Thrombin‐activatable carboxypeptidase B cleavage of osteopontin regulates neutrophil survival and synoviocyte binding in rheumatoid arthritis

Abstract

Objective Osteopontin (OPN) is a pro-inflammatory cytokine important in rheumatoid arthritis (RA). OPN can be cleaved by thrombin, leading to OPN-Arg (OPN-R) and exposing the cryptic C-terminal α4β1 and α9β1 integrin-binding motif (SVVYGLR). Thrombin-activatable carboxypeptidase B (CPB), also termed thrombin-activatable fibrinolysis inhibitor (TAFI), removes the C-terminal arginine from OPN-R, generating OPN-Leu (OPN-L) and abrogating its enhanced cell binding. We investigated the roles of OPN-R and OPN-L in: (i) synoviocyte adhesion, which contributes to formation of invasive pannus, and (ii) neutrophil survival, which affects inflammatory infiltrates, in RA. Methods and Results We developed ELISAs specific for OPN-R and OPN-L, and demonstrate elevations of OPN-R and OPN-L in RA, but not in osteoarthritis or psoriatic arthritis, synovial fluid samples. OPN-R and OPN-L levels correlated with multiple inflammatory cytokines including TNFα and IL-6. Immunohistochemical analyses demonstrated robust expression of OPN-FL, but minimal OPN-R, in RA synovium, suggesting that cleaved OPN is released into the synovial fluid. In cellular assays, OPN-FL, and to a lesser extent OPN-R and OPN-L, had an anti-apoptotic effect on neutrophils. OPN-R, but not OPN-L, augmented RA fibroblast-like synoviocyte binding mediated by SVVYGLR binding to α4β1. Conclusion Thrombin activation of OPN (OPN-R) and its subsequent inactivation by thrombin-activatable CPB (OPN-L) occurs locally within inflamed joints in RA. Our data suggest that thrombin-activatable CPB plays a central homeostatic role in RA, by regulating neutrophil viability and reducing synoviocyte adhesion.

Thrombin-cleaved Fragments of Osteopontin Are Overexpressed in Malignant Glial Tumors and Provide a Molecular Niche with Survival Advantage

“…When apoptosis was induced by thapsigargin in T98G cells, resistance to apoptosis was conferred by the presence of OPN, with the greatest effect observed with OPN-FL and less but still significant protective effect with OPN-R and OPN-L (Fig. 7F); this pattern is similar to that previously observed in cultured synoviocytes (25). However, when apoptosis was induced by WP1066, both OPN-FL and the cleaved forms conferred an equal degree of protection (Fig.…”

“…DBTRG-05MG, T98G, and U-87 MG and 1321N1 cells showed an ϳ1.5-fold increase in binding to immobilized OPN-R and OPN-L compared with OPN-FL, whereas Hs 683 showed no adhesion to any of the OPN isoforms. In contrast to Jurkat cells and synoviocytes (25), there was no significant change in binding when OPN-R was cleaved to OPN-L. Removing the RGD site by mutation in OPN-FL (OPN RAA -FL) abolished cell binding completely in all three GBM cell lines, indicating that cell binding was primarily driven by the RGD .…”

“…Following a 10-min centrifugation at 400 ϫ g at 4°C, samples were appropriately diluted in PBS with 2% BSA, pH 7.4. ELISAs for OPN and its cleaved fragments were performed as described (25). Briefly, a commercial antibody (MAB14331, R&D Systems, Minneapolis, MN) was used for the capture of OPN-FL, OPN-R, and OPN-L. OPN-FL was measured using the R&D Systems Quantikine OPN ELISA kit.…”

“…We recently developed specific ELISAs for OPN-R and OPN-L and demonstrated that these cleaved OPN forms are markedly elevated in the synovial fluid of patients with rheumatoid arthritis but not osteoarthritis or psoriatic arthritis, validating its significance in vivo (25). Thrombin activation is increased in patients with GBM as shown by the elevation of prothrombin fragment F1 ϩ 2 in their plasma (27), and intravascular thrombosis has been suggested to be involved in progression of astrocytomas to GBMs (28).…”

“…We have shown that plasma procarboxypeptidase 2 (pCPB2, also termed thrombin-activatable fibrinolysis inhibitor or TAFI), upon activation to CPB2, cleaves the C-terminal arginine from SVVYGLR, thereby converting OPN-R to OPN-Leu (OPN-L). Jurkat cells and synoviocytes have enhanced cell adhesion to OPN-R compared with full-length OPN (OPN-FL) that is reduced when OPN-R is converted to OPN-L by either CPB2 or CPN, the constitutively active plasma carboxypeptidase (25,26). We postulate that the sequential cleavages of OPN by thrombin and CPN/CPB2 may up-regulate and down-regulate OPNЈs inflammatory properties, respectively.…”