Interestingly we found that high salt also increased

Interestingly, we found that high salt also increased the translocation of p65 as well the degradation of IκB in RAW 264.7 macrophages (Supplementary Fig. 5), which is in parallel with a previous report (Roth et al., 2010). These data indicates that the NF-κB pathway is activated by hypertonicity in these cells. Given the requirement of NF-κB activity for NFAT5 transcription, it is unclear why KRN2 failed to hamper high salt-induced NFAT5 upregulation and its target gene expression, including Ar and Smit. A possible explanation would be the differential activation of co-regulators depending on the kinds of stimuli. We have demonstrated that ROS are essential for NFAT5 transcription, but their source differs depending on the context: mitochondria for high salt and xanthine oxidase for TLR (Kim et al., 2013, 2014). Moreover, the two pathways are mutually exclusive and suppressive (Kim et al., 2013), suggesting that a distinct set of signal regulators can be activated for NFAT5 transcription according to high salt versus LPS. In this regard, other transcriptional factors or co-regulators, in addition to NF-κB, might also be targets of KRN2, since they can bind to the Nfat5 promoter region (−3000 to +1) and interact with each other to result in NFAT5 transcription (Buxadé et al., 2012). Further studies are required to clarify this issue. In arthritic joints, various inflammatory cells, including macrophages, T cells, synoviocytes, and endothelial Triptolide interact with each other via an array of cytokines and/or cell-to-cell contact, leading to prolonged inflammation and destruction of cartilage and bone. As demonstrated in NFAT5 haplo-insufficient mice (Yoon et al., 2011), the NFAT5 blockade by KRN2 and KRN5 successfully repressed experimentally-induced arthritis in mice with AIA and CIA where innate and adaptive immune cells play major roles, respectively, which confirms that NFAT5 is crucial in RA pathogenesis. Given that KRN2 and KRN5 inhibited macrophage activation in both mouse and human systems through suppressing the expression of pro-inflammatory mediators, such inhibition of macrophage activation may explain the therapeutic efficacy of KRN2 and KRN5 in vivo. However, NFAT5 controls antigen-specific T cell proliferation, Th17 cell activation, and Treg cell function (Go et al., 2004; Kleinewietfeld et al., 2013; Wu et al., 2013; Li et al., 2014). NFAT5 is also involved in synoviocyte proliferation and angiogenesis (Yoon et al., 2011), both pathologic hallmarks of RA. We believe that the pharmacological effects of KRN2 and KRN5 are not limited to macrophages in mice with AIA or CIA. Ultimately, the therapeutic effects may be demonstrated to result from the overall action of these small molecules on multiple types of cells, including macrophages, Th17 cells, Treg cells, and synoviocytes. In sum, 13-(2-fluoro)-benzylberberine, KRN2 inhibits NFAT5-dependent reporter activity, NFAT5 mRNA and protein expression, NFAT5 translocation to the nucleus, and NFAT5 promoter activity. KRN2 exhibits 40 times more anti-NFAT5 activity (IC50=0.1μM) than BBR deprived of its 13-fluorobenzyl group. KRN2 inhibition of transcriptional activation of NFAT5 was at least partially due to the decrease in the formation of NF-κB p65-DNA complexes in the Nfat5 promoter region. This compound selectively suppresses the expression of pro-inflammatory genes regulated by NFAT5 in TLR4-stimulated macrophages without hampering ‘osmotic’ NFAT5 and its target gene expressions. Moreover, KRN2 and its oral derivative KRN5 show suppressive effects on the development of experimental arthritis in mice. Given their high efficacy in arthritic mice, KRN2 and KRN5 should be considered as potential therapeutic agents for treating chronic arthritis, including RA. Particularly, KRN5, as an oral agent, seems to be promising since it was stronger in suppressing arthritis than methotrexate, a commonly used anti-rheumatic drug, displaying better potency and safety than its original compound BBR. In addition, our strategy of discovering NFAT5 suppressors using HTS and an NFAT5-depdendent reporter system can be applied to other chemical libraries to discover the ideal anti-NFAT5 drugs with high specificity and less cytotoxicity than KRN2 and KRN5. We anticipate that the resultant anti-NFAT5 small molecules will provide novel candidates in the treatment of other chronic inflammatory diseases and certain types of cancer in which NFAT5 plays a key role.