Endothelial protein C receptor-associated invasiveness of rheumatoid synovial fibroblasts is likely driven by group V secretory phospholipase A2Reportar como inadecuado




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Arthritis Research and Therapy

, 16:R44

First Online: 05 February 2014Received: 11 June 2013Accepted: 28 January 2014

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.

AbbreviationsAPCactivated protein C

DMEMDulbecco’s modified Eagle’s medium

ELISAenzyme-linked immunosorbent assay

EPCRendothelial protein C receptor

FBSfetal bovine serum

ILinterleukin

IPimmunoprecipitation

LysoPChlysophosphotidylcholine

MAPmitogen-activated protein

MMPmatrix metalloproteinase

MTTcolorimetric 3-4,5-dimethylthiazol-2-yl-2,5-diphenyl tetrazolium bromide

NF-κBnuclear factor-kappa-B

OAosteoarthritis

PAFplatelet-activating factor

PCprotein C

RArheumatoid arthritis

RASFrheumatoid synovial fibroblast

sEPCRsoluble endothelial protein C receptor

SFsynovial fibroblast

sGAGsulphated glycosaminoglycan

siRNAsmall interfering RNA

sPLA2Vgroup V secretory phospholipase A2

TNF-αtumor necrosis factor-alpha.

Electronic supplementary materialThe online version of this article doi:10.1186-ar4473 contains supplementary material, which is available to authorized users.

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Autor: Meilang Xue - Kaitlin Shen - Kelly McKelvey - Juan Li - Yee-Ka Agnes Chan - Vicky Hatzis - Lyn March - Christopher B Lit

Fuente: https://link.springer.com/



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