Abstract
BackgroundTNF-α-stimulated gene 6 (TSG-6) protein, a TNF-α-responsive hyaladherin, possesses enzymatic activity that can catalyze covalent crosslinks of the polysaccharide hyaluronic acid (HA) to another protein to form heavy chain-hyaluronic acid (HC-HA) complexes in pathological conditions such as osteoarthritis (OA). Here, we examined HA synthase and inflammatory gene expression; synovial fluid HA, TNF-α, and viscosity; and TSG-6-mediated HC-HA complex formation in an equine OA model. The objectives of this study were to (1) evaluate the TNF-α-TSG-6-HC-HA signaling pathway across multiple joint tissues, including synovial membrane, cartilage, and synovial fluid, and (2) determine the impact of OA on synovial fluid composition and biophysical properties.MethodsHA and inflammatory cytokine concentrations (TNF-α, IL-1β, CCL2, 3, 5, and 11) were analyzed in synovial fluid from 63 OA and 25 control joints, and HA synthase (HAS1-3), TSG-6, and hyaluronan-degrading enzyme (HYAL2, HEXA) gene expression was measured in synovial membrane and cartilage. HA molecular weight (MW) distributions were determined using agarose gel electrophoresis and solid-state nanopore measurements, and HC-HA complex formation was detected via immunoblotting and immunofluorescence. SEC-MALS was used to evaluate TSG-6-mediated HA crosslinking, and synovial fluid and HA solution viscosities were analyzed using multiple particle-tracking microrheology and microfluidic measurements, respectively.ResultsTNF-α concentrations were greater in OA synovial fluid, and TSG6 expression was upregulated in OA synovial membrane and cartilage. TSG-6-mediated HC-HA complex formation was greater in OA synovial fluid and tissues than controls, and HC-HA was localized to both synovial membrane and superficial zone chondrocytes in OA joints. SEC-MALS demonstrated macromolecular aggregation of low MW HA in the presence of TSG-6 and inter-α-inhibitor with concurrent increases in viscosity.ConclusionsSynovial fluid TNF-α concentrations, synovial membrane and cartilage TSG6 gene expression, and HC-HA complex formation were increased in equine OA. Despite the ability of TSG-6 to induce macromolecular aggregation of low MW HA with resultant increases in the viscosity of low MW HA solutions in vitro, HA concentration was the primary determinant of synovial fluid viscosity rather than HA MW or HC-HA crosslinking. The TNF-α-TSG-6-HC-HA pathway may represent a potential therapeutic target in OA.
Highlights
tumor necrosis factor-α (TNF-α)-stimulated gene 6 (TSG-6) protein, a TNF-α-responsive hyaladherin, possesses enzymatic activity that can catalyze covalent crosslinks of the polysaccharide hyaluronic acid (HA) to another protein to form heavy chain-hyaluronic acid (HC-HA) complexes in pathological conditions such as osteoarthritis (OA)
Synovial fluid TNF-α concentrations, synovial membrane and cartilage TNF-αstimulated gene 6 protein (TSG6) gene expression, and HCHA complex formation were increased in equine OA
Despite the ability of TNF-α-stimulated gene 6 (TSG-6) to induce macromolecular aggregation of low molecular weight (MW) HA with resultant increases in the viscosity of low MW HA solutions in vitro, HA concentration was the primary determinant of synovial fluid viscosity rather than HA MW or heavy chains (HC)-HA crosslinking
Summary
TNF-α-stimulated gene 6 (TSG-6) protein, a TNF-α-responsive hyaladherin, possesses enzymatic activity that can catalyze covalent crosslinks of the polysaccharide hyaluronic acid (HA) to another protein to form heavy chain-hyaluronic acid (HC-HA) complexes in pathological conditions such as osteoarthritis (OA). Synovial fluid TNF-α has been associated with radiographic OA progression in human knee OA [5], and increased synovial fluid TNF-α has been reported in both naturally occurring equine carpal OA [6] and in an experimentally induced equine cartilage carpal defect model [7]. Besides variation in HA MW, there are few postsynthetic modifications that occur to HA One of these unique modifications is the covalent crosslinking of HA monomers by the TNF-α-inducible protein TNF-α-stimulated gene 6 protein (TSG-6) [14, 15]. TSG-6 can bind non-covalently and reversibly to HA through its Link domain, resulting in the crosslinking of HA chains [14, 17]; TSG-6 can induce a covalent modification of HA by facilitating the transfer of heavy chains (HC) via a trans-esterification reaction [18] from the proteoglycan inter-alpha trypsin inhibitor (IαI), a protein found in serum and pathological synovial fluid [19]
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