1. Date : 2014.04.30.(Wen) pm 17:00 ~ 18:00
2. Venue : B101 , Institute of Biomedical Science and Technology
3. Speaker : Wan-Uk Kim, M.D./Ph.D.
Department of Internal Medicine, Division of Rheumatology
Catholic University of Korea, Seoul, Korea
Identification of novel targets for rheumatoid arthritis through a systems
Rheumatoid arthritis (RA) is a chronic autoimmune disease that primarily attacks synovial joints. Despite the advances in diagnosis and treatment of RA, novel molecular targets are still needed to improve the accuracy of diagnosis and the therapeutic outcomes. Here, we present a systems approach that can effectively 1) identify core RA-associated genes (RAGs), 2) reconstruct RA-perturbed networks, and 3) select potential targets for diagnosis and treatments of RA. By integrating multiple gene expression datasets previously reported, we first identified 983 core RAGs that show RA dominant differential expression, compared to osteoarthritis (OA), in the multiple datasets. Using the core RAGs, we then reconstructed RA-perturbed networks that delineate key RA associated cellular processes and transcriptional regulation. The networks revealed that synovial fibroblasts play major roles in defining RA-perturbed processes, anti-TNF-a therapy restored many RA-perturbed processes, and 19 transcription factors (TFs) have major contribution to deregulation of the core RAGs in the RA-perturbed networks. Finally, we selected a list of potential molecular targets that can act as metrics or modulators of the RA-perturbed networks. In particular, we identified NFAT5 as a novel target for RA through this approach, and its validity was confirmed by the in vitro and in vivo functional tests.
NFAT5 is originally identified as an osmo-protective transcription factor whose DNA binding domain shares a structural homology with NF-kB and other member of NFAT family. Recently, we identified first that NFAT5 is critical for rheumatoid arthritis pathogenesis (Yoon HJ, at al. Arthritis Rheum 2011), suggesting that NFAT5 may be an effective therapeutic target. However, there is no specific NFAT5 inhibitor identified by now. In the present study, we tried to find out small molecules to specifically block NFAT5 activity. We first identified that NFAT5 is involved in the production of nitric oxide (NO) and IL-6 by RAW 264.7 macrophages stimulated with LPS. Based on this, we screened small molecules (>150,000) to suppress NO production by RAW 264.7 macrophages using high-throughput drug screening. As a result, we found more than 200 candidate chemicals that strongly inhibited the production of NO, a NFAT5-target gene (IC50 < 1 mM). We next tested a direct NFAT5 inhibitory activity of the candidate chemicals using RAW 264.7 macrophages transfected stably with a novel NFAT5-specific GFP reporter in the presence of LPS. Through this way, we could select a couple of chemicals (e.g. compound 2,3,4 and 5) based on the NFAT5 inhibitory potency as well as on in vitro cytotoxicity. The candidate chemicals did not affect NFAT1-4, NF-kB, p38 MAP kinase and CREB activity, indicating the specificity of the candidates molecules for NFAT5. Interestingly, the candidates did not down-regulate the high-salt-induced NFAT5 expression in RAW 264.7 macrophages, suggesting that they may not alter cellular homeostasis in the kidney. We also found that a candidate chemical compound inhibited LPS-induced IL-6, TNF-a, GM-CSF2, iNOS, and NO production by RAW 264.7 macrophages, while it did not down-regulated AR and BGT1 mRNA expressions that are involved in osmo-protection and cellular homeostasis. Finally, we intra-peritonealy injected the chemical compound into mice with experimental arthritis, and found significant suppression of arthritis severity without serious side effects. Moreover, oral feeding of a derivative of the chemical compound successfully inhibited the progression of arthritis in mice. In summary, we identified a specific inhibitor of NFAT5 that potently inhibits the production of pro-inflammatory mediators and that suppresses experimentally-induced arthritis in mice.
Rheumatoid synoviocytes, which consist of fibroblast-like synoviocytes (FLS) and synovial macrophages (SM), are crucial for the progression of RA. Particularly, FLS of RA patients (RA-FLS) exhibit invasive characteristics reminiscent of cancer cells, destroying cartilage and bone. RA-FLS and SM originate differently from mesenchymal and myeloid cells, respectively, but share many pathologic functions. However, the molecular signatures and biological networks representing the distinct and shared features of the two cell types are unknown. Presently, we performed global transcriptome profiling of FLS and SM obtained from RA and osteoarthritis patients. By comparing the transcriptomes, we identified distinct molecular signatures and cellular processes defining invasiveness of RA-FLS and pro-inflammatory properties of RA synovial macrophages (RA-SM), respectively. Interestingly, under the interleukin1b-stimulated condition, the RA FLS newly acquired pro-inflammatory signature dominant in RA-SM without losing invasive properties. We next reconstructed a network model that delineates the shared, RA-FLS-dominant (invasive), and RA-SM-dominant (inflammatory) processes. From the network model, we selected 13 genes, including POSTN and TWIST1, as novel regulator candidates responsible for FLS invasiveness. Of note, POSTN and TWIST1 expressions were elevated in independent RA-FLS and were further instigated by interleukin1β. Functional assays demonstrated the requirement of POSTN and TWIST1 for migration and invasion of RA-FLS stimulated with interleukin1β. Taken together, our systems approach to rheumatoid synovitis provides a basis for identifying novel regulators responsible for pathological features of RA-FLS and RA-SM, demonstrating how a certain type of cells acquires functional redundancy under chronic inflammatory conditions.