Disruption of Proteoglycan 4 (PRG4)-CD44 Signaling Modulates Chronic Synovitis in Conditionally Inactivated Mice.

Background: Proteoglycan-4 (PRG4) is a mucinous glycoprotein secreted by synovial fibroblasts and superficial zone chondrocytes. PRG4 inhibits synovial macrophage (SM) activation via xanthine oxidase (XO) and hypoxia inducible factor alpha (HIF-1α) suppression. We aimed to evaluate the contribution of PRG4-CD44 interaction to synovial homeostasis and investigate PRG4's signaling dysfunction in synovial tissues from patients with osteoarthritis (OA). We hypothesized that CD44 mediates synovitis due to PRG4 loss and that PRG4 signaling dysfunction is associated with high-grade synovitis in OA. Methods: Prg4 FrtloxP/FrtloxP are transgenic mice wherein tamoxifen (TAM) inactivates the Frt allele and creates a knockout state (Prg4 FrtKO/FrtKO ). TAM (Prg4 Off ) or corn oil (Prg4 On ) administration occurred in 4 weeks-old animals (4-6 animals with 2-3 males per group). We crossed this mouse with Cd44 -/- mice to generate Cd44 +/+ & Prg4 On , Cd44 +/+ & Prg4 Off , Cd44 -/- & Prg4 On , and Cd44 -/- & Prg4 Off . XO and HIF-1α immunostaining was conducted. Isolated SMs were activated using LPS + IFNγ and SM glycolytic activation was measured by proton efflux rate (PER). HIF-1α levels were measured by ELISA. Synovial tissues were collected from the medial and lateral joint compartments of OA patients undergoing knee arthroplasty (n = 9; 7 females and 2 males). Specimens were classified by Krenn's synovitis score as low-grade (Score: 2-4) or high-grade (score: 5-9) synovitis. Isolated CD14 + cells were stimulated with LPS ± febuxostat, and glycolytic activation was measured by PER. Immunohistochemistry (IHC) included PRG4, CD44, XO and HIF-1α. Results: CD44 deficiency reduced XO and HIF-1α staining in addition to synovial pathology following Prg4 inactivation (p < 0.05). SMs from Cd44 -/- & Prg4 Off mice were less activated than Cd44 +/+ & Prg4 Off mice (p < 0.001) and had lower HIF-1α levels (p < 0.0001). High-grade synovitis tissues displayed less PRG4 and greater CD44, XO and HIF-1α (p < 0.001) IHC staining compared to low-grade and normal tissues. Febuxostat reduced CD14 + cell activation from medial (p < 0.0001) and lateral (p < 0.05) joint compartments. Conclusions: CD44 loss abrogated chronic synovitis observed following PRG4 loss. Dysfunction in PRG4 signaling, demonstrated by lower tissue levels of PRG4 along with higher CD44, XO and HIF-1α, was associated with high-grade synovitis. Targeting the downstream events of PRG4 loss is potentially therapeutic in OA synovitis.