NFAT5 (nuclear factor of activated T cells), a well-known osmoprotective factor, can be activated by isotonic stimuli such as Toll-like receptor (TLR) triggering. However, it is unclear how NFAT5 discriminates between isotonic and hypertonic stimuli to produce different functional and molecular outcomes. Here, we identified a novel XO-ROS-p38 MAPK-NFAT5 pathway (XO is xanthine oxidase, ROS is reactive oxygen species) that is activated in RAW 264.7 macrophages upon isotonic TLR stimulation. Unlike what is seen under hypertonic conditions, XO-derived ROS were selectively required for the TLR-induced NFAT5 activation and NFAT5 binding to the IL-6 promoter in RAW 264.7 macrophages under isotonic conditions. In mouse peritoneal macrophages and human macrophages, TLR ligation also induced NFAT5 activation, which was dependent on XO and p38 kinase. The involvement of XO in NFAT5 activation by TLR was confirmed in RAW 264.7 macrophages implanted in BALB/c mice. Moreover, allopurinol, an XO inhibitor, suppressed arthritis severity and decreased the expression of NFAT5 and IL-6 in splenic macrophages in C57BL/6 mice. Collectively, these data support a novel function of the XO-NFAT5 axis in macrophage activation and TLR-induced arthritis, and suggest that XO inhibitor(s) could serve as a therapeutic agent for chronic inflammatory arthritis.Keywords: Inflammatory arthritis r Innate immunity r NFAT5 r Reactive oxygen species r Xanthine oxidase Additional supporting information may be found in the online version of this article at the publisher's web-site IntroductionThe nuclear factor of activated T cells (NFAT) family is a group of five versatile transcription factors that are crucial to the develCorrespondence: Dr. Wan-Uk Kim e-mail: wan725@catholic.ac.kr opment and function of the immune system. Among the five members of the NFAT family (NFAT1-5), NFAT5 is unique in that it is the only protein that responds to hypertonicity [1]. NFAT5 is very similar to a Drosophila transcription factor involved * These authors contributed equally to this work. 2722 Nam-Hoon Kim et al. Eur. J. Immunol. 2014. 44: 2721-2736 in the defense against hypertonicity [2], indicating that it may represent an ancient prototype among members of the NFAT family. Indeed, the ability to guard cells against osmotic damage is commonly required in invertebrates as well as in vertebrates including humans. Accordingly, the role of NFAT5 is most pronounced in tissues constantly exposed to osmotic stress, such as kidney medulla epithelia, intestine epithelia, skin epidermis, and cornea [3]. Interestingly, NFAT5 is also activated in nonhypertonic conditions. Biologic processes that accompany NFAT5 activation include integrin-mediated carcinoma invasion [4], migration and differentiation of myoblasts [5], and T-cell proliferation [6]. Additionally, we have demonstrated for the first time that NFAT5 activity is increased by tumor necrosis factor alpha (TNF-α) and interleukin (IL) 1β stimulation under isotonic conditions [7]. NFAT5-deficient mice show a marked r...

Nuclear factor of activated T cells 5 (NFAT5) has been implicated in the pathogenesis of various human diseases, including cancer and arthritis. However, therapeutic agents inhibiting NFAT5 activity are currently unavailable. To discover NFAT5 inhibitors, a library of > 40,000 chemicals was screened for the suppression of nitric oxide, a direct target regulated by NFAT5 activity, through high-throughput screening. We validated the anti-NFAT5 activity of 198 primary hit compounds using an NFAT5-dependent reporter assay and identified the novel NFAT5 suppressor KRN2, 13-(2-fluoro)-benzylberberine, and its derivative KRN5. KRN2 inhibited NFAT5 upregulation in macrophages stimulated with lipopolysaccharide and repressed the formation of NF-κB p65-DNA complexes in the NFAT5 promoter region. Interestingly, KRN2 selectively suppressed the expression of pro-inflammatory genes, including Nos2 and Il6, without hampering high-salt-induced NFAT5 and its target gene expressions. Moreover, KRN2 and KRN5, the latter of which exhibits high oral bioavailability and metabolic stability, ameliorated experimentally induced arthritis in mice without serious adverse effects, decreasing pro-inflammatory cytokine production. Particularly, orally administered KRN5 was stronger in suppressing arthritis than methotrexate, a commonly used anti-rheumatic drug, displaying better potency and safety than its original compound, berberine. Therefore, KRN2 and KRN5 can be potential therapeutic agents in the treatment of chronic arthritis.

GREM1 is highly expressed in RA joints, and functions as a regulator of survival, proliferation, migration, and invasion of RA-FLS.

Fibroblast like synoviocyte plays critical roles is the major cell in the inflammatory processes of Rheumatoid arthritis. We attempted to identify a key regulator that can drive aggressive phenotypes of RA FLS. For this, we performed an integrative transcriptome data analysis using three datasets from independent RA studies, a panel of normal tissues, and NCI60 cancer cell line panel. Through the analysis, we identified Gremlin (GREM1), which is robustly up-regulated in RA and predominantly expressed in FLS. GREM1 is identified as bone morphogenetic proteins antagonist and is known to bind BMPs. To investigate potential roles of GREM1 in RA, we first examined the level of GREM1 expression in synovial tissues, synovial fluid (SF) and FLS from RA. Immunohistochemistry and ELISA revealed up-regulation of GREM1 protein in RA compared to OA. Proinflammatory cytokines also induced the expression of GREM1 in RA FLS and their expressions were significantly correlated with GREM1 expression in RA SF. We next investigated cellular functions of GREM1 using RA FLS. GREM1 deficieny by siRNA elicited significant suppression of FLS proliferation, migration and invasion. In contrast, stimulation with rhGREM1 to RA FLS increased cell survival and invasion. Furthermore, we found that GREM1 activates AKT1 and MAPK signaling cascade, as well as antagonizes BMP2. This study demonstrated that GREM1 is a key regulator responsible for synovial hyperplasia and FLS migration.