Stimulating bone formation is an important challenge for bone anabolism in osteoporotic patients or to repair bone defects. The osteogenic properties of matrix glycosaminoglycans (GAGs) have been explored; however, the functions of GAGs at the surface of bone-forming cells are less documented. Syndecan-2 is a membrane heparan sulfate proteoglycan that is associated with osteoblastic differentiation. We used a transgenic mouse model with high syndecan-2 expression in osteoblasts to enrich the bone surface with cellular GAGs. Bone mass was increased in these transgenic mice. Syndecan-2 overexpression reduced the expression of receptor activator of NF-kB ligand (RANKL) in bone marrow cells and strongly inhibited bone resorption. Osteoblast activity was not modified in the transgenic mice, but bone formation was decreased in 4-month-old transgenic mice because of reduced osteoblast number. Increased proteoglycan expression at the bone surface resulted in decreased osteoblastic and osteoclastic precursors in bone marrow. Indeed, syndecan-2 overexpression increased apoptosis of mesenchymal precursors within the bone marrow. However, syndecan-2 specifically promoted the vasculature characterized by high expression of CD31 and Endomucin in 6-week-old transgenic mice, but this was reduced in 12-week-old transgenic mice. Finally, syndecan-2 functions as an inhibitor of Wnt-β-catenin–T-cell factor signaling pathway, activating glycogen synthase kinase 3 and then decreasing the Wnt-dependent production of Wnt ligands and R-spondin. In conclusion, our results show that GAG supply may improve osteogenesis, but also interfere with the crosstalk between the bone surface and marrow cells, altering the supporting function of osteoblasts.
Background
Heparan sulfate (HS) proteoglycans (PG) may be found at the chondrocyte surface and in the pericellular cartilage matrix, and are involved in cell-cell and cell-matrix interactions. An important function of HS chains is to regulate cell fate through specific interactions with heparin-binding proteins (HBP) modulated by their complex sulfation pattern. Osteoarthritis (OA) is a joint disorder characterized by the degradation of articular cartilaginous extracellular matrix. The aim of this study was to investigate HS structure and functions in osteoarthritic cartilages compared to normal cartilages (controls).
Methods
Glycosaminoglycans (GAG) were extracted from human macroscopically normal cartilages (controls, n = 7) and (OA cartilages n = 11). HS were isolated and quantified using the DMMB quantification method. Their structure and functions were then compared using respectively a HPLC analysis and HBP binding tests and their phenotypic effects on murine chondrocytes were studied by RQ-PCR. Statistical analyzes were performed using a one-way ANOVA followed by a Dunnett’s test or a t test for pairwise comparisons.
Results
In OA, HS were characterized by increased sulfation levels compared to controls. Moreover, the capacity of these HS to bind HBP involved in the OA pathophysiological process such as FGF2 and VEGF was reduced. Chondroitin sulfates and keratan sulfates regulated these binding properties. Finally, HS from OA cartilages induced the mRNA levels of catabolic markers such as MMP3, MMP13, and TS4 and inhibited the mRNA levels of anabolic markers such as COL2, ACAN, SOX9, and VEGF in murine articular chondrocytes.
Conclusion
The sulfation of HS chains was increased in OA cartilages with changes in HBP binding properties and biological effects on chondrocyte phenotypes. Thus, modified HS present in altered cartilages could be a novel therapeutic target in OA.
Background:Interleukin-6 (IL-6) plays an important role in osteoarthritis (OA). Transcriptomic analyses (RNAseq) revealed that SerpinA3N, a serine protease inhibitor, is a key target of IL-6 in chondrocyte.Objectives:This study aimed to examine the role of SerpinA3N and Leukocyte Elastase (Elane), a serine protease targeted by SerpinA3N, in cartilage destruction during OA.Methods:The role of SerpinA3N was investigated in the destabilization of medial meniscus (DMM) model of murine OA with 1) mice with conditional inducible knockdown ofSerpina3nin cartilage (Col2CreER;Serpina3nfl/flmice [ΔSerpina3nCol2]) and 2) C57BL/6 wild type (WT) mice treated with intra-articular injection of SerpinA3N (1,5 or 15 nM/week). OA joint lesions were assessed by histology (OARSI and synovitis scores) and micro-CT analysis (osteophyte volume, subchondral bone remodeling).Because serine proteases targeted by SerpinA3N are not produced by murine chondrocytes,Elaneexpression (qRT-PCR) was determined in murine macrophages (Raw) stimulated or not by IL-6 (100 ng/ml). Recombinant SerpinA3N (30 nM) and a specific Elane inhibitor, Sivelestat (100 µg/ml) were used on cartilage explants treated by conditioned medium of macrophages pre-treated or not by IL-6 (CM–IL-6). Cartilage catabolism was determined by histology and matrix metalloproteinase MMP-3 production was evaluated by Western Blot and immunohistochemistry (IHC). Weekly intra-articular injections of Sivelestat (1mM) were performed in the DMM to determine the role of Elane in OA.Results:ΔSerpina3nCol2mice had more severe OA lesions than control littermates 6 weeks after DMM, with greater cartilage damage (mean±SD OARSI score: 5.6±0.4 vs 3.4±0.5, p=0.01), increased synovitis scores (3.0±0.3 vs 1.9±0.3, p=0.03) and bigger osteophytes (7.2±0.8x107 vs 3.8±0.8x107 µ3, p=0.048). Conversely, WT mice treated with intra-articular injections of SerpinA3N 15nM exhibited less severe cartilage loss than mice treated with PBS after DMM (OARSI score: 2.1±0.4 vs 3.9±0.5, p=0.02). Elane mRNA expression was increased in macrophages upon IL-6 stimulation. In cartilage explants, CM–IL-6 activated cartilage catabolism and MMP-3 production, and effect that was blunted by SerpinA3N and Sivelestat. Finally, mice treated with intra-articular injections of Sivelestat had less severe cartilage damage than those treated with PBS after DMM (OARSI score: 3.3±0.47 vs 5.8±0.53, p=0.0046).Conclusion:SerpinA3N protects against experimental OA via the inhibition of Elane, a pro-catabolic serine protease produced by macrophages. This results highlight the crosstalk between cartilage and surrounding macrophages and open up new therapeutic perspectives.Acknowledgments:This work has been supported by French Society of Rheumatology and ART Viggo association.Disclosure of Interests:None declared
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