Background Synovial fibroblasts (SF) play an important role in chronic inflammation and joint destruction in rheumatoid arthritis (RA). While many anti-apoptotic mechanisms in RA-SF have been demonstrated, various molecules promoting the proliferation of RA-SF were also believed to participate in the pathogenesis of RA.
Objectives The aim of this study was to investigate the expression profile of RA-SF during proliferation and to explore the molecular mechanisms of synovial proliferation in RA.
Methods Cultured RA-SF in passage 6 from one patient were used for cDNA subtraction and RT-PCR. Total RNA was extracted from two culture conditions, 4 days after replating (low-density cells; LD) and 14 days after replating (high-density cells; HD), respectively. One μg of total RNA from each culture was used for cDNA synthesis with the SMART PCR cDNA Synthesis Kit (CLONTECH), then suppression subtraction hybridization was performed with the PCR-Select cDNA Subtraction Kit (CLONTECH). Forward (LD-specific) and reverse (HD-specific) subtracted cDNA was subcloned into PCR-Script Amp SK (+) cloning vector (STRATAGENE). The nucleotide sequences of these clones were analysed by commercial sequencing (Microsynth), then BLAST searches (NCBI) were performed to identify each clone. mRNA expression of distinct clones in RA-SF were confirmed by RT-PCR with primers specific for each gene. The expression and distribution of distinct genes were examined on paraffin-embedded sections of synovial tissues from 7 patients with RA and 3 non-RA patients by in situ hybridization with digoxigenin-labelled RNA probes specific for each gene.
Results 44 clones were upregulated in LD cells and 44 clones were upregulated in HD cells, respectively. 46 of 88 clones were identical to the sequences that have been already identified and characterised, including the sequences of enzymes for metabolic pathways (i.e., aldose reductase, glutamine synthetase), cytoskeletal proteins (i.e., CDC10, EPLIN), proliferation-associated molecules (i.e., S100A4, NAP-1 related protein), matrix proteins (i.e., EFEMP1, versican), and ribosomal proteins. On the other hand, 29 clones were identical to cDNAs that have been already identified, but with unknown functions so far. 13 gene fragments did not show significant homology to any sequence in the GenBank (NCBI). mRNA expression of distinct clones were confirmed by RT-PCR. Moreover, in situ hybridization showed that distinct genes, such as Fbx3 and S100A4, were also expressed on synovial tissues from patients with RA but not from non-RA patients, suggesting that these molecules may contribute to the pathogenesis of RA.
Conclusion In this study, a number of molecules, including several novel genes, were isolated from RA-SF using suppression subtraction hybridization. The expression of these molecules may be involved in synovial activation in the pathogenesis of RA. *K. Masuda is supported by Japan Rheumatism Foundation.
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