The autocrine role of proteoglycan-4 (PRG4) in modulating osteoarthritic synoviocyte proliferation and expression of matrix degrading enzymes

Background: Lubricin/proteoglycan 4 (PRG4) is a mucinous glycoprotein secreted by synovial fibroblasts and superficial zone chondrocytes. Recently, we showed that recombinant human PRG4 (rhPRG4) is a putative ligand for CD44 receptor. rhPRG4-CD44 interaction inhibits cytokine-induced rheumatoid arthritis synoviocyte proliferation. The objective of this study is to decipher the autocrine function of PRG4 in regulating osteoarthritic synoviocyte proliferation and expression of catabolic and pro-inflammatory mediators under basal and interleukin-1 beta (IL-1 beta)stimulated conditions. Methods: Cytosolic and nuclear levels of nuclear factor kappa B (NF kappa B) p50 and p65 subunits in Prg4(+/+) and Prg4(-/-) synoviocytes were studied using western blot. Nuclear translocation of p50 and p65 proteins in osteoarthritis (OA) fibroblast-like synoviocytes (FLS) in response to IL-1 beta stimulation in the absence or presence of rhPRG4 was studied using DNA binding assays. OA synoviocyte (5000 cells per well) proliferation following IL-1 beta (20 ng/ml) treatment in the absence or presence of rhPRG4 (50-200 mu g/ml) over 48 hours was determined using a colorimetric assay. Gene expression of matrix metalloproteinases (MMPs), tissue inhibitor of metallproteinases-1 (TIMP-1), TIMP-2, IL-1 beta, IL-6, IL8, TNF-alpha, cycloxygenae-2 (COX2) and PRG4 in unstimulated and IL-1 beta (1 ng/ml)-stimulated OA synoviocytes, in the presence or absence of rhPRG4 (100 and 200 mu g/ml), was studied following incubation for 24 hours. Results: Prg(4-/-) synoviocytes contained higher nuclear p50 and p65 levels compared to Prg4(+/+) synoviocytes (p < 0. 05). rhPRG4 (100 mu g/ml) reduced p50 and p65 nuclear levels in Prg4(+/+) and Prg4(-/-) synoviocytes (p < 0.001). Similarly, rhPRG4 (200 mu g/ml) inhibited NF kappa B translocation and cell proliferation in OA synoviocytes in a CD44-dependent manner (p < 0.001) via inhibition of I kappa B alpha phosphorylation. IL-1 beta reduced PRG4 expression in OA synoviocytes and rhPRG4 (100 mu g/ml) treatment reversed this effect (p < 0.001). rhPRG4 (200 mu g/ml) reduced basal gene expression of MMP-1, MMP-3, MMP-13, IL-6, IL-8, and PRG4 in OA synoviocytes, while increasing TIMP-2 and cycloxygenase-2 (COX2) expression (p < 0.001). rhPRG4 (200 mu g/ml) reduced IL-1 beta induction of MMP-1, MMP-3, MMP-9, MMP-13, IL-6, IL-8, and COX2 expression in a CD44-dependent manner (p < 0.001). Conclusion: PRG4 plays an important anti-inflammatory role in regulating OA synoviocyte proliferation and reduces basal and IL-1 beta-stimulated expression of catabolic mediators. Exogenous rhPRG4 autoregulates native PRG4 expression in OA synoviocytes.