Endothelial protein C receptor-associated invasiveness of rheumatoid synovial fibroblasts is likely driven by group V secretory phospholipase A2
© Xue et al.; licensee BioMed Central Ltd. 2014
Received: 11 June 2013
Accepted: 28 January 2014
Published: 5 February 2014
Rheumatoid 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.
Human 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.
EPCR 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.
Our 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.
Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by synovial inflammation and hyperplasia, leading to progressive cartilage and bone destruction. Normal synovium forms a thin membrane at the edges of joints and provides lubrication and nutrients for the cartilage. In RA, this thin synovial lining layer dramatically increases and transforms into an inflammatory mass, known as the pannus [1, 2]. This tissue mass expands and attaches to and invades the adjacent cartilage and subchondral bone, causing erosion. The major cell type accounting for the thickened lining layer and resultant pannus is the activated RA synovial fibroblasts (RASFs, also referred to as RA synoviocytes). As well as mediating tissue destruction, RASFs play a major role in catalyzing and sustaining RA by producing inflammatory cytokines such as interleukin-1-beta (IL-1β) and tumor necrosis factor-alpha (TNF-α), proangiogenic factors, and matrix-degrading enzymes [1, 2]. Of equal concern, RASFs collaborate with and support the recruitment, survival, activation, and differentiation of T cells, B cells, macrophages, mast cells, osteoclasts, and endothelial cells throughout the RA synovium [1, 2].
Once activated, the aggressive phenotype of RASFs can exist independent of inflammation. This was demonstrated by studies conducted in the severe combined immunodeficient mouse model of RA, in which implanted human RASFs degraded co-implanted human cartilage in the absence of inflammatory cells , and RASFs migrated via the bloodstream to implanted cartilage at a distant site, spreading RA to unaffected joints . These data clearly indicates that RASFs are not passive bystanders, but are active participants in joint destruction in RA.
Endothelial protein C receptor (EPCR) is an endothelial transmembrane glycoprotein able to bind to a natural anticoagulant, protein C (PC), and its activated form, APC, with similar affinity . Though originally identified as an endothelial cell receptor, EPCR has since been detected on many other cell types , including RA synovial lining cells . As a receptor, EPCR mediates the majority of the anti-apoptotic, anti-inflammatory, and barrier-protective functions of APC . In addition, EPCR itself is a central player in the convergent pathways of homeostasis and inflammation . Recently, EPCR has been found to be overexpressed by some cancer cells and increased cancer cell migration and invasion [9–11]. However, the underlying mechanisms are not clear. EPCR can be cleaved from the cell surface to form soluble EPCR (sEPCR), which binds PC/APC with the same affinity as membrane-bound EPCR but blocks the protective function of APC [12–14]. Increased sEPCR is associated with many inflammatory/autoimmune diseases [15–17]. A recent report shows that group V secretory phospholipase A2 (sPLA2V) prevents APC binding to EPCR and inhibits EPCR/APC function by accommodating lysophosphatidylcholine (LysoPCh) or platelet-activating factor (PAF) in the hydrophobic groove of EPCR . Being a key enzyme in the production of diverse mediators of inflammatory conditions, sPLA2V is present in significantly higher levels in the RA joint [19, 20] and stimulates RA synovial proliferation and joint destruction [20, 21].
The role of EPCR has not been studied in RASFs. We report here that EPCR is highly expressed by RASFs and contributes to the inflammatory and cartilage-degradative actions of these cells in vitro. Importantly, we show that these unexpected destructive effects of EPCR are likely driven by sPLA2V.
Methods and materials
Fetal bovine serum (FBS), anti-cadherin 11 antibody, and TRIzol were purchased from Invitrogen (Carlsbad, CA, USA); anti-ERK, phosphorylated ERK (P-ERK), p38, P-p38 antibodies and anti-fibroblast marker (ER-TR7), A/G Plus-agarose, scrambled control, validated EPCR, PC, and sPLA2V small interfering RNAs (siRNAs) from Santa Cruz Biotechnology (Santa Cruz, CA, USA); recombinant APC from Eli Lilly and Company (Indianapolis, IN, USA); RiboCellin Transfection Reagents from BioCellChallenge (Paris, France); anti-β-actin and PC/APC antibodies and recombinant TNF-α from Sigma-Aldrich (St. Louis, MO, USA); anti-human CD68 from eBioscience (San Diego, CA, USA); human IL-1β, IL-6, and IL-8 enzyme-linked immunosorbent assay (ELISA) DuoSet from R&D Systems (Minneapolis, MN, USA); anti-nuclear factor-kappa B (anti-NF-κB) p65 subunit antibody from Chemicon International (Temecula, CA, USA); recombinant sPLA2V from Abcam (Cambridge, MA, USA); anti-human sPLA2V antibody from Cayman (Ann Arbor, MI, USA); NE-PER nuclear extraction reagents from Pierce Chemical Co. (Rockford, IL, USA). RCR252 (EPCR blocking antibody) and RCR92 (EPCR non-blocking antibody) were kindly provided by Kenji Fukudome (Saga Medical School, Saga, Japan).
Synovial fibroblast isolation, culture, and treatment
Demographics of patients from which synovial fibroblasts were isolated from synovial tissues obtained from knee replacement surgeries
69.2 ± 7.2
71 ± 5.7
68.1 ± 6.2
70.5 ± 4.7
Synovial fluid and cartilage
Demographics of patients from which synovial fluid samples were collected by needle puncture from the knee joints
68.2 ± 5.1
70 ± 4.5
67.0 ± 4.6
69.5 ± 3.6
Usage of human tissue and fluid samples was in accordance with the ethics committee of the Northern Sydney Local Health District. All patients fulfilled the American College of Rheumatology criteria for RA and OA [22, 23] and gave their written informed consent.
SFs were transfected with EPCR, sPLA2V, or scrambled control siRNAs using RiboCellin Transfection Reagent in accordance with the protocol of the manufacturer. The efficacy of siRNA was detected by reverse-transcription real time polymerase chain reaction, ELISA, or Western blot.
EPCR, IL-1β, IL-6, and IL-8 in culture supernatants/whole cell lysates or synovial fluids were measured by ELISA DuoSet in accordance with the instructions of the manufacturer.
Matrix metalloproteinase-2 (MMP-2) and MMP-9 protein secretion and activation in the culture supernatants were measured by using gelatin zymography under non-reducing conditions .
Immunoprecipitation and Western blot
SFs were lysed in cell lysis buffer (20 mM HEPES, 1% Triton X, and 10% Glycerol) supplemented with protease and phosphate inhibitors. Nuclear proteins were extracted with NE-PER nuclear extraction reagents in accordance with the instructions of the manufacturer. Immunoprecipitation (IP) was performed by using A/G Plus-agarose after incubation with anti-human EPCR antibody. Equal amounts of protein were separated on 10% or 15% (for IP) SDS-PAGE. Immunoreactivity was detected by using the ECL detection system and semi-quantified with gel image analysis software. Anti-human β-actin antibody was used to normalize equal loading.
In vitro migration/invasion assay
RASF invasion was measured by using a collagen gel assay  with modifications. Briefly, RASFs (2 × 105 cells/mL in DMEM) were mixed with equal volume of collagen solution (2 mg/mL, pH 7.2) and transferred into 24-well plates (20 μL/drop, 4 drops/well). After polymerization for 2 hours, 0.5 mL DMEM with 10% FBS was added to each well, and plates were incubated at 37°C. After 48 hours, cells and collagen gels were stained with crystal violet. Cells that had migrated out of gels were counted under a light microscope. At least 16 collagen gel drops per experimental group were analyzed.
Immunohistochemistry, immunofluorescence, and toluidine blue staining
Cultured SFs in Falcon culture slides (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) were fixed with 1% paraformaldehyde. Human synovial tissues were fixed in 10% phosphate-buffered saline-buffered formalin. Immunohistochemical and immunofluorescent staining and toluidine blue staining were performed as described previously [26, 27]. Isotype IgG for each antibody was used as a negative control. Images were acquired and processed by using a digital camera and software (Nikon, Tokyo, Japan) and ImageJ.
Cells (4 × 103/well) were seeded into a 96-well plate. After 4 hours’ attachment, cells were treated with different test agents for 24, 48, or 72 hours, and cell viability was detected by the colorimetric MTT assay and confirmed by a trypan blue exclusion test.
1,9-dimethylmethylene blue assay
The level of sulphated glycosaminoglycans (sGAGs) released from the cartilage explants was determined by a 1,9-dimethylmethylene blue (DMMB) assay against a standard curve of chondroitin sulfate .
Significance was determined by using one-way analysis of variance followed by Tukey’s honestly significant difference (HSD) post hoc test or Student t test. P values of less than 0.05 were considered statistically significant.
EPCR is overexpressed by RASFs
To confirm whether SFs express EPCR, dual immunofluorescent staining was performed by using anti-EPCR antibody and the fibroblast marker, ER-TR7. EPCR staining in the lining layer was localized on SFs in both OA and RA synovium (Figure 1C). Isolated RASFs also expressed higher levels of EPCR than OASFs as assessed by immunostaining (Figure 1D). ELISA data using whole cell lysates confirmed that RASFs expressed threefold higher EPCR than OASFs (Figure 1E). sEPCR was not detectable in culture supernatants of RASFs or OASFs. At the gene level, both OASFs and RASFs expressed EPCR mRNA, with RASFs expressing more than 50% higher levels than OASFs at passage 1.
Suppressing EPCR inhibits the aggressive properties of RASFs
RASF survival/growth, a critical step that leads to synovial hyperplasia, was measured at 72 hours after transfection. Suppressing EPCR with siRNA resulted in a more than 30% reduction in RASF viability (Figure 2C). This was confirmed by the blocking antibody RCR252 treatment, which displayed a significant reduction in viable cells, whereas the non-blocking control antibody, RCR92, had no effect (Figure 2C). Another aggressive property of RASFs is their elevated migratory/invasive capacity. To assess this, a collagen gel migration/invasion assay was performed. In basal conditions, 20% to 30% of cells migrated out of a gel after 48 hours of incubation. There were more than 30% fewer cells migrating out of the collagen gel following EPCR siRNA transfection compared with control siRNA (Figure 2D), indicating that suppressing EPCR inhibits RASF migration/invasion. EPCR siRNA transfection also reduced sGAG release, a measure of cartilage degradation , into culture media from co-incubated cartilage explants, by more than 40% (Figure 2E). Toluidine blue staining confirmed that cartilage explants preserved more cartilage when incubated with EPCR siRNA-transfected RASFs when compared with control siRNA (Figure 2E).
Suppressing EPCR reduces MMP-2, IL-1β, and cadherin-11 and inactivates NF-κB and MAP kinases
In response to EPCR siRNA transfection, secretion of IL-1β, the key initiator of inflammation and cartilage breakdown in RA [31, 32], was reduced by approximately 50% from RASFs (Figure 3B). TNF-α stimulated IL-1β production in control but not in EPCR siRNA-transfected RASFs (Figure 3B). This inhibitory effect was cytokine-specific for IL-1β as silencing EPCR by siRNA had no effect on IL-6 in either control or TNF-α-stimulated conditions (Figure 3C).
Silencing EPCR also reduced the activation of NF-κB in whole cell lysates by 50% in control but not after TNF-α stimulation (Figure 3D). This was confirmed by the activation of NF-κB in the nuclear fraction, which displayed a reduction similar to that shown in whole cell lysate (Figure 3E). In addition, cadherin-11, a key molecule that regulates RASF function , was significantly suppressed when RASFs were transfected with EPCR siRNA in both control and TNF-α-stimulated conditions (Figure 3D).
sPLA2V co-localizes with EPCR in synovial tissues and blocks APC binding
sPLA2V promotes the aggressive properties of RASFs via EPCR
Taken together, these data suggest that sPLA2V is mainly responsible for RASF-mediated cartilage degradation and inflammation associated with overexpression of EPCR.
SFs are key effectors in the pathogenesis of RA. In this study, we have demonstrated that RASFs express higher levels of EPCR than OASFs. In contrast to its conventional anti-inflammatory effects, EPCR expressed by RASFs was associated with increased invasiveness and inflammatory responses of these cells. Further investigation revealed that sPLA2V is co-localized with EPCR, prevents APC from interacting with EPCR, and drives EPCR-associated invasiveness, inflammation, and cartilage degradation.
EPCR plays a critical role in augmenting PC activation and mediating the anti-inflammatory and cytoprotective functions of the PC pathway . In this study, however, EPCR expression was associated with the destructive effects of RASFs. Suppressing EPCR decreased RASF viability, invasion, and cartilage degradation ability through inhibition of inflammatory cytokine IL-1β. In RA, IL-1β stimulates the production of MMPs and the maturation of osteoclasts and ultimately promotes cartilage breakdown [31, 32] and the development of bone erosion [40, 41]. In synovium, IL-1β is a major activator of SFs by promoting the activation of NF-κB and MAP kinases . Moreover, IL-1β, but not TNF-α, can markedly induce sPLA2V production by SFs .
Suppression of EPCR also markedly reduced the expression and activation of MAP kinases which regulate cell survival, apoptosis, viability, cellular stress, and inflammatory responses. The three major classes of MAP kinases—ERK, p38, and JNK—are all enhanced in RA synovial tissues [37, 38]. Survival/growth of RASFs is mediated by ERK , which plays an important role in the maintenance of RA by promoting pannus formation . JNK activation is required for the regulation of collagenase production by SFs [35, 36]. ERK and JNK activation predict development of erosive disease in early arthritis [35, 36]. Our results suggest that EPCR promotes RASF viability and inflammation via activation of MAP kinases. Inhibition of NF-κB and cadherin-11 by suppression of EPCR also likely contributes to reduced invasion and cartilage degradation by RASFs [33, 42]. Our data also show that suppressing EPCR in RASFs inhibits production of MMP-2, a function of EPCR that is present in endothelial cells . Although MMP-2 may promote cartilage degradation, it suppresses the development of inflammatory joint disease in a mouse arthritis model . MMP-2 is constitutively expressed by RASFs, and the exact role of MMP-2 activity by these cells is still unclear.
Our unexpected finding that EPCR is not cytoprotective in RASFs has precedent in cancer cells. EPCR increases cell migration and invasion of breast cancer cells in vitro and is a possible biomarker of ovarian cancer onset . EPCR also promotes metastasis and correlates with clinical outcome in lung adenocarcinoma . However, vascular wall EPCR inhibits cancer cell adhesion and transmigration . The reasons for these contradictory findings are unclear but may reflect the different regulatory mechanisms of EPCR in different cell types and tissues.
EPCR can be regulated by proteolytic release from the cell surface to form sEPCR [12, 13]. sEPCR binds PC to inhibit APC generation or binds APC to block the protective function of APC . Pro-inflammatory cytokines IL-1β and TNF-α enhance EPCR shedding from the endothelial cell surface [16, 46]. Accordingly, higher levels of sEPCR have been reported in patients with systemic inflammatory diseases [15, 17, 47]. sEPCR produced by ovarian cancer cells is a possible biomarker of cancer onset  and is likely to be a biomarker of cancer-associated hypercoagulability in human hematologic malignancies . However, in the present study, we found that cell-associated EPCR is three times higher in cultured RASFs than in OASFs and that there is no difference in sEPCR, either in cultured supernatants of OASFs and RASFs or in synovial fluids from patients with OA and RA (Figure 1). These data suggest that it is not sEPCR, but the membrane bound form, that exerts the inflammatory and cartilage-degradative actions of RASFs. This destructive property of EPCR differs from its cytoprotective actions in other settings . The present study explored the reasons for this diametrical role of EPCR in RA. Although RASFs express PC/APC, neither silencing endogenous PC by siRNA nor adding recombinant APC significantly changed RASF viability (Additional file 1 shows this in more detail), indicating that this function of EPCR is not due to PC/APC. We found that this paradox may be explained by the actions of sPLA2V, which generates bioactive lipids LysoPCh and PAF. These two lipids can substitute for PCh, which normally resides in the deep groove of EPCR and is required for normal EPCR function. When LysoPCh or PAF substitutes for PCh, they impair the ability of EPCR to interact with PC or APC, thus inhibiting EPCR’s cytoprotective function in endothelial cells .
In the present study, RASFs expressed high levels of sPLA2V, which promoted the aggressive properties of RASFs. Suppressing endogenous sPLA2V reduced RASF viability, cartilage degradation ability, and IL-1β, whereas recombinant sPLA2V enhanced RASF-mediated cartilage degradation and NF-κB activation (Figure 6). Further study using dual immunostaining and co-immunoprecipitation indicated that sPLA2V and EPCR are spatially associated with each other on RASFs. We found that sPLA2V not only blocks APC binding to RASFs but also uses EPCR to promote its inflammatory effects on RASFs. This was evidenced by the fact that suppressing endogenous sPLA2V enhanced but that recombinant sPLA2V inhibited APC binding to RASFs (Figure 5). Moreover, suppression of EPCR reduced the stimulatory effect of sPLA2V on cartilage degradation and NF-κB activation by RASFs (Figure 6). We propose that, similar to its effect on endothelial cells , sPLA2V impairs the ability of EPCR to interact with APC in RASFs, inhibiting EPCR’s cytoprotective function.
In summary, this study demonstrated that elevated EPCR promotes the inflammatory responses and invasiveness of RASFs, which are likely driven by sPLA2V. These results provide new insights into the mechanisms underlying SF-mediated joint inflammation in RA and may inspire new targeted therapeutic approaches.
activated protein C
Dulbecco’s modified Eagle’s medium
enzyme-linked immunosorbent assay
endothelial protein C receptor
fetal bovine serum
colorimetric 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide
rheumatoid synovial fibroblast
soluble endothelial protein C receptor
small interfering RNA
group V secretory phospholipase A2
tumor necrosis factor-alpha.
We wish to thank Benjamin Chan from Raymond Purves Laboratory for his help in 1,9-dimethylmethylene blue (DMMB) assay. Financial support was provided by National Health and Medical Research Council, Royal North Shore Hospital Staff Specialist Trust fund, Arthritis Australia, and Henry Langley Research Fellowship.
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