Novel tumor necrosis factor α–regulated genes in rheumatoid arthritis

Abstract

Objective. To determine novel genes regulated by tumor necrosis factor ␣ (TNF␣) signaling in primary rheumatoid arthritis synovial fibroblasts (RASFs).Methods. Oligonucleotide microarrays were used to measure gene expression levels in 6 independent replicate samples of RASFs. RASFs were transfected for 18 hours with AdIB-dominant negative (AdIB-DN) (n ‫؍‬ 3) or with control AdTet expressing the reverse tetracycline trans-activator (n ‫؍‬ 3). The cells were stimulated for 3 hours with TNF␣, and total RNA was prepared. Several novel parametric and nonparametric methods were used to rank genes in terms of the magnitude and significance of intergroup differences. Microarray expression differences were confirmed by real-time quantitative reverse transcription-polymerase chain reaction. Small interfering RNA (siRNA) was used to specifically down-modulate microarrayidentified genes to demonstrate their role in the promotion of apoptosis, proliferation, or matrix metalloproteinase (MMP) expression. Rheumatoid arthritis (RA) is associated with inflammation of the synovium and development of RA synovial fibroblasts (RASFs) that undergo hyperplasia and invade cartilage and bone (1). RASFs exhibit an increased ability to enter into the cell cycle and, therefore, to undergo hyperplasia (2,3), and they show a decreased ability to undergo apoptosis (4,5). RASFs produce proinflammatory cytokines, such as interleukin-1 (IL-1) and tumor necrosis factor ␣ (TNF␣), which provide further stimulation for the inflammation in RA (6), as well as enzymes, including stromelysin and collagenase, which are capable of invading cartilage and bone (7,8). Thus, RASFs are unique, and their altered properties of growth and production of proinflammatory and matrix-degrading proteins are central to the erosion of cartilage and bone that is seen in RA. Results. Blocking of NF-B byThe cytokine TNF␣ is central to the development and continued growth and invasion of RASFs (9).

Synovial fibroblasts promote osteoclast formation by RANKL in a novel model of spontaneous erosive arthritis

“…We also have demonstrated that these fibroblasts exhibit abnormal up-regulation of numerous genes, including cytokines, chemokines, inflammation mediators, and members of the TNF superfamily of receptors, including RANKL (18). Other investigators have observed that survival genes, such as FLIP and SUMO-1, are expressed by synovial fibroblasts (33,34).…”

“…The close association of the macrophages and MSFs, together with the abnormal osteoclast formation at the sites of bone erosion, suggests that the fibroblastmacrophage interaction may lead to osteoclast formation. To determine whether the spontaneous erosion and osteoclast formation in BXD2 mice is due to either an abnormal MSF phenotype, an abnormal macrophage phenotype, or both, MSFs were derived from BXD2 mice as well as B6 and D2 mice, as described previously (18). After 7 days of culture, primary cultures of MSFs were established, as indicated by the expression of vimentin ( Figure 3A), an intermediate filament that is expressed in fibroblasts but not macrophages (19).…”

“…RA synovial fibroblasts exhibit various abnormalities, including increased proliferation, increased production (28), and resistance to apoptosis (25,(29)(30)(31)(32). We have previously shown that fibroblasts from patients with RA exhibit increased signaling through the phosphatidylinositol 3-kinase, Akt, and NF-B pathways (18).…”

Molecular signatures and new candidates to target the pathogenesis of rheumatoid arthritis

“…More relevant to RA disease in particular was the attempt to generate a molecular profile of synovial tissue from RA patients using primary synovial fibroblasts (38,50). The analysis of gene expression patterns of primary as well as cultured synovial fibroblasts revealed the involvement of the TNF-␣ pathway in the pathogenesis of RA (24,79). Array-based approaches in synovial tissues, being the key target of the disease, showed lower variation of gene expres-sion differences in different synovial biopsies from the same patient compared with those of different patients (46).…”

Differential Gene Expression Modulated by the Cytoplasmic Domain of FcγRIa (CD64) α-Chain

“…Accordingly, we performed experiments at two time points (1.5 and 3 h after receptor-specific cross-linking) to screen for genes differentially regulated by the WT and TL receptors. To identify target genes putatively regulated by the CY of Fc␥RIa ␣-chain, we analyzed the hybridization data to identify genes with a hybridization signal with Ն2-fold difference in signal intensity between the WT and TL cell lines in three independent experiments and with a statistically significant differences (regular t test with a Bonferroni correction; p Ͻ 0.05) as described (22). Based on these criteria, we identified five genes which are immunologically related to the functions of macrophages (Table II).…”