Alteration of Wnt5a expression and of the non-canonical Wnt/PCP and Wnt/PKC-Ca2+ pathways in human osteoarthritis osteoblasts.

OBJECTIVE: Clinical and in vitro studies suggest that subchondral bone sclerosis due to abnormal osteoblasts (Ob) is involved in the progression and/or onset of osteoarthritis (OA). Human Ob isolated from sclerotic subchondral OA bone tissue show an altered phenotype, a decreased canonical Wnt/ß-catenin signaling pathway (cWnt), and a reduced mineralization in vitro. In addition to the cWnt pathway, at least two non-canonical signaling pathways, the Wnt/PKC and Wnt/PCP pathway have been described. However, there are no reports of either pathway in OA Ob. Here, we studied the two non-canonical pathways in OA Ob and if they influence their phenotype. METHODS: Human primary subchondral Ob were isolated from the subchondral bone plate of tibial plateaus of OA patients undergoing total knee arthroplasty, or of normal individuals at autopsy. The expression of genes involved in non-canonical Wnt signaling was evaluated by qRT-PCR and their protein production by Western blot analysis. Alkaline phosphatase activity and osteocalcin secretion (OC) were determined with substrate hydrolysis and EIA, respectively. Mineralization levels were evaluated with Alizarin Red Staining, Wnt/PKC and Wnt/PCP pathways by target gene expression and their respective activity using the NFAT and AP-1 luciferase reporter assays. RESULTS: OA Ob showed an altered phenotype as illustrated by an increased alkaline phosphatase activity and osteocalcin release compared to normal Ob. The expression of the non-canonical Wnt5a ligand was increased in OA Ob compared to normal. Whereas, the expression of LGR5 was significantly increased in OA Ob compared to normal Ob, the expression of LGR4 was similar. Wnt5a directly stimulated the expression and production of LGR5, contrasting, Wnt5a did not stimulate the expression of LGR4. Wnt5a also stimulated the phosphorylation of both JNK and PKC, as well as the activity of both NFAT and AP-1 transcription factors. The inhibition of Wnt5a expression partially corrects the abnormal mineralization, OC secretion and ALPase activity of OA Ob. CONCLUSION: These data indicate that the alteration of Wnt5a, a non-canonical Wnt signaling activator, is implicated in the modified signalisation and phenotype observed in OA Ob.

Beneficial effect of resveratrol on phenotypic features and activity of osteoarthritic osteoblasts.

BACKGROUND: Osteoarthritis (OA) is a complex disease, which affects multiple tissues, namely the subchondral bone, articular cartilage and synovial membrane. Alterations of the subchondral bone include an increased, yet under mineralized osteoid matrix, abnormal osteoblast cell phenotype including elevated alkaline phosphatase (ALP) activity, increased release of osteocalcin (OC) and transforming growth factor ß-1 (TGF-ß1). Previous studies have demonstrated an inhibition of the canonical Wnt signaling (cWnt) pathway in OA osteoblasts (Ob). As resveratrol (RSV) has been shown to upregulate the Wnt signaling pathway in different cell systems, we hypothesized that RSV could be beneficial for OA Ob. METHOD: We prepared primary human Ob using the subchondral bone plate of tibial plateaus of OA patients undergoing total knee arthroplasty, or tibial plateaus of normal individuals at autopsy. Sirtuin 1 (Sirt1) expression in normal and OA subchondral bone tissue was evaluated by immunohistochemical analysis. Expression of genes was evaluated by qRT-PCR and protein production by western blot analysis. ALP activity and osteocalcin secretion were evaluated respectively with substrate hydrolysis and enzyme immunoassay. Mineralization levels were evaluated with alizarin red staining. Wnt/ß-catenin signaling was evaluated by target gene expression using the TOPflash TCF/lef luciferase reporter assay and intracellular signaling using ß-catenin levels in western blot analysis. Extracellular signal-regulated kinase (Erk)1/2 and the Smad1/5/8 pathways were evaluated by western blot analysis. RESULTS: Sirt1 expression and production were reduced in OA subchondral bone tissue compared to normal tissue. RSV upregulated Sirt1 and its activity, and reduced the expression of leptin. RSV increased Erk1/2 phosphorylation in OA Ob; however, it had no effect on Smad 1/5/8 phosphorylation. RSV had little effect on cell proliferation and only slightly affected the Bax/Bcl2 ratio. The expression of Runx2/Cbfa1 and peroxisome proliferator-activated receptor (PPAR)γ were not affected by increasing doses of RSV. The endogenous increased ALP activity and OC release observed in OA Ob compared to normal Ob were partly corrected only for ALP at high RSV levels but not for OC release. In contrast, RSV increased the mineralization of OA Ob. Moreover, whereas Wnt3a stimulates the Wnt/ß-catenin pathway in these cells, RSV further increased the response to Wnt3a. CONCLUSION: These data indicate that RSV promotes Sirt1 levels, inhibits the endogenous expression of leptin by OA osteoblasts and can promote the Wnt/ß-catenin and Erk1/2 signaling pathways, which are altered in these cells.

Platelet rich plasma (PRP) induces chondroprotection via increasing autophagy, anti-inflammatory markers, and decreasing apoptosis in human osteoarthritic cartilage.

OBJECTIVES: Autophagy constitutes a defense mechanism to overcome aging and apoptosis in osteoarthritic cartilage. Several cytokines and transcription factors are linked to autophagy and play an important role in the degradative cascade in osteoarthritis (OA). Cell therapy such as platelet rich plasma (PRP) has recently emerged as a promising therapeutic tool for many diseases including OA. However, its mechanism of action on improving cartilage repair remains to be determined. The purpose of this study is to investigate the effect of PRP on osteoarthritic chondrocytes and to elucidate the mechanism by which PRP contributes to cartilage regeneration. METHODS: Osteoarthritic chondrocytes were co-cultured with an increasing concentration of PRP obtained from healthy donors. The effect of PRP on the proliferation of chondrocytes was performed using cell counting and WST8 proliferation assays. Autophagy, apoptosis and intracellular level of IL-4, IL-10, and IL-13 were determined using flow cytometry analyses. Autophagy markers BECLIN and LC3II were also determined using quantitative polymerase chain reaction (qPCR). qPCR and ELISA were used to measure the expression of ADAMDTS-5, MMP3, MMP13, TIMP-1-2-3, aggregan, Collagen type 2, TGF-ß, Cox-2, Il-6, FOXO1, FOXO3, and HIF-1 in tissues and co-cultured media. RESULTS: PRP increased significantly the proliferation of chondrocytes, decreased apoptosis and increased autophagy and its markers along with its regulators FOXO1, FOXO3 and HIF-1 in osteoarthritic chondrocytes. Furthermore, PRP caused a dose-dependent significant decrease in MMP3, MMP13, and ADAMTS-5, IL-6 and COX-2 while increasing TGF-ß, aggregan, and collagen type 2, TIMPs and intracellular IL-4, IL-10, IL-13. CONCLUSION: These results suggest that PRP could be a potential therapeutic tool for the treatment of OA.

PPARγ deficiency results in severe, accelerated osteoarthritis associated with aberrant mTOR signalling in the articular cartilage.

OBJECTIVES: We have previously shown that peroxisome proliferator-activated receptor gamma (PPARγ), a transcription factor, is essential for the normal growth and development of cartilage. In the present study, we created inducible cartilage-specific PPARγ knockout (KO) mice and subjected these mice to the destabilisation of medial meniscus (DMM) model of osteoarthritis (OA) to elucidate the specific in vivo role of PPARγ in OA pathophysiology. We further investigated the downstream PPARγ signalling pathway responsible for maintaining cartilage homeostasis. METHODS: Inducible cartilage-specific PPARγ KO mice were generated and subjected to DMM model of OA. We also created inducible cartilage-specific PPARγ/mammalian target for rapamycin (mTOR) double KO mice to dissect the PPARγ signalling pathway in OA. RESULTS: Compared with control mice, PPARγ KO mice exhibit accelerated OA phenotype with increased cartilage degradation, chondrocyte apoptosis, and the overproduction of OA inflammatory/catabolic factors associated with the increased expression of mTOR and the suppression of key autophagy markers. In vitro rescue experiments using PPARγ expression vector reduced mTOR expression, increased expression of autophagy markers and reduced the expression of OA inflammatory/catabolic factors, thus reversing the phenotype of PPARγ KO mice chondrocytes. To dissect the in vivo role of mTOR pathway in PPARγ signalling, we created and subjected PPARγ-mTOR double KO mice to the OA model to see if the genetic deletion of mTOR in PPARγ KO mice (double KO) can rescue the accelerated OA phenotype observed in PPARγ KO mice. Indeed, PPARγ-mTOR double KO mice exhibit significant protection/reversal from OA phenotype. SIGNIFICANCE: PPARγ maintains articular cartilage homeostasis, in part, by regulating mTOR pathway.

Identification of two populations of osteoarthritic osteoblasts according to the 1,25[OH]2 vitamin D3 potency to stimulate osteocalcin.

INTRODUCTION: Few studies have tried to discriminate a differential behavior between osteoarthritic (OA) osteoblasts (Obs). Based on osteocalcin level, we aimed, in the present study, to evaluate the capacity of OA Obs for producing molecules of the Wnt/ß-catenin signaling pathway. METHODS: Human primary OA Obs (n=11) were exposed or not to 50 nM of 1,25 dihydroxyvitamin D3 (VitD3) for 24 h. Osteocalcin (OCN), TGF-ß1, Dickkopf-related protein 2 (DKK2), R-spondin 2 (Rspo2), Wnt5b, and low density lipoprotein related-receptor 1 (LRP1) were evaluated by real time RT-PCR. RESULTS: All samples responded to VitD3 as validated by the increase in OCN expression. However two populations of Obs were discriminated; one called "high responders" whose OCN stimulation was higher than 100 fold (mean 881 fold, p<0.01, n=5) and the second one whose stimulation was inferior to 100 fold (mean 47 fold, p<0.01, n=6), namely "low responders". In fact, high responders have a weaker basal expression of OCN. With regards to these two cell populations and in absence of VitD3 challenge, the expression level of TGF-ß1 (15 fold, p<0.001), DKK2 (2.5 fold, p<0.002) and Wnt5b (5.5 fold, p<0.003) was higher in "high responders", meanwhile Rspo2 and LRP1 expression was unchanged. VitD3 exacerbated this pattern but corrected OCN expression and favored Wnt agonist expression. CONCLUSION: We identified 2 populations of OA Obs according to the OCN expression under the control of VitD3. In addition under basal conditions, these 2 populations expressed differently TGF-ß1, Wnt5b, DKK2, suggesting a heterogeneous differentiation and phenotype in Obs among OA patients.

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

INTRODUCTION: Bone remodelling and increased subchondral densification are important in osteoarthritis (OA). Modifications of bone vascularisation 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 since they are up-regulated 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. METHODS: Obs from the sclerotic portion of OA tibial plateaus were cultured either under 20% or 2% oxygen tension in the presence or not of 50 nM of 1,25-dihydroxyvitamin D3 (VitD3). The expression of leptin, osteocalcin, DKK2, hypoxia-inducible factors (Hif)-1α and -2α was measured by real-time polymerase chain reaction and leptin production by enzyme-linked immunosorbent assay (ELISA). The expression of Hif-1α, Hif-2α, leptin and DKK2 was reduced using silencing (si) RNA technique. Signalling pathway of hypoxia-induced leptin was investigated by western blotting and mitogen-activated protein kinase (MAPK) inhibitors. RESULTS: As expected, hypoxia stimulated the expression of Hif-1 and Hif-2. The expression of leptin and DKK2 in Obs was also 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 under normoxia, it further stimulated it to 28- and 6.2-fold under hypoxia (p<0.05). The hypoxia-induced leptin production was confirmed by ELISA, particularly in presence of VitD3 (p<0.02). Compared to Obs incubated in the presence of siScramble RNAs, siHif-2α inhibited VitD3-stimulated leptin mRNA and protein levels by 70% (p=0.004) and 60% (p<0.02), respectively while it failed to significantly alter the expression of DKK2. SiHif-1α has no effect on these genes. Immunoblotting showed that VitD3 greatly stabilized Hif-2α under hypoxic condition. The increase in leptin expression under hypoxia was also regulated, in addition to the role of Hif-2α, by p38 MAPK (p<0.03) and PI 3-kinase (p<0.05). Finally, we demonstrated that the expression of leptin and DKK2 were not related to each other under hypoxia. CONCLUSIONS: Hypoxic conditions via Hif-2 regulation trigger Obs to produce leptin particularly under VitD3 stimulation whereas DKK2 is mainly regulated by VitD3 rather than hypoxia.

Low sirtuin 1 levels in human osteoarthritis subchondral osteoblasts lead to abnormal sclerostin expression which decreases Wnt/ß-catenin activity.

INTRODUCTION: Wnt/ß-catenin (cWnt) signaling plays a key role in osteogenesis by promoting the differentiation and mineralization of osteoblasts, activities altered in human osteoarthritic subchondral osteoblast (OA Ob). Sclerostin (SOST) has been shown to alter cWnt signaling. Sirtuin 1 (SIRT1) acts as a novel bone regulator and represses SOST levels in Ob. However the role of SIRT1 and SOST in OA Ob remains unknown. Herein, we explored the role played by SIRT1 and SOST on the abnormal mineralization and cWnt signaling in OA Ob. METHODS: Primary human normal and OA Ob were prepared from tibial plateaus. SOST levels were evaluated by immunohistochemistry, the expression and production of genes by qRT-PCR and WB analysis. Their inhibitions were performed using siRNA. cWnt signaling was measured by the TOPflash TCF/lef luciferase reporter assay. Mineralization was determined by alizarin red staining. RESULTS: SOST levels were significantly increased in OA Ob compared to normal and were linked with elevated TGF-ß1 levels in these cells. SIRT1 expression was significantly reduced in OA Ob compared to normal yet not modified by TGF-ß1. Specific inhibition of SIRT1 increased TGF-ß1 and SOST expressions in OA Ob, while stimulating SIRT1 activity with ß-Nicotinamide mononucleotide reduced the expression of TGF-ß1 and SOST, and increased mineralization in OA Ob. Resveratrol also reduced SOST expression in OA Ob. Reduced cWnt signaling, ß-catenin levels, and mineralization in OA Ob were all corrected via reducing SOST expression. CONCLUSION: These data indicate that high level of SOST is responsible, in part, for the reduced cWnt and mineralization of human OA Ob, which in turn is linked with abnormal SIRT1 levels in these pathological cells.

Strontium ranelate reduces the progression of experimental dog osteoarthritis by inhibiting the expression of key proteases in cartilage and of IL-1ß in the synovium.

OBJECTIVE: To explore the disease-modifying effect, under therapeutic conditions, of strontium ranelate (SrRan) on the progression of joint structural changes and on the major pathophysiological pathways in an experimental osteoarthritis dog model. METHODS: Dogs underwent sectioning of the anterior cruciate ligament, and 4 weeks after surgery received oral treatment of SrRan 25, 50 or 75 mg/kg per day, or placebo for 12 weeks. Methods included macroscopy, picrosirius red staining, histology, subchondral bone histomorphometry, quantitative PCR, and ELISA for CTX-II level in serum. Strontium plasma and synovial fluid levels were also measured. RESULTS: At steady state, strontium blood exposures were within the clinical therapeutic range of osteoarthritis patients and correlated with strontium concentrations in synovial fluid. SrRan treatment significantly reduced the osteoarthritis cartilage lesions at all doses tested (p≤0.05). Significantly better preservation of the collagen network was also found in SrRan-treated dogs at 50 and 75 mg/kg per day (p=0.03). The osteoarthritis subchondral bone thickening observed in osteoarthritis-placebo dogs was significantly reduced by SrRan at 50 mg/kg per day (p=0.02). The increased gene expression levels of MMP-1, MMP-13 and cathepsin K in osteoarthritis cartilage were all significantly reduced by SrRan at 75 mg/kg per day (p≤0.03) as were, in osteoarthritis synovium, IL-1ß at 50 and 75 mg/kg per day (p=0.05) and MMP-3 at all doses tested (p≤0.02). The serum level of CTX-II was reduced (p≤0.04) by SrRan at 16 weeks in dogs treated with 50 and 75 mg/kg per day. CONCLUSIONS: This study is the first to demonstrate in vivo in an animal model that SrRan reduced the progression of osteoarthritis structural changes. The inhibition of several key proteases as well as IL-1ß may have contributed to the beneficial effect of SrRan.

R-spondins are newly recognized players in osteoarthritis that regulate Wnt signaling in osteoblasts.

OBJECTIVE: Clinical and in vitro studies suggest that altered osteogenesis or bone remodeling is involved in the progression and/or onset of osteoarthritis (OA). Wnt signaling plays a key role in osteogenesis via the canonical Wnt/ß-catenin signaling pathway. Two of the R-spondins, Rspo-1 and Rspo-2, a family of 4 proteins unrelated to other Wnt ligands that act as Wnt agonists, are present in bone tissues. The purpose of this study was to investigate the potential role of Rspo-1 and Rspo-2 in OA osteoblasts. METHODS: Primary human normal and OA osteoblasts were prepared from tibial plateaus. The expression of Rspo-1 and Rspo-2 was evaluated by quantitative reverse transcription-polymerase chain reaction analysis. Western blot analysis was used to determine Rspo-2, ß-catenin, and phospho-ß-catenin levels. Wnt/ß-catenin signaling was evaluated using the TOPflash T cell factor (TCF)/lymphoid enhancer factor (LEF) luciferase reporter assay. Mineralization was evaluated by alizarin red staining. RESULTS: The expression of Rspo-1 was similar in normal and OA osteoblasts, whereas the expression and production of Rspo-2 were reduced in OA osteoblasts due to elevated levels of transforming growth factor ß1 in these cells. The reduced Wnt-3a-dependent TOPflash TCF/LEF luciferase reporter activity in OA osteoblasts as compared to normal osteoblasts was corrected by the addition of recombinant human Rspo-2. Wnt-3a-dependent ß-catenin levels were also corrected in OA osteoblasts by Rspo-2 addition. Wnt-3a alone increased the mineralization of OA osteoblasts, which was further increased by Rspo-2. CONCLUSION: Reduced Rspo-2 levels in OA osteoblasts are responsible, at least in part, for their reduced Wnt/ß-catenin signaling and abnormal mineralization. As Rspo-2 is a secreted soluble protein, this could lead to potential new avenues of treatment of OA.

Tiludronate treatment improves structural changes and symptoms of osteoarthritis in the canine anterior cruciate ligament model.

INTRODUCTION: The aim of this prospective, randomized, controlled, double-blind study was to evaluate the effects of tiludronate (TLN), a bisphosphonate, on structural, biochemical and molecular changes and function in an experimental dog model of osteoarthritis (OA). METHODS: Baseline values were established the week preceding surgical transection of the right cranial/anterior cruciate ligament, with eight dogs serving as OA placebo controls and eight others receiving four TLN injections (2 mg/kg subcutaneously) at two-week intervals starting the day of surgery for eight weeks. At baseline, Week 4 and Week 8, the functional outcome was evaluated using kinetic gait analysis, telemetered locomotor actimetry and video-automated behaviour capture. Pain impairment was assessed using a composite numerical rating scale (NRS), a visual analog scale, and electrodermal activity (EDA). At necropsy (Week 8), macroscopic and histomorphological analyses of synovium, cartilage and subchondral bone of the femoral condyles and tibial plateaus were assessed. Immunohistochemistry of cartilage (matrix metalloproteinase (MMP)-1, MMP-13, and a disintegrin and metalloproteinase domain with thrombospondin motifs (ADAMTS5)) and subchondral bone (cathepsin K) was performed. Synovial fluid was analyzed for inflammatory (PGE(2) and nitrite/nitrate levels) biomarkers. Statistical analyses (mixed and generalized linear models) were performed with an α-threshold of 0.05. RESULTS: A better functional outcome was observed in TLN dogs than OA placebo controls. Hence, TLN dogs had lower gait disability (P = 0.04 at Week 8) and NRS score (P = 0.03, group effect), and demonstrated behaviours of painless condition with the video-capture (P < 0.04). Dogs treated with TLN demonstrated a trend toward improved actimetry and less pain according to EDA. Macroscopically, both groups had similar level of morphometric lesions, TLN-treated dogs having less joint effusion (P = 0.01), reduced synovial fluid levels of PGE(2) (P = 0.02), nitrites/nitrates (P = 0.01), lower synovitis score (P < 0.01) and a greater subchondral bone surface (P < 0.01). Immunohistochemical staining revealed lower levels in TLN-treated dogs of MMP-13 (P = 0.02), ADAMTS5 (P = 0.02) in cartilage and cathepsin K (P = 0.02) in subchondral bone. CONCLUSION: Tiludronate treatment demonstrated a positive effect on gait disability and joint symptoms. This is likely related to the positive influence of the treatment at improving some OA structural changes and reducing the synthesis of catabolic and inflammatory mediators.

Elevated Dickkopf-2 levels contribute to the abnormal phenotype of human osteoarthritic osteoblasts.

The Wnt signaling pathway is crucial for osteogenesis and regulates terminal osteoblast differentiation. Although osteoarthritic (OA) osteoblasts show an abnormal phenotype and poor in vitro mineralization, the mechanism leading to this situation still remains unknow. Recent evidence indicates that Wnt signaling may be altered in OA osteoblasts. In this study we determined whether an alteration of the Wnt/ß-catenin signaling pathway is responsible for the abnormal phenotype of OA osteoblasts. Expression of the Wnt signaling antagonist Dickkopf-1 (DKK1) was similar in normal and OA osteoblasts, whereas DKK2 expression was higher in OA osteoblasts than in normal osteoblasts. OA osteoblasts showed a decrease of Wnt3a-dependent Wnt/ß-catenin signaling, measured by the TOPflash reporter assay and by Western blot analysis, compared with normal osteoblasts. Correcting DKK2 levels in OA osteoblasts by siRNA techniques enhanced Wnt/ß-catenin signaling. Elevated DKK2 levels could be explained by elevated transforming growth factor ß1 (TGF-ß1) in OA osteoblasts, and exogenous TGF-ß1 increased DKK2 expression in normal osteoblasts, whereas ablating TGF-ß1 expression in OA osteoblasts reduced DKK2 expression. Inhibiting TGF-ß1 or DKK2 expression corrected the abnormal phenotype of OA osteoblasts. In vitro mineralization of OA osteoblasts also was increased by DKK2 siRNA. We conclude that elevated TGF-ß1 levels in OA osteoblasts can stimulate DKK2 expression, which, in turn, is responsible, at least in part, for their abnormal phenotype.

Role of proinflammatory cytokines in the pathophysiology of osteoarthritis.

Osteoarthritis (OA) is associated with cartilage destruction, subchondral bone remodeling and inflammation of the synovial membrane, although the etiology and pathogenesis underlying this debilitating disease are poorly understood. Secreted inflammatory molecules, such as proinflammatory cytokines, are among the critical mediators of the disturbed processes implicated in OA pathophysiology. Interleukin (IL)-1ß and tumor necrosis factor (TNF), in particular, control the degeneration of articular cartilage matrix, which makes them prime targets for therapeutic strategies. Animal studies provide support for this approach, although only a few clinical studies have investigated the efficacy of blocking these proinflammatory cytokines in the treatment of OA. Apart from IL-1ß and TNF, several other cytokines including IL-6, IL-15, IL-17, IL-18, IL-21, leukemia inhibitory factor and IL-8 (a chemokine) have also been shown to be implicated in OA and could possibly be targeted therapeutically. This Review discusses the current knowledge regarding the role of proinflammatory cytokines in the pathophysiology of OA and addresses the potential of anticytokine therapy in the treatment of this disease.

Local leptin production in osteoarthritis subchondral osteoblasts may be responsible for their abnormal phenotypic expression.

INTRODUCTION: Leptin is a peptide hormone with a role in bone metabolism and rheumatic diseases. The subchondral bone tissue plays a prominent role in the pathophysiology of osteoarthritis (OA), related to abnormal osteoblast (Ob) differentiation. Although leptin promotes the differentiation of Ob under normal conditions, a role for leptin in OA Ob has not been demonstrated. Here we determined if endogenous leptin produced by OA Ob could be responsible for the expression of the abnormal phenotypic biomarkers observed in OA Ob. METHODS: We prepared primary normal and OA Ob from subchondral bone of tibial plateaus removed for knee surgery of OA patients or at autopsy. We determined the production of leptin and of the long, biologically active, leptin receptors (OB-Rb) using reverse transcriptase-polymerase chain reaction, ELISA and Western blot analysis. We determined the effect of leptin on cell proliferation by BrdU incorporation and 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays, and we determined by Western blot analysis phospho 42/44 MAPK (p42/44 Erk1/2) and phospho p38 levels. We then determined the effect of the addition of exogenous leptin, leptin receptor antagonists, inhibitors of leptin signaling or siRNA techniques on the phenotypic features of OA Ob. Phenotypic features of Ob were determined by measuring alkaline phosphatase activity (ALP), osteocalcin release (OC), collagen type 1 production (CICP) and of Transforming Growth Factor-beta1 (TGF-beta1). RESULTS: Leptin expression was increased approximately five-fold and protein levels approximately two-fold in OA Ob compared to normal. Leptin stimulated its own expression and the expression of OB-Rb in OA Ob. Leptin dose-dependently stimulated cell proliferation of OA Ob and also increased phosphorylated p42/44 Erk1/2 and p38 levels. Inactivating antibodies against leptin reduced ALP, OC, CICP and TGF-beta1 levels in OA Ob. Tyrphostin (AG490) and piceatannol (Pce), inhibitors of leptin signaling, reproduced this effect. Inhibition of endogenous leptin levels using siRNA for leptin or inhibiting leptin signaling using siRNA for OB-Rb expression both reduced ALP and OC about 60%. Exogenous leptin addition stimulated ALP, yet this failed to further increase OC or CICP. CONCLUSIONS: These results suggest that abnormal production of leptin by OA Ob could be responsible, in part, for the elevated levels of ALP, OC, collagen type 1 and TGF-beta1 observed in these cells compared to normal. Leptin also stimulated cell proliferation, and Erk 1/2 and p38 signaling. Taken together, these data suggest leptin could contribute to abnormal osteoblast function in OA.

Targeting subchondral bone for treating osteoarthritis: what is the evidence?

Over the past few decades, significant progress has been made with respect to new concepts about the pathogenesis of osteoarthritis (OA). This article summarises some of the knowledge we have today on the involvement of the subchondral bone in OA. It provides substantial evidence that changes in the metabolism of the subchondral bone are an integral part of the OA disease process and that these alterations are not merely secondary manifestations, but are part of a more active component of the disease. Thus, a strong rationale exists for therapeutic approaches that target subchondral bone resorption and/or formation, and data evaluating the drugs targeting bone remodelling raise the hope that new treatment options for OA may become available.

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

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.

Altered mineralization of human osteoarthritic osteoblasts is attributable to abnormal type I collagen production.

OBJECTIVE: Bone tissue in osteoarthritis (OA) is composed of abundant undermineralized osteoid matrix. The aim of this study was to investigate the mechanisms responsible for this abnormal matrix, using in vitro OA subchondral osteoblasts. METHODS: Primary normal and OA osteoblasts were prepared from tibial plateaus. Phenotype was determined by alkaline phosphatase activity, and osteocalcin, osteopontin, prostaglandin E2 (PGE2), and transforming growth factor beta1 (TGFbeta1) were assessed by enzyme-linked immunosorbent assay. Expression of COL1A1 and COL1A2 was determined by real-time polymerase chain reaction. The production of type I collagen was determined by the release of its C-terminal propeptide and Western blot analysis. In vitro mineralization was evaluated by alizarin red staining. Inhibition of TGFbeta1 expression was performed using a small interfering RNA technique. RESULTS: Mineralization of OA osteoblasts was reduced compared with mineralization of normal osteoblasts, even in the presence of bone morphogenetic protein 2 (BMP-2). Alkaline phosphatase and osteocalcin levels were elevated in OA osteoblasts compared with normal osteoblasts, whereas osteopontin levels were similar. The COL1A1-to-COL1A2 messenger RNA ratio was 3-fold higher in OA osteoblasts compared with normal osteoblasts, and the production of collagen by OA osteoblasts was increased. Because TGFbeta1 inhibits BMP-2-dependent mineralization, and because TGFbeta1 levels are approximately 4-fold higher in OA osteoblasts than in normal osteoblasts, inhibiting TGFbeta1 levels in OA osteoblasts corrected the abnormal COL1A1-to-COL1A2 ratio and increased alizarin red staining. CONCLUSION: Elevated TGFbeta1 levels in OA osteoblasts are responsible, in part, for the abnormal ratio of COL1A1 to COL1A2 and for the abnormal production of mature type I collagen. This abnormal COL1A1-to-COL1A2 ratio generates a matrix that blunts mineralization in OA osteoblasts.

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-1 beta (100 pg/ml), TNF-alpha (5 ng/ml), TGF-beta1 (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-1 beta, TNF-alpha 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.

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

OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

An active role for soluble and membrane intercellular adhesion molecule-1 in osteoclast activity in vitro.

In osteoclastogenesis, the intercellular adhesion molecule (ICAM)-1 provides a high-affinity adhesion between the osteoblast and the osteoclast precursor, thereby facilitating the interaction between receptor activator nuclear factor kappaB ligand (RANKL) and its receptor RANK. However, the role of soluble ICAM (sICAM) in that process remains obscure. Therefore, the purpose of this study was to determine whether sICAM and ICAM-1 play an active role in the formation and maturation of osteoclasts. Monocytes isolated from healthy donors and cultured alone or with human osteoblast were stimulated with macrophage colony-stimulating factor, sRANKL, ICAM-1 monoclonal antibody (mAb), leucocyte function antigen (LFA)-1 mAb, and/or sICAM to produce mature osteoclasts. Release of TRAP 5b and resorption area were analyzed as markers of osteoclast formation and function, respectively. The effect of ICAM-1 and sICAM stimulation on apoptosis, cathepsin K, alphavbeta3, collagen-1, and on RANKL/osteoprotegerin (OPG)/RANK expression was evaluated. sICAM did not modify the release of TRAP 5b from osteoclast precursors in both mono and co-culture, but induced a significant increase in resorption area in both culture systems, as well as a positive effect on cathepsin K and alphavbeta3 protein expression. Cross-linking ICAM-1 on osteoblast resulted in increased RANKL mRNA and caspase-3 protein expression, decreased collagen-1 mRNA expression, and decreased osteoblast survival. Stimulation of preosteoclast with sICAM produced a significant increase in preosteoclast survival and a decrease in caspase-3 expression. These results indicate that ICAM-1 and sICAM have a dual effect on bone homeostasis, increasing osteoclast activity while lowering osteoblast anabolic activity.

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 PGE2 [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 D3 (50 nM), IL-1beta (100 pg/ml), TNF-alpha (5 ng/ml), PGE2 (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.