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 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. 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 a-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 (Prg4GT) and recombined Prg4GTR animals was studied at 2 and 9 months of age. Synovial expression of α-SMA and PLOD2 was determined in 2-months old Prg4GT/GT&Cd44-/- and Prg4GTR/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 Prg4GT/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. Conclusion: PRG4 is an endogenous antifibrotic modulator in the joint and its effect on myofibroblast formation is partially mediated by CD44, but CD44 is not required to demonstrate an antifibrotic effect in vivo.

Proteoglycan-4 is an Essential Regulator of Synovial Macrophage Polarization and Inflammatory Macrophage Joint Infiltration

“…025740, JAX, USA) is born lacking Prg4 expression which can be restored via CRE-mediated recombination [26]. The Prg GT/GT animal displays significant synovial hyperplasia and subintimal fibrosis and recombination appeared to attenuate these changes [23]. In this study, Prg4 recombination (Prg4 GTR/GTR ) occurred in 3-weeks old animals via intraperitoneal injection of tamoxifen (0.1mg/g in 100L corn oil vehicle) daily for 10 days, using vehicle-administered gene-trap animals as controls.…”

“…Throughout our studies, we utilized Prg4 GT/GT (n=102), Prg4 GTR/GTR (n=32) and Prg4 +/+ animals (n=56; stock # 101045, JAX; B6/129S background) (animals were studied at 2 or 6 months of age and depletion of SMs was performed when animals were 4 months-old), and we included randomly assigned litter and agematched males and females in our experimental groups. The number of animals per group was based on our previous analysis of the effect of Prg4 recombination on synovial fibrosis using alpha smooth muscle actin (-SMA) staining as our primary endpoint [23].…”

“…Statistical analyses were conducted as we previously described [23]. We used unpaired, paired Student's t-test, one-way or two-way ANOVAs followed by Tukey's or Bonferroni post-hoc tests, with statistical significance set at p<0.05.…”

“…PRG4-CD44 interaction inhibits nuclear factor kappa B (NF-B) nuclear translocation in human synovial fibroblasts and attenuates IL-1-induced synoviocyte proliferation and expression of matrixdegrading enzymes [22]. PRG4 regulates synovial fibroblast to myofibroblast transition and reduces the expression of fibrotic markers: alpha smooth muscle actin (-SMA) and collagen type I [23,24]. PRG4 also suppresses the activation of TLR2 and TLR4 receptors by DAMPs in OA synovial fluid aspirates [21].…”

“…PRG4 also suppresses the activation of TLR2 and TLR4 receptors by DAMPs in OA synovial fluid aspirates [21]. Synovial hyperplasia and fibrosis that progressed with age are evident in murine knee joints lacking Prg4 expression [23,25,26]. The biological role of PRG4 helps explain its disease-modifying activity in rodent and pig models of posttraumatic osteoarthritis (PTOA), where PRG4 reduced cartilage degeneration and enhanced its repair [27,28].…”