Objective To investigate the role of nuclear factor of activated T cells 5 (NFAT5), which is known as an osmoprotective transcription factor, in synovial hyperplasia and angiogenesis in rheumatoid arthritis (RA) Methods Expression of NFAT5 was examined in the synovial tissues and synoviocytes of RA patients using immunohistochemistry and Western blot analysis, respectively. The mRNAs of RA synoviocytes and human umbilical vein endothelial cells (HUVEC) transfected with dummy siRNA or NFAT5 siRNA were profiled using microarray technology. Assays to determine synoviocyte apoptosis and proliferation were performed in the presence of NFAT5 siRNA.VEGF165-induced angiogenesis was assessed by measuring the proliferation, tube formation, and wounding migration of HUVEC. Experimental arthritis was induced in mice by injection of anti-type II collagen antibody. Results NFAT5 was highly expressed in the rheumatoid synovium and its activity was increased by proinflammatory cytokines, such as IL-1β and TNF-α. The mRNA profiling of synoviocytes and HUVEC transfected with NFAT5-targeted siRNA revealed three major changes in cellular processes associated with the pathogenesis of RA: cell cycle and survival, angiogenesis, and cell migration. Consistent with these results, NFAT5 knock-down in RA synoviocytes and HUVEC inhibited their proliferation/survival and impeded angiogenic processes in HUVEC. Mice with NFAT5 haplo-insufficiency (NFAT5+/-) developed very limited degree of synovial proliferation in histological analysis, decreased angiogenesis, and exhibited a nearly complete suppression of experimentally induced arthritis. Conclusion NFAT5 regulates synovial proliferation and angiogenesis in chronic arthritis.

Tonicity-responsive enhancer binding protein (TonEBP) plays a key role in protecting renal cells from hypertonic stress by stimulating transcription of specific genes. Under hypertonic conditions, TonEBP activity is enhanced via increased nuclear translocation, transactivation, and abundance. It was reported previously that hypertonicity exerted a dual, time-dependent effect on vasopressin-inducible aquaporin-2 (AQP2) expression in immortalized mouse collecting duct principal cells (mpkCCD cl4 ). Whereas AQP2 abundance decreased after 3 h of hyperosmotic challenge, it increased after 24 h of hypertonic challenge. This study investigated the role that TonEBP may play in these events by subjecting mpkCCD cl4 cells to 3 or 24 h of hypertonic challenge. Hypertonic challenge increased TonEBP mRNA and protein content and enhanced TonEBP activity as illustrated by both increased TonEBP-dependent luciferase activity and mRNA expression of several genes that are targeted by TonEBP. Irrespective of the absence or presence of vasopressin, decreased TonEBP activity in cells that were transfected with either TonEBP small interfering RNA or an inhibitory form of TonEBP strongly reduced AQP2 mRNA and protein content under iso-osmotic conditions and blunted the increase of AQP2 abundance that was induced after 24h of hypertonic challenge. Conversely, decreased TonEBP activity did not significantly alter reduced expression of AQP2 mRNA that was induced by 3 h of hypertonic challenge. Mutation of a TonE enhancer element located 489 bp upstream of the AQP2 transcriptional start site abolished the hypertonicity-induced increase of luciferase activity in cells that expressed AQP2 promoter-luciferase plasmid constructs, indicating that TonEBP influences AQP2 transcriptional activity at least partially by acting directly on the AQP2 promoter. These findings demonstrate that in collecting duct principal cells, TonEBP plays a central role in regulating AQP2 expression by enhancing AQP2 gene transcription.

Tonicity responsive binding protein (TonEBP) is a transcription factor that plays a key role in osmoprotection. Here, we demonstrate enhanced activity of prosurvival NF-κB—at the onset of hypertonic challenge that depends on p38 kinase—and Akt-dependent formation of p65-TonEBP complexes that bind to elements of NF-κB-responsive genes.

Defective apoptotic death of activated macrophages has been implicated in the pathogenesis of rheumatoid arthritis (RA). However, the molecular signatures defining apoptotic resistance of RA macrophages are not fully understood. Here, global transcriptome profiling of RA macrophages revealed that the osmoprotective transcription factor nuclear factor of activated T cells 5 (NFAT5) critically regulates diverse pathologic processes in synovial macrophages including the cell cycle, apoptosis, and proliferation. Transcriptomic analysis of NFAT5-deficient macrophages revealed the molecular networks defining cell survival and proliferation. Proinflammatory M1-polarizing stimuli and hypoxic conditions were responsible for enhanced NFAT5 expression in RA macrophages. An in vitro functional study demonstrated that NFAT5-deficient macrophages were more susceptible to apoptotic death. Specifically, CCL2 secretion in an NFAT5-dependent fashion bestowed apoptotic resistance to RA macrophages in vitro. Injection of recombinant CCL2 into one of the affected joints of Nfat5+/- mice increased joint destruction and macrophage infiltration, demonstrating the essential role of the NFAT5/CCL2 axis in arthritis progression in vivo. Moreover, after intra-articular injection, NFAT5-deficient macrophages were more susceptible to apoptosis and less efficient at promoting joint destruction than were NFAT5-sufficient macrophages. Thus, NFAT5 regulates macrophage survival by inducing CCL2 secretion. Our results provide evidence that NFAT5 expression in macrophages enhances chronic arthritis by conferring apoptotic resistance to activated macrophages.