P. Reboul

Oxidative stress-induced expression of HSP70 contributes to the inhibitory effect of 15d-PGJ2 on inducible prostaglandin pathway in chondrocytes

The inhibitory effect of 15-deoxy-Δ(12,14)-prostaglandin J2 (15d-PGJ2) on proinflammatory gene expression has been extensively documented and frequently ascribed to its ability to prevent NF-κB pathway activation. We and others have previously demonstrated that it was frequently independent of the peroxisome proliferator activated receptor (PPAR)γ activation. Here, we provide evidence that induction of intracellular heat shock protein (HSP)70 by oxidative stress is an additional regulatory loop supporting the anti-inflammatory effect of 15d-PGJ2 in chondrocytes. Using real-time quantitative PCR and Western blotting, we showed that 15d-PGJ2 stimulated HSP70, but not HSP27 expression while increasing oxidative stress as measured by spectrofluorimetry and confocal spectral imaging. Using N-acetylcysteine (NAC) as an antioxidant, we demonstrated further that oxidative stress was thoroughly responsible for the increased expression of HSP70. Finally, using an HSP70 antisense strategy, we showed that the inhibitory effect of 15d-PGJ2 on IL-1-induced activation of the NF-κB pathway, COX-2 and mPGES-1 expression, and PGE2 synthesis was partly supported by HSP70. These data provide a new anti-inflammatory mechanism to support the PPARγ-independent effect of 15d-PGJ2 in chondrocyte and suggest a possible feedback regulatory loop between oxidative stress and inflammation via intracellular HSP70 up-regulation. This cross talk is consistent with 15d-PGJ2 as a putative negative regulator of the inflammatory reaction.

Calcium Input Potentiates the Transforming Growth Factor (TGF)-β1-dependent Signaling to Promote the Export of Inorganic Pyrophosphate by Articular Chondrocyte

Transforming growth factor (TGF)-β1 stimulates extracellular PPi (ePPi) generation and promotes chondrocalcinosis, which also occurs secondary to hyperparathyroidism-induced hypercalcemia. We previously demonstrated that ANK was up-regulated by TGF-β1 activation of ERK1/2 and Ca2+-dependent protein kinase C (PKCα). Thus, we investigated mechanisms by which calcium could affect ePPi metabolism, especially its main regulating proteins ANK and PC-1 (plasma cell membrane glycoprotein-1). We stimulated articular chondrocytes with TGF-β1 under extracellular (eCa2+) or cytosolic Ca2+ (cCa2+) modulations. We studied ANK, PC-1 expression (quantitative RT-PCR, Western blotting), ePPi levels (radiometric assay), and cCa2+ input (fluorescent probe). Voltage-operated Ca2+-channels (VOC) and signaling pathways involved were investigated with selective inhibitors. Finally, Ank promoter activity was evaluated (gene reporter). TGF-β1 elevated cCa2+ and ePPi levels (by up-regulating Ank and PC-1 mRNA/proteins) in an eCa2+ dose-dependent manner. TGF-β1 effects were suppressed by cCa2+ chelation or L- and T-VOC blockade while being mostly reproduced by ionomycin. In the same experimental conditions, the activation of Ras, the phosphorylation of ERK1/2 and PKCα, and the stimulation of Ank promoter activity were affected similarly. Activation of SP1 (specific protein 1) and ELK-1 (Ets-like protein-1) transcription factors supported the regulatory role of Ca2+. SP1 or ELK-1 overexpression or blockade experiments demonstrated a major contribution of ELK-1, which acted synergistically with SP1 to activate Ank promoter in response to TGF-β1. TGF-β1 promotes input of eCa2+ through opening of L- and T-VOCs, to potentiate ERK1/2 and PKCα signaling cascades, resulting in an enhanced activation of Ank promoter and ePPi production in chondrocyte.

Fibroblast Growth Factor 23 drives MMP13 expression in human osteoarthritic chondrocytes in a Klotho-independent manner

OBJECTIVE Fibroblast Growth Factor 23 (FGF23) may represent an attractive candidate that could participate to the osteoarthritic (OA)-induced phenotype switch of chondrocytes. To address this hypothesis, we investigated the expression of FGF23, its receptors (FGFRs) and co-receptor (Klotho) in human cartilage and studied the effects of rhFGF23 on OA chondrocytes. METHOD Gene expression or protein levels were analysed by RT-PCR and immunohistochemistry. Collagenase 3 (MMP13) activity was measured by a fluorescent assay. MAPK signalling pathways were investigated by phosphoprotein array, immunoblotting and the use of selective inhibitors. RNA silencing was performed to confirm the respective contribution of FGFR1 and Klotho. RESULTS We showed that the expression of FGF23, FGFR1 and Klotho was up-regulated at both mRNA and protein levels in OA chondrocytes when compared to healthy ones. These overexpressions were markedly elevated in the damaged regions of OA cartilage. When stimulated with rhFGF23, OA chondrocytes displayed an extended expression of FGF23 and of markers of hypertrophy such as MMP13, COL10A1, and VEGF. We demonstrated that FGF23 auto-stimulation was both FGFR1-and Klotho-dependent, whereas the expression of markers of hypertrophy was mainly dependent on FGFR1 alone. Finally, we showed that FGF23-induced MMP13 expression was strongly regulated by the MEK/ERK cascade and to a lesser extent, by the PI-3K/AKT pathway. CONCLUSION These results demonstrate that FGF23 sustains differentiation of OA chondrocytes in a Klotho-independent manner.

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.

Basic science of osteoarthritis.

Osteoarthritis (OA) is a prevalent, disabling disorder of the joints that affects a large population worldwide and for which there is no definitive cure. This review provides critical insights into the basic knowledge on OA that may lead to innovative end efficient new therapeutic regimens. While degradation of the articular cartilage is the hallmark of OA, with altered interactions between chondrocytes and compounds of the extracellular matrix, the subchondral bone has been also described as a key component of the disease, involving specific pathomechanisms controlling its initiation and progression. The identification of such events (and thus of possible targets for therapy) has been made possible by the availability of a number of animal models that aim at reproducing the human pathology, in particular large models of high tibial osteotomy (HTO). From a therapeutic point of view, mesenchymal stem cells (MSCs) represent a promising option for the treatment of OA and may be used concomitantly with functional substitutes integrating scaffolds and drugs/growth factors in tissue engineering setups. Altogether, these advances in the fundamental and experimental knowledge on OA may allow for the generation of improved, adapted therapeutic regimens to treat human OA.

Elevated hepatocyte growth factor levels in osteoarthritis osteoblasts contribute to their altered response to bone morphogenetic protein-2 and reduced mineralization capacity

PURPOSE Clinical and in vitro studies suggest that subchondral bone sclerosis due to abnormal osteoblasts is involved in the progression of osteoarthritis (OA). Human osteoblasts isolated from sclerotic subchondral OA bone tissue show an altered phenotype, a decreased canonical Wnt/ß-catenin pathway, and a reduced mineralization in vitro as well as in vivo. These alterations were linked with an abnormal response to BMP-2. OA osteoblasts release factors such as the hepatocyte growth factor (HGF) that contribute to cartilage loss whereas chondrocytes do not express HGF. HGF can stimulate BMP-2 expression in human osteoblasts, however, the role of HGF and its effect in OA osteoblasts remains unknown. Here we investigated whether elevated endogenous HGF levels in OA osteoblasts are responsible for their altered response to BMP-2. METHODS We prepared primary human subchondral osteoblasts using the sclerotic medial portion of the tibial plateaus of OA patients undergoing total knee arthroplasty, or from tibial plateaus of normal individuals obtained at autopsy. The expression of HGF was evaluated by qRT-PCR and the protein production by western blot analysis. HGF expression was reduced with siRNA technique whereas its activity was inhibited using the selective inhibitor PHA665752. Alkaline phosphatase activity (ALPase) and osteocalcin release were measured by substrate hydrolysis and EIA respectively. Canonical Wnt/β-catenin signaling (cWnt) was evaluated both by target gene expression using the TOPflash TCF/lef luciferase reporter assay and western blot analysis of β-catenin levels in response to Wnt3a stimulation. Mineralization in response to BMP-2 was evaluated by alizarin red staining. RESULTS The expression of HGF was increased in OA osteoblasts compared to normal osteoblasts and was maintained during their in vitro differentiation. OA osteoblasts released more HGF than normal osteoblasts as assessed by western blot analysis. HGF stimulated the expression of TGF-β1. BMP-2 dose-dependently (1 to 100 ng/ml) stimulated both ALPase and osteocalcin in normal osteoblasts whereas, it inhibited them in OA osteoblasts. HGF-siRNA treatments reversed this response in OA osteoblasts and restored the BMP-2 response. cWnt is reduced in OA osteoblasts compared to normal, and HGF-siRNA treatments increased cWnt in OA osteoblasts almost to normal. Smad1/5/8 phosphorylation in response to BMP-2, which is reduced in OA osteoblasts, was corrected when these cells were treated with PHA665752. The BMP-2-dependent mineralization of OA osteoblasts, which is also reduced compared to normal, was only partially restored by PHA665752 treatment whereas 28 days treatment with HGF reduced the mineralization of normal osteoblasts. CONCLUSION OA osteoblasts expressed more HGF than normal osteoblasts. Increased endogenous HGF production in OA osteoblasts stimulated the expression of TGF-β1 and reduced their response to BMP-2. Inhibiting HGF expression or HGF signaling restored the response to BMP-2 and Smad1/5/8 signaling. In addition, decreased HGF signaling partly corrects the abnormal mineralization of OA osteoblasts while increased HGF prevents the normal mineralization of normal osteoblasts. In summary, we hypothesize that sustained elevated HGF levels in OA osteoblasts drive their abnormal phenotype and is implicated in OA pathophysiology.

Galectin-3: A key player in arthritis

Arthritis is more and more considered as the leading reason for the disability in the world, particularly regarding its main entities, rheumatoid arthritis and osteoarthritis. The common feature of arthritis is inflammation, which is mainly supported by synovitis (synovial inflammation), although the immune system plays a primary role in rheumatoid arthritis and a secondary one in osteoarthritis. During the inflammatory phase of arthritis, many pro-inflammatory cytokines and mediators are secreted by infiltrating immune and resident joint cells, which are responsible for cartilage degradation and excessive bone remodeling. Amongst them, a β-galactoside-binding lectin, galectin-3, has been reported to be highly expressed and secreted by inflamed synovium of rheumatoid arthritis and osteoarthritis patients. Furthermore, galectin-3 has been demonstrated to induce joint swelling and osteoarthritis-like lesions after intra-articular injection in laboratory animals. However, the mechanisms underlying its pathophysiological role in arthritis have not been fully elucidated. This review deals with the characterization of arthritis features and galectin-3 and summarizes our current knowledge of the contribution of galectin-3 to joint tissue lesions in arthritis.

CRDSAT Generated by pCARGHO: A New Efficient Lectin-Based Affinity Tag Method for Safe, Simple, and Low-Cost Protein Purification

Purification of recombinant proteins remains a bottleneck for downstream processing. The authors engineered a new galectin 3 truncated form (CRDSAT ), functionally and structurally characterized, with preserved solubility and lectinic activity. Taking advantage of these properties, the authors designed an expression vector (pCARGHO), suitable for CRDSAT -tagged protein expression in prokaryotes. CRDSAT binds to lactose-Sepharose with a high specificity and facilitates solubilization of fusion proteins. This tag is structurally stable and can be easily removed from fusion proteins using TEV protease. Furthermore, due to their basic isoelectric point (pI), CRDSAT , and TEV are efficiently eliminated using cationic exchange chromatography. When pI of the protein of interest (POI) and CRDSAT are close, other chromatographic methods are successfully tested. Using CRDSAT tag, the authors purified several proteins from prokaryote and eukaryote origin and demonstrated as examples, the preservation of both Escherichia coli Thioredoxin 1 and human CDC25Bcd activities. Overall, yields of proteins obtained after tag removal are about 5-50 mg per litre of bacterial culture. Our purification method displays various advantages described herein that may greatly interest academic laboratories, biotechnology, and pharmaceutical companies.

FRI0017 Pharmacological Blockade of CCR3 Reduces Collagen-Induced Arthritis Severity

Background Chemokines are key pathophysiological mediators in the early phase of arthritis. Eotaxin-1 (Paquet and al., 2012) and its receptor, CCR-3 (CC Chemokine Receptor 3) (Haas and al., 2005), were previously shown to be highly expressed in the course of experimental arthritis. In human, CCR-3 positive monocytes are more abundant in the synovial fluid of arthritic patients than in healthy ones (Katschke and al., 2001). Moreover, RANTES, another chemokine with pathophysiological relevance to arthritis, is also a ligand of CCR-3, further suggesting that CCR-3 could be a potential therapeutic target for joint diseases. Objectives The main obective of this project was to study consequences of CCR-3 inactivation by a pharmacological antagonist in collagen-induced arthritis. Methods CIA was induced in 20 DBA/1J mice by intradermal injection of 100mg of bovine type II collagen in CFA at the basis of the tail, with a booster injection of 50mg by day 21. From day 15, mice were administered daily with antagonist of CCR-3 at a concentration of 10 mg/kg/day (i.p). Mice were regularly weighed and evaluated for the severity of arthritis with an arthritic score and by measuring hindpaw oedema by plethysmography. Mice were sacrificed at day 41, and histological analysis was performed on ankle joints after HES and safranin-O fast green staining. At that time, serum levels of IL-6 and IL-17F were measured by the multiplex technology. Results The clinical parameters showed that mice treated with a specific antagonist of CCR-3 develop less severe arthritis (respective clinical score 3.89 ± 1.25 vs 8.57 ± 1.63). Histological analyzes indicated that antagonist reduced the intensity of inflammatory process and limits cartilage degradation in arthritic joints. Blood level of IL-6 and IL-17F cytokines were reduced in mice treated with antagonist of CCR-3 compared to untreated arthritic mice. Conclusions Thus, our study shows that CCR-3 has an important role in arthritis development and designates it as a potential therapeutic target. References Haas C.S., Martinez R.J., Attia N., Kenneth H., Campbell P.L., Koch A.E., Chemokine receptor expression in rat adjuvant-induced arthritis. Arthritis and Rheumatism, 2005, 52, 3718–1730. Katschke K.J., Jr., Rottman J.B., Ruth J.H., Qin S., Wu L., LaRosa G., Ponath P., Park C.C., Pope R.M., Koch A.E. Differential expression of chemokine receptors on peripheral blood, synovial fluid, and synovial tissue monocytes/macrophages in rheumatoid arthritis. Arthritis and Rheumatism, 2001, 44: 1022–1032. Paquet J., Goebel, J-C, Delaunay C., Pinzano A., Grossin L., Cournil-Henrionnet C., Gillet P., Netter P., Jouzeau J-Y., Moulin D. Cytokines profiling by multiplex analysis in experimental arthritis: which pathophysiological relevance for articular versus systemic mediators? Arthritis Research and Therapy, 2012, 14: 60–75 Acknowledgement The authors thank fondation Arthritis for granting this study. Disclosure of Interest None declared

SAT0569 Galectin 3 Exerts A Protective Role in A Murine Model of Osteoarthritis

Background Osteoarthritis (OA) is the most common adult joint disease characterized by cartilage destruction, subchondral bone remodelling and mild synovial inflammation. Pathogenesis of OA involves overload, ageing, genetic factors and chondrocyte dysregulation. Galectin 3 (Gal3) expression is increased in OA cartilage and synovial fluid. Gal3 is involved in multiple cellular functions including proliferation, differentiation and cell death. During embryogenesis, the growth plate of Gal3 mutant mice has a reduction of hypertrophic zone and many lacunae due to a loss of chondrocytes. These results suggest a role of Gal3 in chondrocyte dedifferentiation. Objectives To determine the role of Gal3 in osteoarthritis and to assess its functions in chondrocyte biology. Methods In vivo, murine OA was induced in Wild type (WT) 129 and Gal3 mutant mice by partial medial meniscectomy (MNX) of the right knee. A sham operation was performed on the left knee. Animals were sacrificed 4 weeks after surgery. Cartilages lesions were assessed according to OARSI recommendations. Chondrocyte apoptosis was assessed by TUNEL labelling and chondrocyte catabolism by immunostaining for aggrecanases and metalloproteases. Subchondral bone modifications were evaluated by microCT. In vitro, primary murine chondrocytes, isolated from new born WT and Gal3 mutant mice, were cultured and stimulated by inflammatory cytokines (IL-1β and TNF-α). Anabolism, catabolic and differentiation responses were assessed by RT-qPCR Results In vivo, MNX induced in Gal3 mutant mice more severe OA cartilage lesions compared to WT as assessed by OARSI scoring (4.4±1.0 vs 1.3±0.8, p<0.05). Expression of Gal3 decreased in WT cartilages after MNX compared to sham operated cartilage (34.5% ±4.9 vs 53.0% ±2.8; p<0.01). Cartilages lesions were associated with higher ratio of chondrocyte positive-TUNEL staining in Gal3 mutant cartilages compared to WT (17.7% ±6.2 vs 10.6% ±3.3 p<0.02). Similarly, immunostaining for ADAMTS-5 (26.7% ±0.5 vs 16.0% ±1.4, p<0.02) and VDIPEN (52.0% ±4.0 vs 30.0% ±2.0, p<0.05) was increased in Gal3 mutant cartilages. Moreover, collagen type X expression was increased in Gal3 mutant cartilage. No difference was observed in subchondral bone modifications. In vitro, IL-1β stimulation increased ADAMTS-4 (4.6±2.0 vs; 1.6±0.3, p<0.05) and ADAMTS-5 (4.5±2.2 vs 1.8±1.0, p<0.05) mRNA expressions in Gal3 mutant chondrocytes compared to WT chondrocytes. Conclusions Gal3 deletion exacerbated murine OA lesions induced by mechanical stress. OA lesions were associated with increased chondrocyte catabolism, differentiation and death. These results suggested Gal3 had a protective role in OA and chondrocyte homeostasis. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.4750