drugs also showed a decrease in RANKL level, which, however, did not reach statistical significance. Importantly, under basal conditions, CS and both compounds combined significantly upregulated the expression ratio of OPG/RANKL. Vitamin D3 decreased this ratio, and GS further decreased it. Both drugs reduced the resorption activity, and statistical significance was reached for GS and when CS and GS were incubated together. Our data indicate that CS and GS do not overly affect cell integrity or bone biomarkers. Yet CS and both compounds together increase the expression ratio of OPG/RANKL, suggesting a positive effect on OA subchondral bone structural changes. This was confirmed by the decreased resorptive activity for the combination of CS and GS. These data are of major significance and may help to explain how these two drugs exert a positive effect on OA pathophysiology.

P336 The differential expression of OPG/RANKL in human osteoarthritic subchondral bone osteoblasts is an indicator of the metabolic state of these disease cells

P336 The differential expression of OPG/RANKL in human osteoarthritic subchondral bone osteoblasts is an indicator of the metabolic state of these disease cells