and 2 other(s)
INTRODUCTION: Synovial fibrosis is a common feature in advanced OA that contributes to joint pain and stiffness. In synovial fibroblasts, TGF-β1 induces fibrotic changes characterized by cell proliferation and collagen type I accumulation. Additionally, TGF-β1 promotes the differentiation of OA synoviocytes into a myofibroblast-like phenotype, characterized by the expression of alpha smooth muscle actin (α-SMA). Myofibroblasts are effector cells in fibrosis characterized by enhanced migration and production of extracellular matrix components. Synoviocytes produce proteoglycan-4 (PRG4; lubricin), a heavily glycosylated mucinous glycoprotein, and hyaluronic acid (HA). Both PRG4 and HA play important roles in joint lubrication. PRG4 and HA also compete upon binding to the CD44 receptor and the downstream response of PRG4 binding to CD44 reduces synoviocyte proliferation and expression of proteolytic enzymes. Our objective is to study the role of PRG4 in modulating myofibroblast differentiation of OA fibroblast-like synoviocytes (OA FLS) and whether this effect is mediated by interaction with CD44. METHODS: OA FLS were harvested from patients undergoing total knee replacement and used between the third and sixth passages. OA FLS (300,000 cells per well) were treated with TGF-β1 (1ng/ml)±native synovial PRG4, recombinant human PRG4 (rhPRG4; ~240kDa) or high molecular weight hyaluronic acid (HA; ~1,200 kDa) (100μg/ml for all treatments) for 24 hours followed by RNA isolation, cDNA synthesis and qPCR using TaqMan Fast Advanced Master Mix (n=4 independent patients). The cycle threshold (Ct) value of α-SMA (ACTA2) was normalized to the Ct value of GAPDH in the same sample, and the relative expression was calculated using the 2-ΔΔCt method. OA FLS were treated with TGF-β1±PRG4 or HA for 48 hours and probing was performed using FITC-conjugated anti α-SMA antibody and Alexa Fluor 594 conjugated anti-alpha tubulin antibody overnight at 4oC. Corrected total cell fluorescence (CTCF) was quantified. Synovial tissues were isolated from 2 months old male Prg4+/+, Prg4-/- and 2 and 9 months old Prg4 gene-trap animals. The Prg4 gene-trap allele is a loss of function allele (Prg4GT/GT) whose function is restored with Cre-excision (Prg4GTR/GTR). Prg4 expression was restored at 3 weeks of age (n=5 animals per group). RNA isolation, cDNA synthesis and qPCR of ACTA2 was performed using GAPDH as a reference gene. Synovial tissues were probed with a FITC-conjugated anti α-SMA antibody. Rhodamine labeled rhPRG4 (25μg/ml) was incubated with OA FLS±anti-CD44 or isotype control (IC) antibodies for 30 min and CTCF was quantified. OA FLS were treated with TGF-β1±rhPRG4 (100μg/ml)±anti-CD44 or IC antibodies (2μg/ml) for 24 hours followed by determining ACTA2 expression. Statistical significance was performed using ANOVA and Tukey’s post-hoc test. RESULTS: Synovial PRG4 and HA treatments reduced ACTA2 expression (p<0.001) and reduced α-SMA protein content (p<0.001 for PRG4 and p<0.01 for HA) in TGF-β stimulated OA FLS. PRG4 treatment also reduced stress fiber formation (Fig. 1). ACTA2 expression was higher in Prg4-/- synovia compared to Prg4+/+ synovia (p<0.05). Older gene-trap animals had higher ACTA2 expression and re-expression of Prg4 reduced ACTA2 expression (p<0.001) (Fig. 2). Synovial tissues in Prg4-/- and gene-trap mice had more detectable α-SMA protein compared to recombined littermates. rhPRG4 was internalized by OA FLS mediated by the CD44 receptor (Fig. 3). CD44 neutralization reversed the reduction in ACTA2 expression by rhPRG4 in TGF-β1 stimulated OA FLS (p<0.05). DISCUSSION: PRG4 reduced the differentiation of OA FLS into myofibroblasts as determined by α-SMA expression. Lack of Prg4 expression in vivo resulted in induction of α-SMA expression and accumulation in murine synovia and this was reversed by re-expression of Prg4. PRG4’s antifibrotic role was potentially due to its interaction with the CD44 receptor and internalization by OA FLS. SIGNIFICANCE AND RELEVANCE: OA fibrosis is an important feature of advanced OA and reversing OA fibrosis can have a disease-modifying effect. PRG4 may have a potential therapeutic application in mitigating synovial fibrosis in patients with advanced OA. ACKNOWLEDGEMENTS: This work is supported by R01AR067748 to KE and GJ.
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