Abstract
BackgroundLubricin/proteoglycan-4 (PRG4) is a mucinous glycoprotein secreted by synovial fibroblasts and superficial zone chondrocytes. PRG4 has a homeostatic multifaceted role in the joint. PRG4 intra-articular treatment retards progression of cartilage degeneration in pre-clinical posttraumatic osteoarthritis models. The objective of this study is to evaluate the binding of recombinant human PRG4 (rhPRG4) and native human PRG4 (nhPRG4) to toll-like receptors 2 and 4 (TLR2 and TLR4) and whether this interaction underpins a PRG4 anti-inflammatory role in synovial fluid (SF) from patients with osteoarthritis (OA) and rheumatoid arthritis (RA).MethodsrhPRG4 and nhPRG4 binding to TLR2 and TLR4 was evaluated using a direct enzyme linked immunosorbent assay (ELISA). Association of rhPRG4 with TLR2 and TLR4 overexpressing human embryonic kidney (HEK) cells was studied by flow cytometry. Activation of TLR2 and TLR4 on HEK cells by agonists Pam3CSK4 and lipopolysaccharide (LPS) was studied in the absence or presence of nhPRG4 at 50, 100 and 150 μg/ml. Activation of TLR2 and TLR4 by OA SF and RA SF and the effect of nhPRG4 SF treatment on receptor activation was assessed. PRG4 was immunoprecipitated from pooled OA and RA SF. TLR2 and TLR4 activation by pooled OA and RA SF with or without PRG4 immunoprecipitation was compared.ResultsrhPRG4 and nhPRG4 exhibited concentration-dependent binding to TLR2 and TLR4. rhPRG4 associated with TLR2- and TLR4-HEK cells in a time-dependent manner. Co-incubation of nhPRG4 (50, 100 and 150 μg/ml) and Pam3CSK4 or LPS reduced TLR2 or TLR4 activation compared to Pam3CSK4 or LPS alone (p <0.05). OA SF and RA SF activated TLR2 and TLR4 and nhPRG4 treatment reduced SF-induced receptor activation (p <0.001). PRG4 depletion by immunoprecipitation significantly increased TLR2 activation by OA SF and RA SF (p <0.001).ConclusionPRG4 binds to TLR2 and TLR4 and this binding mediates a novel anti-inflammatory role for PRG4.
Highlights
Lubricin/proteoglycan-4 (PRG4) is a mucinous glycoprotein secreted by synovial fibroblasts and superficial zone chondrocytes
Wells that were coated with toll-like receptor 2 (TLR2) or toll-like receptor 4 (TLR4) or corresponding uncoated control wells were blocked with 2 % bovine serum albumin (BSA; 300 μL per well) for 2 h at room temperature. recombinant human PRG4 (rhPRG4) or native human PRG4 (nhPRG4) were added at 50, 10, 1, and 0.1 μg/mL (100 μL per well) and incubated for 1 h at room temperature. rhPRG4 is a full-length product produced by CHO-M cells (Lubris, Framingham, MA, USA) [21]. nhPRG4 is purified from culture supernatants of human fibroblast-like synoviocytes (FLS) as previously described [13]. rhPRG4 and nhPRG4 have similar immunoreactivity towards anti
Binding of rhPRG4 and nhPRG4 to immobilized TLR2 and TLR4 The concentration-dependent binding of rhPRG4 and nhPRG4 to TLR2 is shown in Fig. 1a. rhPRG4 and nhPRG4 concentrations are reported in μg/Ml and PRG4 in pmoles, based on a predicted molecular weight of 240 kDa [18]. rhPRG4 (1, 10, and 50 μg/mL), corresponding to 0.4, 4 and
Summary
Lubricin/proteoglycan-4 (PRG4) is a mucinous glycoprotein secreted by synovial fibroblasts and superficial zone chondrocytes. The objective of this study is to evaluate the binding of recombinant human PRG4 (rhPRG4) and native human PRG4 (nhPRG4) to toll-like receptors 2 and 4 (TLR2 and TLR4) and whether this interaction underpins a PRG4 anti-inflammatory role in synovial fluid (SF) from patients with osteoarthritis (OA) and rheumatoid arthritis (RA). Lubricin/proteoglycan-4 (PRG4) is a glycoprotein secreted from synovial fibroblasts and superficial zone chondrocytes that has a multifaceted function including boundary lubrication, resulting in lowering of friction between apposed cartilage surfaces [1,2,3,4,5,6,7,8,9]. PRG4 from RA SF binds L-selectin and coats the surface of polymorphonuclear cells (PMNs) [19, 20]
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