Research article

Key regulatory molecules of cartilage destruction in rheumatoid arthritis: an in vitro study

Kristin Andreas¹ , Carsten Lübke² , Thomas Häupl² , Tilo Dehne² , Lars Morawietz³ , Jochen Ringe¹ , Christian Kaps⁴ and Michael Sittinger²

¹  Tissue Engineering Laboratory and Berlin – Brandenburg Center for Regenerative Therapies, Department of Rheumatology, Charité – Universitätsmedizin Berlin, Tucholskystrasse 2, 10117 Berlin, Germany

²  Tissue Engineering Laboratory, Department of Rheumatology, Charité – Universitätsmedizin Berlin, Tucholskystrasse 2, 10117 Berlin, Germany

³  Institute for Pathology, Charité – Universitätsmedizin Berlin, Charitéplatz 1, 10117 Berlin, Germany

⁴  TransTissueTechnologies GmbH, Tucholskystrasse 2, 10117 Berlin, Germany

author email corresponding author email

Arthritis Research & Therapy 2008, 10:R9doi:10.1186/ar2358

Published:	18 January 2008

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-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).

Conclusion

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.