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 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. 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 fiberpositive 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. 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.

Synovial Fibrosis Involvement in Osteoarthritis

“…Their further findings supported this hypothesis and demonstrated that ADAMTS-5 was blocked by a CD44-dependent mechanism ( 84 ). As PRG4 is a ligand of the CD44 receptor, Qadri et al examined the role of the PRG4-CD44 interaction in regulating SF in OA and demonstrated that PRG4 inhibited fibroblast-to-myofibroblast transition, thus downregulating the expression of fibrotic genes in the OA synovium ( 85 ).…”

Proteoglycan-4 is an essential regulator of synovial macrophage polarization and inflammatory macrophage joint infiltration

“…PRG4-CD44 interaction inhibits nuclear factor kappa B (NF-κB) nuclear translocation in human synovial fibroblasts and attenuates IL-1β-induced synoviocyte proliferation and expression of matrix-degrading enzymes [ 22 ]. PRG4 regulates synovial fibroblast to myofibroblast transition and reduces the expression of fibrotic markers : alpha smooth muscle actin (α-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 ].…”

RNA Sequencing of LX-2 Cells Treated with TGF-β1 Identifies Genes Associated with Early Hepatic Stellate Cell Activation.

“…Similarly, the reduced expression of VIP correlates with progressive cardiac brosis in murine models, which can be reversed by VIP overexpression [28]. PRG4 is associated with protection functions in the connective tissues and reduced broblast activation in the synovial tissue [29].…”