Session Title: Rheumatoid Arthritis - Pathogenetic Pathways
Session Type: Abstract Submissions (ACR)
Long noncoding RNAs (lncRNAs) are critical regulators of gene expression and of major biological processes, such as cell migration, proliferation and invasion. The lncRNA NRON, Noncoding Repressor of Nuclear factor of activated T cells (NFAT), was recently shown to repress the cytoplasmic-nuclear translocation and function of NFAT1-4 transcription factors. It is unknown, however, whether NRON can regulate the function of NFAT5, which was reported to influence the migration and proliferation of rheumatoid arthritis synovial fibroblasts (RASF).
Here we investigated the expression and regulation of NRON in RASF and analyzed its role in regulating the function of NFAT5.
The expression of NFAT5 protein in RA (n=3) and osteoarthritis (OA) (n=3) synovial tissues was analyzed by immunohistochemistry. RASF were transfected with siRNA targeting NRON (n=4) or NFAT5 (n=4) using Lipofectamine 2000. Gene expression in SF was measured by quantitative real-time PCR with normalization to GAPDH or β2-microglobulin. Successful silencing of NFAT5 in SF was confirmed by Western blotting using α-tubulin for normalization. The secretion of IL-6 was measured by ELISA. Immunofluorescence microscopy was used to investigate the cytoplasmic- nuclear trafficking of NFAT5 in RASF transfected with siNRON or stimulated with TNFα (10ng/ml). The formation of stress fibers in SF after silencing of NRON was assessed by phalloidin staining. Migration of SF was analyzed by scratch assay.
The expression of NFAT5 protein was increased in RA compared to OA synovial tissues. The constitutive levels of NFAT5 mRNA (mean dCt±SD: 5.1±0.5 vs 5.8±0.7, p= 0.01) and NRON (mean dCt±SD: 11.7±0.7 vs 13.0±0.5, p= 0.01) were significantly up regulated in RASF (n=15) compared with OASF (n=11). The expression of NRON significantly correlated with the levels of NFAT5 mRNA in RASF (R=0.71, p=0.003). Furthermore, silencing of NFAT5 in RASF resulted in a down regulation of NRON (x-fold±SD: 0.57±0.3, p=0.07, n=4), indicating that NFAT5 regulates the expression of NRON in RASF. Stimulation of RASF with TNFα (2h) also significantly decreased the expression of NRON (by 26.9±25%, p= 0.02, n=7). Importantly, down regulation of NRON in RASF after silencing or TNFα stimulation was accompanied by the translocation of NFAT5 protein from the cytoplasm to the nucleus and by up regulation of NFAT5 target genes, including IL-6 (x-fold±SD: 3.0±2.2, p=0.03, n=4) and MMP-13 (x-fold±SD: 4.7±2.0, p= 0.03) mRNAs. The levels of IL-6 protein also significantly increased by silencing of NRON compared to control (mean±S.D: 1676±479 vs 2433±504 pg/mL, p= 0.007, n=5). Furthermore, silencing of NFAT5 resulted in a significant down regulation of MMP-13 mRNA (x-fold±SD: 0.58±0.2, p=0.02, n=4). The formation of stress fibers (n=3) and the migratory abilities of RASF (n=4) were increased after silencing of NRON.
Our data indicate that the lncRNA NRON regulates gene expression profiles, stress fiber formation and migration of RASF in a NFAT5-dependent manner and can therefore significantly contribute to the pathogenic characteristics of activated synovial fibroblasts in RA.
Japan Rheumatism Fundation,
IAR, EURO-TEAM, IMI BTCure,
EURO-TEAM, IMI BTCure, IAR Epalinges, KFSP USZ,
B. A. Michel,
R. E. Gay,
EURO-TEAM, IMI BTCure, IAR Epalinges,
EURO-TEAM, IMI BTCure, IAR Epalinges, ,
M. Frank Bertoncelj,
IAR, EURO-TEAM, IMI BTCure,
« Back to 2013 ACR/ARHP Annual Meeting
ACR Meeting Abstracts - https://acrabstracts.org/abstract/long-noncoding-rna-nron-regulates-the-cytoplasmic-nuclear-shuttling-and-activity-of-nfat5-in-rheumatoid-arthritis-synovial-fibroblast/