Key regulatory molecules of cartilage destruction in rheumatoid arthritis: an in vitro study.
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.
Andreas
Kristin
K
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
Lübke
Carsten
C
Häupl
Thomas
T
Dehne
Tilo
T
Morawietz
Lars
L
Ringe
Jochen
J
Kaps
Christian
C
Sittinger
Michael
M
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