Endothelial protein C receptor-associated invasiveness of rheumatoid synovial fibroblasts is likely driven by group V secretory phospholipase A2

Abstract

IntroductionRheumatoid synovial fibroblasts (RASFs) mediate joint inflammation and destruction in rheumatoid arthritis (RA). Endothelial protein C receptor (EPCR) is a specific receptor for the natural anticoagulant activated protein C (APC). It mediates the cytoprotective properties of APC and is expressed in rheumatoid synovial tissue. A recent report shows that group V secretory phospholipase A2 (sPLA2V) prevents APC from binding to EPCR in endothelium and inhibits EPCR/APC function. The aim of this study was to investigate the expression and function of EPCR on RASFs.MethodsHuman synovial fibroblasts (SFs) were isolated from RA or osteoarthritis (OA) synovial tissues and treated with control, EPCR, or sPLA2V small interfering RNA (siRNA); recombinant human APC, tumor necrosis factor-alpha (TNF-α), or sPLA2V. RASF viability and migration/invasion were measured by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) and collagen gel migration/invasion assays, respectively, and cartilage degradation by 1,9-dimethylmethylene blue (DMMB) assay in the presence of human OA articular cartilage explants. The expression or activation of cytokines, EPCR, cadherin-11, mitogen-activated protein (MAP) kinases, and nuclear factor-kappa-B (NF-κB) or both were detected by enzyme-linked immunosorbent assay, Western blotting, or immunostaining.ResultsEPCR was expressed by both OASFs and RASFs but was markedly increased in RASFs. When EPCR was suppressed by siRNA or blocking antibody cell viability, cell invasion and cartilage degradation were reduced by more than 30%. Inflammatory mediators interleukin-1-beta (IL-1β), cadherin-11, and NF-κB were significantly reduced by EPCR suppression under control or TNF-α-stimulated conditions. The expression or activation (or both) of MAP kinases ERK, p38, and JNK were also markedly decreased in cells transfected with EPCR siRNA. Further analysis revealed that sPLA2V co-localized with EPCR on RASFs. Suppression of sPLA2V reduced cell viability and cartilage degradation and increased APC binding to RASFs. Conversely, recombinant sPLA2V increased cartilage degradation, blocked APC binding to RASFs, and could not rescue the effects induced by EPCR suppression.ConclusionsOur results demonstrate that EPCR is overexpressed by RASFs and mediates the aggressive behavior of RASFs. This function of EPCR is contrary to its cytoprotective role in other settings and is likely driven by sPLA2V.

Nuclear factor‐κB in rheumatoid arthritis

“…In addition, intracellular factors ( Figure 1D), NIK, NOD2, ROK and NLRC5, RANKL, FSTL-1, 15-(s)-HETE, Spla2V, endothelial protein C receptor (EPCR), CIA4, CD40-CD154 and other molecules ( Figure 1E) can also activate NF-kB. Figure 2A); when NF-κB was activated by group V secretory phospholipase (A2sPLA2V), EPCR, Toll-like receptor 2/3/4 (TLR2/3/4) and pre-B cell colony-enhancing factor (PBEF), FLS invasion ability would be promoted as IL-1β/6/8, MMP-1/3 were secreted 24,27,28 ( Figure 2B); when activated by TNF-α, secretion of VCAM-1 can enhance FLS infiltration capability 29 ( Figure 2C). In addition, when NF-κB activated by Ang II and AT1, caspase 3 secretion decreased, and activated by CRT or TNF-α, c-FLIP protein secretion increased; both of them cause FLS cell apoptosis decreases [30][31][32] ( Figure 2D).…”

“…Activated NF‐κB promotes FLS proliferation, enhances its invasion ability, infiltration ability and reduces apoptosis. E2F2, IL‐1α/1β/6, PEG2, ki67, MMP‐1/2/3/9/13, F‐Actin and TNF‐α would be secreted and then promote cell proliferation, after NF‐κB activated by TNF‐α, IL‐36, CARD domain containing 5 (NLRC5), RhoA, Extra‐terminal (BET) family of the bromodomain (Brd) proteins signals 19,24‐26 (Figure 2A); when NF‐κB was activated by group V secretory phospholipase (A2sPLA2V), EPCR, Toll‐like receptor 2/3/4 (TLR2/3/4) and pre‐B cell colony‐enhancing factor (PBEF), FLS invasion ability would be promoted as IL‐1β/6/8, MMP‐1/3 were secreted 24,27,28 (Figure 2B); when activated by TNF‐α, secretion of VCAM‐1 can enhance FLS infiltration capability 29 (Figure 2C). In addition, when NF‐κB activated by Ang II and AT1, caspase 3 secretion decreased, and activated by CRT or TNF‐α, c‐FLIP protein secretion increased; both of them cause FLS cell apoptosis decreases 30‐32 (Figure 2D).…”

Analysis of Novel Immunological Biomarkers Related to Rheumatoid Arthritis Disease Severity

“…This is the case with endothelial protein C receptor (EPCR), which is part of the EPCR-activated protein C (APC)-protease activated receptor (PAR1) system and exhibits a marked anti-inflammatory and immunosuppressive ability [11,12]. However, some authors have described the proinflammatory role of this receptor in RA [13]. In addition, protein C (PC) is, among others, a ligand of the epidermal growth factor receptor (EGFR), whose serum levels are highly increased in this disease [14].…”

Harmful and protective roles of group V phospholipase A2: Current perspectives and future directions

“…Another study investigated the role of PLA2G5 in synovial fibroblasts obtained from a small group of patients with rheumatoid arthritis. In this report, PLA2G5 increased cartilage degradation and proinflammatory effects of endothelial protein C receptor (EPCR) activation [81].…”