title: Key regulatory molecules of cartilage destruction in rheumatoid arthritis: an in vitro study.
abstract: BACKGROUND
  Rheumatoid 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.
  
  
  METHODS
  Human 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.
  
  
  RESULTS
  Antibody-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-kappaB signaling pathway (toll-like receptor 2, spermine synthase, receptor-interacting serine-threonine kinase 2), cytokines/chemokines and receptors (CXCL1-3, CXCL8, CCL20, CXCR4, IL-1beta, IL-6), cartilage degradation (matrix metalloproteinase (MMP)-10, MMP-12) and suppressed matrix synthesis (cartilage oligomeric matrix protein, chondroitin sulfate proteoglycan 2).
  
  
  CONCLUSIONS
  Differential 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.
authors.affiliation: Tissue Engineering Laboratory and Berlin - Brandenburg Center for Regenerative Therapies, Department of Rheumatology, Charité - Universitätsmedizin Berlin, Tucholskystrasse 2, 10117 Berlin, Germany. kristin.andreas@charite.de
authors.forename: Kristin
authors.initials: K
authors.lastname: Andreas
authors.forename: Carsten
authors.initials: C
authors.lastname: Lübke
authors.forename: Thomas
authors.initials: T
authors.lastname: Häupl
authors.forename: Tilo
authors.initials: T
authors.lastname: Dehne
authors.forename: Lars
authors.initials: L
authors.lastname: Morawietz
authors.forename: Jochen
authors.initials: J
authors.lastname: Ringe
authors.forename: Christian
authors.initials: C
authors.lastname: Kaps
authors.forename: Michael
authors.initials: M
authors.lastname: Sittinger
mesh_terms: D001172:Arthritis, Rheumatoid / Q000378:metabolism* / Q000473:pathology*; D002358:Cartilage, Articular / Q000378:metabolism* / Q000473:pathology*; D002478:Cells, Cultured; D019902:Chondrocytes / Q000378:metabolism*; D003199:Computer Systems; D005347:Fibroblasts / Q000378:metabolism; D020869:Gene Expression Profiling; D006801:Humans; D066298:In Vitro Techniques; D020411:Oligonucleotide Array Sequence Analysis; D040901:Proteomics; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013583:Synovial Membrane / Q000378:metabolism / Q000473:pathology