Session Title: Rheumatoid Arthritis - Pathogenetic Pathways
Session Type: Abstract Submissions (ACR)
A recent genome wide association study identified the variant rs26232 in the first intron of an uncharacterized gene C5orf30 as a rheumatoid arthritis susceptibility variant. In addition, it has been associated with severity of radiological joint damage suggesting a role in tissue breakdown1. To date there is no function assigned for C5orf30 and neither the gene or protein show homology to any known functional sequences. However, C5orf30 is highly conserved in chimpanzee, dog, cow, mouse, chicken, and zebrafish (orthologs). The aim of this study is to determine the biological role of C5orf30 in rheumatoid arthritis synovial fibroblast.
Methods: Immunohistochemistry on synovial samples was used to determine expression of C5orf30 including co-localisation using antibodies to macrophages (CD68), fibroblasts (5B5), T (CD3) & B (CD19) cells. Real time PCR and western blotting were used to examine C5orf30 transcript and protein levels in fibroblast-like synovial cells (FLS stimulated with TNF & hypoxia). To investigate gene function siRNA was used to knockdown either C5orf30 or a non-targeting control (NTC) in synovial FLS in vitro.
Confocal microscopy revealed C5orf30 to be strongly expressed in both the nuclear and cytoplasmic compartment of synovial lining cells including macrophages and fibroblasts, but not T & B cells. C5orf30 was undetectable in arthroscopy sections obtained from osteoarthritis or control synovium. C5orf30 was expressed in FLS and was found to be up-regulated by hypoxia (8-fold) and down-regulated by TNF (0.5-fold). We found that C5orf30KD compared to the NTC increased the number of invading FLS using the Matrigel invasion assay (p=0.01) and increased FLS migration using a scratch assay (p=0.02) (n=6). Preliminary gene profiling studies suggest that multiple gene sets involved in cell proliferation, intracellular signal transduction, angiogenesis, and immune and inflammatory pathways were significantly modified following C5orf30KD.
C5orf30 knockdown increased FLS migration and invasion into matrigel suggesting C5orf30 is negatively regulating cellular invasion. Together this identifies a potentially novel pathway mediating tissue damage in RA. We are currently performing proteomic and gene profiling (after C5orf30 KD) studies in order to work out the biology of this important marker in the pathogenesis and severity of RA.
1Teare, M.D., et al (in press). Allele dose association of the C5orf30 rs26232 variant with joint damage in rheumatoid arthritis, Arthritis and Rheumatism.
G. A. Adeleke,
A. G. Wilson,
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ACR Meeting Abstracts - https://acrabstracts.org/abstract/biological-roles-of-c5orf30-in-rheumatoid-arthritis-synovial-fibroblasts/