Lubricin/Proteoglycan 4 Binding to CD44 Receptor: A Mechanism of the Suppression of Proinflammatory Cytokine–Induced Synoviocyte Proliferation by Lubricin

Objective To evaluate the binding of recombinant human proteoglycan 4 (rhPRG4) to CD44 receptor and its consequences on cytokine‐induced synoviocyte proliferation. Methods The binding of rhPRG4 to CD44 and competition with high molecular weight (HMW) hyaluronic acid (HA) was evaluated using a direct enzyme‐linked immunosorbent assay (ELISA) and surface plasmon resonance. Sialidase A and O‐glycosidase digestion of rhPRG4 was performed, and CD44 binding was evaluated using ELISA. Rheumatoid arthritis (RA) fibroblast‐like synoviocytes (FLS) were stimulated with interleukin‐1β (IL‐1β) or tumor necrosis factor α (TNFα) for 48 hours in the presence or absence of rhPRG4 or HMW HA at 20, 40, and 80 μg/ml, and cell proliferation was measured. The contribution of CD44 was assessed by coincubation with a CD44 antibody (IM7). The antiproliferative effect of rhPRG4 was investigated following treatment of PRG4–/– mouse synoviocytes with IL‐1β or TNFα in the presence or absence of IM7. Results Recombinant human PRG4 bound CD44 and interfered with the binding of HMW HA to CD44. Removal of sialic acid and O‐glycosylations significantly increased CD44 binding by rhPRG4 (P