Transcription profiling of human 3D cultured chrondrocytes treated with supernatant of synovial fibroblasts from an RA patients vs controls to investigate key regulatory molecules of cartilage destruction in rheumatoid arthritis

We have studied the expression profile of 3D cultured human chondrocytes that were stimulated with supernatant of synovial fibroblasts derived from a RA patient (RASF=HSE cell line) and from a normal donor (NDSF=K4IM cell line), respectively. For this purpose, passage 2 human chondrocytes were cultured for 14 days in alginate beads and subsequently stimulated for 48 hours with supernatant of RASF and NDSF. Baseline expression was determined of unstimulated chondrocytes. Differential genome-wide microarray analysis of RASF and NDSF stimulated chondrocytes disclosed a distinct expression profile related to cartilage destruction involving marker genes of inflammation (COX-2), NF-kappa B signaling pathway (TLR2), cytokines/chemokines and receptors (CXCL1-3, CCL20, CXCL8, CXCR4, IL-6, IL-1beta), matrix degradation (MMP-10, MMP-12) and suppressed matrix synthesis (COMP). Thus, transcriptome profiling of RASF and NDSF stimulated chondrocytes revealed a disturbed catabolic-anabolic homeostasis of chondrocyte function. This study provides a comprehensive insight into the molecular regulatory processes induced in human chondrocytes during RA-related cartilage destruction. Experiment Overall Design: To reveal the RA-related chondrocyte gene expression profile, genome-wide microarray analysis of RASF stimulated human chondrocytes compared to NDSF stimulation was performed. Unstimulated chondrocytes were analyzed for baseline gene expression. For microarray analysis of RASF and NDSF stimulated and unstimulated chondrocytes, respectively, two RNA pools were analyzed, each pool consisting of equal amounts of RNA from three different donors.