Drug induced differential gene expression in normal and RA synovial fibroblasts cell lines
© The Author(s) 2004
Received: 16 January 2004
Published: 25 February 2004
Rheumatoid arthritis (RA) synovial fibroblasts destructively invade into joint cartilage. We could demonstrate in our pannus model of interactive three-dimensional tissue culture that the immortalized synovial fibroblast cells from a normal donor (K4IM) and a patient with RA (HSE) provide an in vitro system for differential invasiveness into artificial cartilage.
In this study, we investigated the molecular differences between these two cell lines and the influence of antirheumatic drugs on their expression profiles to gain insight into molecular pathways of joint destruction.
HSE and K4IM cells were characterized by immunostaining with Fibroblast-AK, CD3, CD31, CD68 and MAC3 antibodies. MTS-assay provided information for kinetics and effective concentration of MTX, decortin and diclofenac. Gene expression profiles were generated with HG-U133A Affymertrix GeneChips, analyzed by RMA, dCHIP and MAS5.0 software, and validated by RT-PCR, ELISA.
Positive fibroblast-AK staining confirmed the fibroblastoid phenotype of both cell lines. RA fibroblasts reacted more sensitively towards MTX-mediated cytotoxicity compared to K4IM fibroblasts. Gene expression profiling revealed 1303 differentially expressed genes between HSE and K4IM. MTX affected 191/1365, Decortin 84/172 and Diclofenac 19/0 genes in HSE/K4IM, respectively, as identified by all three softwares in common. Only 41, 26, or 8 genes were influenced in HSE towards the K4IM profile by MTX, Decortin or Diclofenac, respectively. Regulation of ten selected genes was confirmed by RT-PCR. Proteins in the supernatant from three of four genes revealed similar ratios by ELISA compared to transcription levels.
Only a minority of differentially regulated genes in the RA fibroblasts was positively influenced by current therapeutics. Those genes, which were not influenced, may be new potential candidates for therapeutic intervention.