Synovial fluid glycosaminoglycans signature involved 3-O-sulfated heparan sulfate modulated by TGF-β pathway in OA

Abstract

Osteoarthritis (OA) involved extracellular matrix (ECM) remodeling of cartilage associated to inflammatory activation of fibroblastic like synoviocytes (FLS) from the synovium, but the molecular crosstalk between these two compartments remains poorly understood. Among Glycosaminoglycans (GAG) compound from ECM, heparan sulfate (HS) specifically regulate the bioavailability and activity of Heparin Binding Proteins (HBP), including cytokines and growth factors, such as TGF-{beta}, whose aberrant bioavailability in the synovial fluid (SF) could drive the catabolic process. Here, we investigated the composition, sulfation patterns and biological activities of GAG from OA SF that are currently uncharacterized and explored the regulation of 3-O-sulfated HS in TGF-{beta} mediated response in FLS. Total GAG from 52 OA-SF SF were extracted and quantitatively characterized by DMMB assay and HPLC. They are functional since they are able to bind several HBP with pleiotropic inflammatory and angiogenic properties according to ELISA competitive binding assay. They were able also to transactivate HBP signaling in cellular tests in-vitro, since they potentiated proliferative properties on fibroblastic and endothelial cells, and induced altered metabolic and inflammatory markers expressions in murine chondrocytes and human FLS. Although HS represented a minor fraction of SF-GAG, they contained a rare tri-sulfated disaccharidic fraction distinct from GAG from cartilage. Same GAG characterization was performed on FLS expanded from SF and SM samples. FLS exhibited also a rare sulfation pattern of 3-O-Sulfated HS on their surface analyzed by immunofluorescent staining, associated with the expression of 4 isoforms of 3-O-sulfotransferase (HS3ST) identified by RT-qPCR. TGF-{beta}1 treatment of FLS culture increases HS3ST expression profiles and HS sulfation pattern through the SMAD2/3 pathway, as observed by RT-qPCR, IF and western blot respectively. Finally, combined siRNA mediated silencing of these HS3ST enzymes affects FLS inflammatory secretome, according to proteome profiler cytokine arrays. among pro-inflammatory CXCL10 and CCL5 chemokines. Altogether, these data identify OA SF-GAG as new effective modulator of joint cell responses and reveal for the first time 3-O-sulfated HS and HS3ST patterns of expression in OA FLS associated with TGF-{beta}-Smad2/3-signaling pathway. Such specific HS fractions may contribute to the regulation of synovial inflammatory secretome, according to HBP partners that need to be further identified. However, this new glycanic mechanistic pathway sustaines the involvement of HS in OA progression and subsequently their interest as glycanic therapeutic targets.