Activation of proteinase-activated receptor 2 in human osteoarthritic cartilage upregulates catabolic and proinflammatory pathways capable of inducing cartilage degradation: a basic science study

Abstract

Proteinase-activated receptors (PARs) belong to a family of G protein-coupled receptors. PARs are activated by a serinedependent cleavage generating a tethered activating ligand. PAR-2 was shown to be involved in inflammatory pathways. We investigated the in situ levels and modulation of PAR-2 in human normal and osteoarthritis (OA) cartilage/chondrocytes. Furthermore, we evaluated the role of PAR-2 on the synthesis of the major catabolic factors in OA cartilage, including metalloproteinase (MMP)-1 and MMP-13 and the inflammatory mediator cyclooxygenase 2 (COX-2), as well as the PAR-2-activated signalling pathways in OA chondrocytes. PAR-2 expression was determined using real-time reverse transcription-polymerase chain reaction and protein levels by immunohistochemistry in normal and OA cartilage. Protein modulation was investigated in OA cartilage explants treated with a specific PAR-2-activating peptide (PAR-2-AP), SLIGKV-NH 2 (1 to 400 μM), interleukin 1 beta (IL-1β) (100 pg/mL), tumor necrosis factor-alpha (TNF-α) (5 ng/mL), transforming growth factor-beta-1 (TGF-β1) (10 ng/mL), or the signalling pathway inhibitors of p38 (SB202190), MEK1/2 (mitogenactivated protein kinase kinase) (PD98059), and nuclear factorkappa B (NF-κB) (SN50), and PAR-2 levels were determined by immunohistochemistry. Signalling pathways were analyzed on OA chondrocytes by Western blot using specific phosphoantibodies against extracellular signal-regulated kinase 1/2 (Erk1/2), p38, JNK (c-jun N-terminal kinase), and NF-κB in the presence or absence of the PAR-2-AP and/or IL-1β. PAR-2-induced MMP and COX-2 levels in cartilage were determined by immunohistochemistry. PAR-2 is produced by human chondrocytes and is significantly upregulated in OA compared with normal chondrocytes (p < 0.04 and p < 0.03, respectively). The receptor levels were significantly upregulated by IL-1β (p < 0.006) and TNF-α (p < 0.002) as well as by the PAR-2-AP at 10, 100, and 400 μM (p < 0.02) and were downregulated by the inhibition of p38. After 48 hours of incubation, PAR-2 activation significantly induced MMP-1 and COX-2 starting at 10 μM (both p < 0.005) and MMP-13 at 100 μM (p < 0.02) as well as the phosphorylation of Erk1/2 and p38 within 5 minutes of incubation (p < 0.03). Though not statistically significant, IL-1β produced an additional effect on the activation of Erk1/2 and p38. This study documents, for the first time, functional consequences of PAR-2 activation in human OA cartilage, identifies p38 as the major signalling pathway regulating its synthesis, and demonstrates that specific PAR-2 activation induces Erk1/2 and p38 in OA chondrocytes. These results suggest PAR-2 as a potential new therapeutic target for the treatment of OA.COX-2 = cyclooxygenase 2; C T = threshold cycle; DMEM = Dulbecco's modified Eagle's medium; Erk1/2 = extracellular signal-regulated kinase 1/ 2; FCS = fetal calf serum; GAPDH = glyceraldehydes-3-phosphate dehydrogenase; IL-1β = interleukin 1 beta; JNK = c-jun N-terminal kinase; MAP = mitogen-activated protein; MEK1...

Activation of cartilage matrix metalloproteinases by activated protein C

“…Of the 4 PARs, the one most strongly implicated in the pathogenesis and progression of inflammatory arthritis is PAR-2 (41,42). Importantly, PAR-2 is also increased in OA human cartilage, can be up-regulated by IL-1␣ and TNF␣, and its activation leads to increased MMP-1 and MMP-13 synthesis (10,11). Unlike the other family members, PAR-2 is not cleaved/activated by thrombin, but it can be activated through canonical cleavage by APC (43).…”

“…In the context of arthritis, PAR-1 activation may play a role in promoting joint inflammation and associated cartilage damage (9). Significantly, APC, unlike thrombin, can also cleave PAR-2, which is increased in osteoarthritic (OA) cartilage and, when activated, leads to the up-regulation of matrix metalloproteinases (MMPs) implicated in cartilage degradation (10,11).…”

Emerging roles of serine proteinases in tissue turnover in arthritis

“…A study in PAR-2 Ϫ/Ϫ mice confirmed PAR-2 as a key mediator of chronic joint inflammation (158), and this is likely to be a consequence of activation by mast cell-derived tryptase (86,87). PAR-2 is associated with the perpetuation of inflammation in both RA and OA, because PAR-2 levels are elevated by proinflammatory cytokines and growth factors (159,160), while PAR-2 activation in peripheral blood monocytes and chondrocytes enhances proinflammatory cytokine production (161,162). Thus, increased expression of serine proteinases capable of activating PARs (Figure 1) has the potential to exacerbate inflammation.…”

Proteinase-Activated Receptors and Arthritis

“…Boileau et al confirmed that the expression of PAR 2 is enhanced in OA chondrocytes compared to normal and this could be further increased by stimulation with IL-1b, TNFa and most significantly by PAR 2 activating peptides [96], suggesting a positive feedback mechanism upon receptor activation. PAR 2 activation in these cells led to an increase in MMP-1, MMP-13 and COX-2, all key enzymes in OA pathology [96].…”