Proteoglycan 4 (PRG4/lubricin) is secreted by cells that reside in articular cartilage and line the synovial joint. Lubricin may play a role in modulating inflammatory responses through interaction with CD44. This led us to examine if lubricin could be playing a larger role in the modulation of inflammation/immunity through interaction with Toll-like receptors (TLRs). Human Embryonic Kidney (HEK) cells overexpressing TLRs 2, 4 or 5 and surface plasmon resonance were employed to determine if full length recombinant human lubricin was able to bind to and activate TLRs. Primary human synovial fibroblasts were also examined using flow cytometry and Luminex multiplex ELISA. A rat destabilization model of osteoarthritis (OA) was used to determine if lubricin injections were able to regulate pain and/or inflammation in vivo. Lubricin can bind to and regulate the activity of TLRs, leading to downstream changes in inflammatory signalling independent of HA. We confirmed these findings in vivo through intra-articular injections of lubricin in a rat OA model where the inhibition of systemic inflammatory signaling and reduction in pain were observed. Lubricin plays an important role in regulating the inflammatory environment under both homeostatic and tissue injury states.

These results suggest alterations in HA MW could significantly affect synovial fluid's cartilage boundary lubricating ability, yet this diminishment in function could be circumvented by physiological levels of PRG4 forming a complex, potentially in solution, with HA.

Proteoglycan 4 (PRG4) is a mucin-like glycoprotein present in synovial fluid and at the surface of articular cartilage. The objectives of this study were to (1) assess the articular cartilage surface adsorption and in vitro cartilage boundary lubricating ability of full-length recombinant human PRG4 (rhPRG4), and (2) cartilage boundary lubricating ability of purified rhPRG4, both alone and in combination with hyaluronan (HA). rhPRG4 adsorption onto articular cartilage explants was assessed by immunohistochemistry and dot blot. An in vitro cartilage-cartilage friction test was used to assess rhPRG4's cartilage boundary lubricating ability compared to bovine PRG4, and that of purified rhPRG4 both alone and in combination with HA. rhPRG4 was able to adsorb to the articular surface, as well as the cut surface, of cartilage explants. The kinetic coefficient of friction of rhPRG4 was similar to that of PRG4 (p = 0.16) and lower than phosphate-buffered saline (p < 0.05), while that of purified rhPRG4 + HA was significantly lower than rhPRG4 alone (p < 0.05). This study demonstrates that rhPRG4 can adsorb to an intact articular cartilage surface and functions as an effective boundary lubricant, both alone and with HA, and provides the foundation for in vivo evaluation of this clinically relevant full-length rhPRG4 for treatment of osteoarthritis.

Purpose The objectives of this study were to assess the cartilage boundary lubricating ability of (1) non reduced (NR) disulfide-bonded proteoglycan 4 (PRG4) multimers versus PRG4 monomers, (2) NR versus reduced and alkylated (R/A) PRG4 monomers, and (3) assess the ability of NR PRG4 multimers versus monomers to adsorb to an articular cartilage surface. Materials and Methods PRG4 was separated into two preparations, PRG4 multimer enriched (PRG4Multi+) and PRG4 multimer deficient (PRG4Multi−), using size exclusion chromatography (SEC) and characterised by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The cartilage boundary lubricating ability of PRG4Multi+ and PRG4Multi− was compared at a physiological concentration (450 μg/mL) and assessed over a range of concentrations (45, 150 and 450 μg/mL). R/A and NR PRG4Multi− were evaluated at 450 μg/mL. Immunohistochemistry with anti-PRG4 antibody 4D6 was performed to visualise the adsorption of PRG4 preparations to the surface of articular cartilage explants. Results Separation into enriched populations of PRG4Multi+ and PRG4Multi− was achieved using SEC and was confirmed by SDS-PAGE. PRG4Multi+ and PRG4Multi− both functioned as effective friction-reducing cartilage boundary lubricants at 450 μg/mL; with PRG4Multi+ being more effective than PRG4Multi−. PRG4Multi+ lubricated in a dose-dependent manner, however PRG4Multi− did not. R/A PRG4Multi− lubricated similar to NR PRG4Multi−. PRG4 containing solutions showed 4D6 immunoreactivity at the articular surface; the immunoreactive intensity of PRG4Multi+ appeared to be similar to SF, whereas PRG4Multi− appeared to have less intensity. Conclusions These results demonstrate that the inter-molecular disulfide-bonded multimeric structure of PRG4 is important for its ability to adsorb to a cartilage surface and function as a boundary lubricant. These findings contribute to a greater understanding of the molecular basis of cartilage boundary lubrication of PRG4. Elucidating the PRG4 structure-lubrication function relationship will further contribute to the understanding of PRG4's role in diarthrodial joint homeostasis and disease.