Session Title: Genetics, Genomics and Proteomics I
Session Type: Abstract Submissions (ACR)
Background/Purpose: Long noncoding RNAs (lncRNAs) are increasingly recognized as master regulators of gene expression. The lncRNA NRON, noncoding repressor of nuclear factor of activated T cells (NFAT) can repress the cytoplasmic-nuclear translocation and function of NFAT1-4 transcriptional factors. Recently, we have reported that NRON regulates also the activity of NFAT5, affecting thereby the function of rheumatoid arthritis synovial fibroblasts (RASF). In addition, the ubiquitin-independent proteasome system has been shown to play an important role in regulating the cellular levels of NFAT5. Our objective was to investigate the regulation of NRON levels in RASF and to explore the role of NRON in protein turnover of NFAT5.
Methods: The levels and subcellular localization of NFAT5 protein in RASF were analyzed by Western blotting using α-tubulin for normalization. RASF were transfected with siRNA targeting NRON or scrambled siRNA using Lipofectamine 2000. RASF were treated with TNFα (10ng/ml), p38MAPK inhibitor (p38i, 10uM) SB202190, and/or proteasome inhibitor MG132 (1.0uM). Gene expression was measured by quantitative real-time PCR with normalization to GAPDH or β2-microglobulin. ELISA was used to measure IL-6 secretion from RASF.
Results: The levels of NRON were significantly decreased and the levels of NFAT5 protein were increased in RASF after 2hr of TNFα stimulation, while the levels of NFAT5 mRNA were not changed. Down regulation of NRON after silencing or TNFα stimulation was accompanied by the translocation of NFAT5 from the cytoplasm to the nucleus of RASF, increasing the transcription of known NFAT5 target genes, such as IL-6 (x-fold±SD: 3.0±2.2, p=0.03, n=4;) and MMP13 (x-fold±SD: 4.7±2.0, p=0.03, n=4). The secretion of IL-6 in the culture medium of RASF was also significantly increased (mean±S.D: 1676±479 vs 2433±504 pg/mL, p=0.007, n=5). The treatment of RASF with p38i significantly repressed not only the TNFα-induced up regulation of IL-6 but also the TNFα-induced down regulation of NRON (p= 0.001 and p= 0.04, n=5, respectively). Additionally, the proteasome-dependent degradation of NFAT5 was significantly enhanced by p38i (p= 0.01, n=5). Blocking the proteasome activity by MG132 inhibited the p38-induced degradation of NFAT5. Furthermore, the p38i-induced degradation of NFAT5 was inhibited also after silencing of NRON in RASF.
Conclusion: Our data show that TNFα down regulates the expression of lncRNA NRON in RASF by enhancing the activity of p38MAPK. Down regulation of NRON not only enhances the nuclear translocation and transcriptional activity of NFAT5 but also increases the total amount of NFAT5 in RASF by influencing the turnover of NFAT5 via ubiquitin-independent proteasome system. This novel data show the complex and multilevel capacities of the lncRNA NRON in regulating the function of NFAT5, thereby promoting proinflammatory and matrix-destructive responses of RASF.
IMI BTCure, EuroTEAM, IAR,
M. Frank Bertoncelj,
IMI BTCure, EuroTEAM, IAR,
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ACR Meeting Abstracts - https://acrabstracts.org/abstract/long-noncoding-rna-nron-regulates-the-activity-of-nfat5-through-ubiquitin-independent-proteasome-pathway-in-rheumatoid-arthritis/