WNT5A is a novel regulator of cytokine and chemokine expression in bone marrow stromal cells

Rheumatoid arthritis (RA) is a chronic inflammatory disease that negatively affects bone mass by enhancing cytokine-driven osteoclastogenesis and suppressing osteoblast functions. Recently, Dickkopf-1, an inhibitor of WNT signaling, has been found to be induced by TNFa and suppress bone formation in RA. Here, we aimed to identify other members of the WNT signaling pathway that are regulated by inflammatory stimuli and might affect bone remodelling. Of several WNT ligands investigated, only WNT5A was consistently induced up to 3-fold at mRNA and protein level in human bone marrow stromal cells (BMSCs) after stimulation with 1 μg/ml lipopolysaccharide (LPS). In addition, an increased expression of WNT5A was found in the synovial tissue of 10 weeks-old TNFtransgenic mice compared to wild-type controls as assessed by immunohistochemistry. The LPS-induced WNT5A expression in BMSCs was completely blocked when cells were pretreated with an antibody against toll-like receptor 4 (10 μg/ml) or with specific inhibitors of the NFkB (BAY-7011, 1 μM) and MAPK (UO126, 10 μM) pathways. Of note, the WNT5A receptor ROR2 was up-regulated 6-fold by LPS and signalled through similar pathways. BMSCs respond to LPS stimulation by highly up-regulating the expression of several cytokines and chemokines including IL-1b, IL-6, TNFa, CCL2, CCL5, and CXCL12. Knock-down of WNT5A using siRNA potently suppressed the basal and LPS-induced expression of IL-1b, IL-6, CCL2, and CCL5, but not that of TNFa or CXCL12. In line with these data, BMSCs treated with 500 ng/ml human recombinant WNT5A for 48 h induced the expression of IL-1b, IL-6, CCL2, and CCL5. To gain more insight into the underlying signaling pathways, cells were pretreated for 1 h with pathway blockers (NFkB: BAY-7011, 1 μM; MAPK: UO126, 10 μM; PI-3 K: LY-294002, 50 μM, NFAT: VIVIT, 1 μM). Interestingly, while WNT5A-induced IL-1b expression was solely regulated by the MAPK pathway, the induction of IL-6 expression was dependent on the NFkB and MAPK pathways. The chemokines CCL2 and CCL5 were regulated by multiple pathways including NFkB, MAPK, and Ca/NFAT. Of note, WNT5A also induced its own expression, which was dependent on the Ca/NFAT pathway. Taken together, WNT5A is induced under inflammatory conditions in BMSCs and controls the expression of cytokines and chemokines. The relevance of this regulatory circuit needs to be further explored in experimental arthritis models. This article is part of a Special Issue entitled ECTS 2011. Disclosure of interest: None declared. doi:10.1016/j.bone.2011.03.149 OPB12 (recipient of a 2011 ECTS/IBMS New Investigator Award) Localised thickening of subchondral bone in a model of loading-induced articular cartilage lesions: Product of cartilage damage or not? B. Poulet ⁎, L. Saxon, O. Barker, R. de Souza, A.A. Pitsillides Veterinary Basic Sciences, The Royal Veterinary College, London, UK Abstract: Changes in subchondral bone (SCB) thickness are a well known characteristic of osteoarthritis (OA). Increases have been described in human OA and in animal OA Changes in subchondral bone (SCB) thickness are a well known characteristic of osteoarthritis (OA). Increases have been described in human OA and in animal OA models. Despite this, there remains controversy regarding the aetiology of SCB changes: do they precede or follow articular cartilage degeneration which defines OA? The aim of the current work was to analyse SCB changes in a mouse model of knee joint loading, in which localised articular cartilage lesions are generated in only the lateral femur where it becomes closely opposed to the lateral tibia during loading. These joints can subsequently be subjected to solely habitual use or to further repetitive loading and the SCB changes examined. Right knees of 8 week-old CBA mice was loaded 3 times each week for 2 (± 3 weeks of normal use with no loads applied) or 5 weeks at a magnitude of 9 N as described previously (1,2). Micro-CT scanning was performed on left (non-loaded) and right (loaded) knee joints and SCB thickness measured in the posterior half of each condyle using CTAn software, and in 0.1 mm sections within this posterior half. Paired t-test was used for statistical analysis. Examination of the lateral femur, inwhich localised cartilage lesions develop in response to loading, revealed intimately associated increases in SCB thickness, with no changes in regions remote from the articular cartilage lesion. Extended loading for 5 weeks gave rise to the most obvious thickening of SCB. In addition, there were increases in SCB thickness in the most posterior region of the lateral tibia. Although no cartilage lesions were observed in this condyle, this region was directly opposed to the area of contact between the femur and tibia during loading. This thickening was again most prominent in joints loaded for 5 weeks. In conclusion, we have described very focal thickening of the SCB associated with loadinduced articular cartilage lesion formation in the lateral femur, as well as thickening of the SCB in areas exposed to direct mechanical loads (without cartilage lesions) in the lateral tibia. This indicates that SCB changes can be induced by loading independently of articular cartilage lesions, and that altered load distribution associated with the presence of articular cartilage lesions acts to enhance load-induced SCB thickening. These data suggest that SCB thickening is due to altered mechanical loads in OA joints. 1. de Souza et al (2005) Bone; 37:810–818. 2. Poulet et al. Arth Rheum ahead of print. This article is part of a Special Issue entitled ECTS 2011. Disclosure of interest: None declared. doi:10.1016/j.bone.2011.03.150 OPB13 Bag-1 haploinsufficiency affects cartilage development and osteogenic differentiation R.S. Tare ⁎, P.A. Townsend , G.K. Packham , R.O. Oreffo a a Bone and Joint Research Group, UK b Human Genetics, UK c Cancer Sciences, University of Southampton, Southampton, UK Abstract: The Bcl-2-associated athanogene-1 (Bag-1) co-chaperone is expressed by chondrocytes and osteoblasts, and functions as a transcriptional regulator mediating The Bcl-2-associated athanogene-1 (Bag-1) co-chaperone is expressed by chondrocytes and osteoblasts, and functions as a transcriptional regulator mediating chondrocyte differentiation and apoptosis in vitro. By utilising cell populations from a mouse model of Bag-1 haploinsufficiency (i.e. one Bag-1 null allele and one normal Bag-1 allele), this study aims to define effects of Bag-1 heterozygosity on formation of cartilage templates and osteoblast development. Micromass cultures of E11.5 Bag-1 and Bag-1+/− mouse embryonic fibroblasts (MEFs), differentiated in presence of BMP-2 (100 ng/ml), ascorbate and beta-glycerophosphate, were utilised to model developmental stages in the formation of cartilaginous primordia in vivo. Cultureswere analysed at days 7,14, 21, 28, 35 and 42 for expression of Bag-1 anddevelopmentof cartilage nodules. Bone marrow cells (BMCs) from 14-week Bag-1 and Bag-1+/− mice were cultured for 21 days inmedia supplementedwith either 10 nMDexamethasone, 25 nMCalcitriol or 100 ng/ml BMP-2, to analyse their osteogenic response. During the initial 14-day period, significantly high Bag-1 expression was observed in micromass cultures of Bag-1 MEFs compared to Bag-1+/− MEFs. The 14-day period of micromass culture is comparable to in vivo murine embryonic limb development at E10.5 and E11.5, associated with robust Bag-1 expression and formation of vital mesenchymal condensations prefiguring future skeletal elements. Significantly, days 7 and 14 of Bag-1 MEF micromass cultures were characterised by the presence of cellular condensations and mineralised cartilaginous nodules were observed by day 42 of culture. Bag-1 haploinsufficiency compromised the ability of Bag-1+/− MEFs to undergo condensations and organise into defined cartilaginous nodules by day 42 of micromass culture. Moreover, morphologically distinct hypertrophic chondrocytes (characterised by increase in cell volume and hence, enlarged cell size) in lacunae were absent in day 42 Bag-1+/− micromass cultures.