Synovial tissue fibrosis is common in advanced OA with features including the presence of stress fiber-positive myofibroblasts and deposition of cross-linked collagen type-I. Proteoglycan-4 (PRG4) is a mucinous glycoprotein secreted by synovial fibroblasts and is a major component of synovial fluid. PRG4 is a ligand of the CD44 receptor. Our objective was to examine the role of PRG4-CD44 interaction in regulating synovial tissue fibrosis in vitro and in vivo.
OA synoviocytes were treated with TGF-β ± PRG4 for 24 h and α-SMA content was determined using immunofluorescence. Rhodamine-labeled rhPRG4 was incubated with OA synoviocytes ± anti-CD44 or isotype control antibodies and cellular uptake of rhPRG4 was determined following a 30-min incubation and α-SMA expression following a 24-h incubation. HEK-TGF-β cells were treated with TGF-β ± rhPRG4 and Smad3 phosphorylation was determined using immunofluorescence and TGF-β/Smad pathway activation was determined colorimetrically. We probed for stress fibers and focal adhesions (FAs) in TGF-β-treated murine fibroblasts and fibroblast migration was quantified ± rhPRG4. Synovial expression of fibrotic markers: α-SMA, collagen type-I, and PLOD2 in Prg4 gene-trap ( Prg4 GT ) and recombined Prg4 GTR animals were studied at 2 and 9 months of age. Synovial expression of α-SMA and PLOD2 was determined in 2-month-old Prg4 GT/GT & Cd44 −/− and Prg4 GTR/GTR & Cd44 −/− animals.
PRG4 reduced α-SMA content in OA synoviocytes ( p < 0. 001). rhPRG4 was internalized by OA synoviocytes via CD44 and CD44 neutralization attenuated rhPRG4’s antifibrotic effect ( p < 0. 05). rhPRG4 reduced pSmad3 signal in HEK-TGF-β cells ( p < 0. 001) and TGF-β/Smad pathway activation ( p < 0. 001). rhPRG4 reduced the number of stress fiber-positive myofibroblasts, FAs mean size, and cell migration in TGF-β-treated NIH3T3 fibroblasts ( p < 0. 05). rhPRG4 inhibited fibroblast migration in a macrophage and fibroblast co-culture model without altering active or total TGF-β levels. Synovial tissues of 9-month-old Prg4 GT/GT animals had higher α-SMA, collagen type-I, and PLOD2 ( p < 0. 001) content and Prg4 re-expression reduced these markers ( p < 0. 01). Prg4 re-expression also reduced α-SMA and PLOD2 staining in CD44-deficient mice.