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).

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.

• Authors: Kristin Andreas, Carsten Lübke, Thomas Häupl, Tilo Dehne, Lars Morawietz, Jochen Ringe, Christian Kaps, Michael Sittinger
• Journal: Arthritis research & therapy (Arthritis Res Ther) Vol. 10 Issue 1 Pg. R9 ( 2008) ISSN: 1478-6362 [Electronic] England
• PMID: 18205922 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Topics

• Arthritis, Rheumatoid (metabolism, pathology)
• Cartilage, Articular (metabolism, pathology)
• Cells, Cultured
• Chondrocytes (metabolism)
• Computer Systems
• Fibroblasts (metabolism)
• Gene Expression Profiling
• Humans
• In Vitro Techniques
• Oligonucleotide Array Sequence Analysis
• Proteomics
• Reproducibility of Results
• Reverse Transcriptase Polymerase Chain Reaction
• Synovial Membrane (metabolism, pathology)