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Arthritis Research and Therapy

, 10:R9

First Online: 18 January 2008Received: 13 July 2007Revised: 28 December 2007Accepted: 18 January 2008


BackgroundRheumatoid arthritis RA is a chronic, inflammatory and systemic autoimmune disease that leads to progressive cartilage destruction. Advances in the treatment of RA-related destruction of cartilage require profound insights into the molecular mechanisms involved in cartilage degradation. Until now, comprehensive data about the molecular RA-related dysfunction of chondrocytes have been limited. Hence, the objective of this study was to establish a standardized in vitro model to profile the key regulatory molecules of RA-related destruction of cartilage that are expressed by human chondrocytes.

MethodsHuman chondrocytes were cultured three-dimensionally for 14 days in alginate beads and subsequently stimulated for 48 hours with supernatants from SV40 T-antigen immortalized human synovial fibroblasts SF derived from a normal donor NDSF and from a patient with RA RASF, respectively. To identify RA-related factors released from SF, supernatants of RASF and NDSF were analyzed with antibody-based protein membrane arrays. Stimulated cartilage-like cultures were used for subsequent gene expression profiling with oligonucleotide microarrays. Affymetrix GeneChip Operating Software and Robust Multi-array Analysis RMA were used to identify differentially expressed genes. Expression of selected genes was verified by real-time RT-PCR.

ResultsAntibody-based protein membrane arrays of synovial fibroblast supernatants identified RA-related soluble mediators IL-6, CCL2, CXCL1–3, CXCL8 released from RASF. Genome-wide microarray analysis of RASF-stimulated chondrocytes disclosed a distinct expression profile related to cartilage destruction involving marker genes of inflammation adenosine A2A receptor, cyclooxygenase-2, the NF-κB signaling pathway toll-like receptor 2, spermine synthase, receptor-interacting serine-threonine kinase 2, cytokines-chemokines and receptors CXCL1–3, CXCL8, CCL20, CXCR4, IL-1β, IL-6, cartilage degradation matrix metalloproteinase MMP-10, MMP-12 and suppressed matrix synthesis cartilage oligomeric matrix protein, chondroitin sulfate proteoglycan 2.

ConclusionDifferential transcriptome profiling of stimulated human chondrocytes revealed a disturbed catabolic–anabolic homeostasis of chondrocyte function and disclosed relevant pharmacological target genes of cartilage destruction. This study provides comprehensive insight into molecular regulatory processes induced in human chondrocytes during RA-related destruction of cartilage. The established model may serve as a human in vitro disease model of RA-related destruction of cartilage and may help to elucidate the molecular effects of anti-rheumatic drugs on human chondrocyte gene expression.

AbbreviationsADORA2A= adenosine A2A receptor

BCL2A1= BCL2-related protein A1

CMKOR= chemokine orphan receptor

COMP= cartilage oligomeric matrix protein

COX= cyclooxygenase

CSPG= chondroitin sulfate proteoglycan

ECM= extracellular matrix

GCOS= GeneChip Operating Software

Gro= growth-related oncogene

IFI-6–16= interferon-α inducible protein-6–16

IL= interleukin

MCP= monocyte chemoattractant protein

MMP= matrix metalloproteinase

NDSF= synovial fibroblast cell line derived from normal donor

NDSFsn= supernatant of NDSF

NF= nuclear factor

OAS1 = 2-5-oligoadenylate synthetase 1

PGES= prostaglandin E synthase

RA= rheumatoid arthritis

RASF= synovial fibroblast cell line derived from patient with RA

RASFsn= supernatant of RASF

RIPK= receptor-interacting serine-threonine kinase

RMA= Robust Multi-array Analysis

RT-PCR= polymerase chain reaction with reverse transcription

SF= synovial fibroblasts

SMS= spermine synthase

STAT= signal transduction and activators of transcription

STS= steroid sulfatase

THBS= thrombospondin

TLR= toll-like receptor

TNF= tumor necrosis factor

TXNIP= thioredoxin interacting protein.

Electronic supplementary materialThe online version of this article doi:10.1186-ar2358 contains supplementary material, which is available to authorized users.

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Autor: Kristin Andreas - Carsten Lübke - Thomas Häupl - Tilo Dehne - Lars Morawietz - Jochen Ringe - Christian Kaps - Michael S


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