Proteoglycan-4 Regulates Fibroblast to Myofibroblast Transition and Expression of Fibrotic Genes in the Synovium
Abstract Background: Synovial tissue fibrosis is common in advanced OA with features including presence of stress fiber-positive myofibroblasts and deposition of cross-linked collagen type-I. An antifibrotic effect in OA synoviocytes was associated with PRG4 secretion and native synovial PRG4 reduced collagen content in OA synoviocytes. 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 . Methods: OA synoviocytes were treated with TGF-β ± PRG4 for 24 hours 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-hour 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 was studied at 2 and 9 months of age. Synovial expression of α-SMA and PLOD2 was determined in 2-months old Prg4 GT/GT & Cd44 -/- and Prg4 GTR/GTR & Cd44 -/- animals . Results: 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-months 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 in CD44-deficient mice. Conclusion: PRG4 is an endogenous antifibrotic modulator in the joint and its effect on myofibroblast formation is mediated by CD44, but CD44 is not required to demonstrate an antifibrotic effect in vivo .