Transcription Factor NFAT5 Promotes Migration and Invasion of Rheumatoid Synoviocytes via Coagulation Factor III and CCL2.

Fibroblast-like synoviocytes (FLSs) play a key role in the progression of rheumatoid arthritis (RA) as a primary component of invasive hypertrophied pannus. FLSs of RA patients (RA-FLSs) exhibit cancer-like features, including promigratory and proinvasive activities that largely contribute to joint cartilage and bone destruction. In this study, we hypothesized that the NF of activated T cell 5 (NFAT5), a transcription factor involving tumor invasiveness, would control the migration and invasion of RA-FLSs. Analyses of transcriptomes demonstrated the significant involvement of NFAT5 in locomotion of RA-FLSs and that tissue factor (TF; also known as coagulation factor III) and CCL2 were the major downstream target genes of NFAT5 involving FLS migration and invasion. In cultured RA-FLSs, IL-1ß and TGF-ß increased TF and CCL2 expression by upregulating NFAT5 expression via p38 MAPK. Functional assays demonstrated that NFAT5- or TF-deficient RA-FLSs displayed decreased lamellipodia formation, cell migration, and invasion under IL-1ß- or TGF-ß-stimulated conditions. Conversely, factor VIIa, a specific activator of TF, increased migration of RA-FLSs, which was blocked by NFAT5 knockdown. Recombinant CCL2 partially restored the decrease in migration and invasion of NFAT5-deficient RA-FLSs stimulated with IL-1ß. NFAT5-knockout mouse FLSs also showed decreased expressions of TF and CCL2 and reduced cell migration. Moreover, KRN2, a specific inhibitor of NFAT5, suppressed migration of FLSs stimulated with TGF-ß. Conclusively, to our knowledge, this is the first study to provide evidence of a functional link between osmoprotective NFAT5 and TF in the migration and invasion of RA-FLSs and supports a role for NFAT5 blockade in the treatment of RA.

The xanthine oxidase-NFAT5 pathway regulates macrophage activation and TLR-induced inflammatory arthritis.

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.

Tonicity-responsive enhancer binding protein regulates the expression of aldose reductase and protein kinase C δ in a mouse model of diabetic retinopathy.

Recent studies revealed that Tonicity-responsive enhancer binding protein (TonEBP) directly regulates the transcription of aldose reductase (AR), which catalyzes the first step of the polyol pathway of glucose metabolism. Activation of protein kinase C δ (PKCδ) is dependent on AR and it has been linked to diabetic complications. However, whether TonEBP affects expressions of AR and PKCδ in diabetic retinopathy was not clearly shown. In this study, we used TonEBP heterozygote mice to study the role of TonEBP in streptozotocin (STZ)-induced diabetic retinopathy. We performed immunofluorescence staining and found that retinal expressions of AR and PKCδ were significantly reduced in the heterozygotes compared to wild type littermates, particularly in ganglion cell layer. To examine further the effect of TonEBP reduction in retinal tissues, we performed intravitreal injection of TonEBP siRNA and confirmed the decrease in AR and PKCδ levels. In addition, we found that a proapoptotic factor, Bax level was reduced and a survival factor, Bcl2 level was increased after injection of TonEBP siRNA, indicating that TonEBP mediates apoptotic cell death. In parallel, TonEBP siRNA was applied to the in vitro human retinal pigment epithelial (ARPE-19) cells cultured in high glucose media. We have consistently found the decrease in AR and PKCδ levels and changes in apoptotic factors for survival. Together, these results clearly demonstrated that hyperglycemia-induced TonEBP plays a crucial role in increasing AR and PKCδ levels and leading to apoptotic death. Our findings suggest that TonEBP reduction is an effective therapeutic strategy for diabetic retinopathy.

Serum amyloid A expression in liver promotes synovial macrophage activation and chronic arthritis via NFAT5.

Nuclear factor of activated T-cells 5 (NFAT5), an osmo-sensitive transcription factor, can be activated by isotonic stimuli, such as infection. It remains unclear, however, whether NFAT5 is required for damage-associated molecular pattern-triggered (DAMP-triggered) inflammation and immunity. Here, we found that several DAMPs increased NFAT5 expression in macrophages. In particular, serum amyloid A (SAA), primarily generated by the liver, substantially upregulated NFAT5 expression and activity through TLR2/4-JNK signalling pathway. Moreover, the SAA-TLR2/4-NFAT5 axis promoted migration and chemotaxis of macrophages in an IL-6- and chemokine ligand 2-dependent (CCL2-dependent) manner in vitro. Intraarticular injection of SAA markedly accelerated macrophage infiltration and arthritis progression in mice. By contrast, genetic ablation of NFAT5 or TLR2/4 rescued the pathology induced by SAA, confirming the SAA-TLR2/4-NFAT5 axis in vivo. Myeloid-specific depletion of NFAT5 also attenuated SAA-accelerated arthritis. Of note, inflammatory arthritis in mice strikingly induced SAA overexpression in the liver. Conversely, forced overexpression of the SAA gene in the liver accelerated joint damage, indicating that the liver contributes to bolstering chronic inflammation at remote sites by secreting SAA. Collectively, this study underscores the importance of the SAA-TLR2/4-NFAT5 axis in innate immunity, suggesting that acute phase reactant SAA mediates mutual interactions between liver and joints and ultimately aggravates chronic arthritis by enhancing macrophage activation.

Phospholipase Cgamma1 negatively regulates growth hormone signalling by forming a ternary complex with Jak2 and protein tyrosine phosphatase-1B.

Growth hormone binds to its membrane receptor (GHR), whereby it regulates many cellular functions, including proliferation, differentiation and chemotaxis. However, although the activation of growth hormone-mediated signalling is well understood, the precise mechanism responsible for its regulation has not been elucidated. Here, we demonstrate that phospholipase Cgamma1 (PLCgamma1) modulates the action of growth hormone-mediated signalling by interacting with tyrosine kinase Jak2 (janus kinase 2) in a growth hormone-dependent manner. In the absence of PLCgamma1 (PLCgamma1(-/-)), growth hormone-induced JAK2 and STAT5 phosphorylation significantly increased in mouse embryonic fibroblasts (MEFs). Furthermore, the re-expression of PLCgamma1 reduced growth hormone-induced Jak2 activation. Growth hormone-induced Jak2 phosphorylation was enhanced by siRNA-specific knockdown of PLCgamma1. Interestingly, PLCgamma1 physically linked Jak2 and protein tyrosine phosphatase-1B (PTP-1B) by binding to both using different domains, and this process was implicated in the modulation of cytokine signalling through Jak2. In addition, in PLCgamma1(-/-) MEFs, growth hormone-dependent c-Fos activation was upregulated and growth hormone-induced proliferation was potentiated. These results suggest that PLCgamma1 has a key function in the regulation of growth hormone-mediated signalling by negatively regulating Jak2 activation.

Tonicity-independent regulation of the osmosensitive transcription factor TonEBP (NFAT5).

Tonicity-responsive enhancer binding protein (TonEBP/nuclear factor of activated T-cells 5 [NFAT5]) is a Rel homology transcription factor classically known for its osmosensitive role in regulating cellular homeostasis during states of hypo- and hypertonic stress. A recently growing body of research indicates that TonEBP is not solely regulated by tonicity, but that it can be stimulated by various tonicity-independent mechanisms in both hypertonic and isotonic tissues. Physiological and pathophysiological stimuli such as cytokines, growth factors, receptor and integrin activation, contractile agonists, ions, and reactive oxygen species have been implicated in the positive regulation of TonEBP expression and activity in diverse cell types. These new data demonstrate that tonicity-independent stimulation of TonEBP is critical for tissue-specific functions like enhanced cell survival, migration, proliferation, vascular remodeling, carcinoma invasion, and angiogenesis. Continuing research will provide a better understanding as to how these and other alternative TonEBP stimuli regulate gene expression in both health and disease.

Nuclear factor of activated T cells 5 regulates vascular smooth muscle cell phenotypic modulation.

OBJECTIVE: The tonicity-responsive transcription factor, nuclear factor of activated T cells 5 (NFAT5/tonicity enhancer binding protein [TonEBP]), has been well characterized in numerous cell types; however, NFAT5 function in vascular smooth muscle cells (SMCs) is unknown. Our main objective was to determine the role of NFAT5 regulation in SMCs. METHODS AND RESULTS: We showed that NFAT5 is regulated by hypertonicity in SMCs and is upregulated in atherosclerosis and neointimal hyperplasia. RNAi knockdown of NFAT5 inhibited basal expression of several SMC differentiation marker genes, including smooth muscle α actin (SMαA). Bioinformatic analysis of SMαA revealed 7 putative NFAT5 binding sites in the first intron, and chromatin immunoprecipitation analysis showed NFAT5 enrichment of intronic DNA. Overexpression of NFAT5 increased SMαA promoter-intron activity, which requires an NFAT5 cis element at +1012, whereas dominant-negative NFAT5 decreased SMαA promoter-intron activity. Because it is unlikely that SMCs experience extreme changes in tonicity, we investigated other stimuli and uncovered 2 novel NFAT5-inducing factors: angiotensin II, a contractile agonist, and platelet-derived growth factor-BB (PDGF-BB), a potent mitogen in vascular injury. Angiotensin II stimulated NFAT5 translocation and activity, and NFAT5 knockdown inhibited an angiotensin II-mediated upregulation of SMαA mRNA. PDGF-BB increased NFAT5 protein, and loss of NFAT5 inhibited PDGF-BB-induced SMC migration. CONCLUSIONS: We have identified NFAT5 as a novel regulator of SMC phenotypic modulation and have uncovered the role of NFAT5 in angiotensin II-induced SMαA expression and PDGF-BB-stimulated SMC migration.

NF-AT5 is a critical regulator of inflammatory arthritis.

OBJECTIVE: To investigate the role of NF-AT5, an osmoprotective transcription factor, in synovial hyperplasia and angiogenesis in patients with rheumatoid arthritis (RA). METHODS: The expression of NF-AT5 in synovial tissue and synoviocytes from RA patients was examined by immunohistochemistry and Western blot analysis, respectively. Messenger RNA (mRNA) in RA synoviocytes and human umbilical vein endothelial cells (HUVECs) transfected with dummy small interfering RNA (siRNA) or NF-AT5 siRNA were profiled using microarray technology. Assays to determine synoviocyte apoptosis and proliferation were performed in the presence of NF-AT5 siRNA. VEGF165-induced angiogenesis was assessed by measuring the proliferation, tube formation, and wound migration of HUVECs. Experimental arthritis was induced in mice by injection of anti-type II collagen antibody. RESULTS: NF-AT5 was highly expressed in rheumatoid synovium, and its activity was increased by proinflammatory cytokines, such as interleukin-1ß and tumor necrosis factor α. The mRNA profiling of synoviocytes and HUVECs transfected with NF-AT5-targeted siRNA revealed 3 major changes in cellular processes associated with the pathogenesis of RA: cell cycle and survival, angiogenesis, and cell migration. Consistent with these results, NF-AT5 knockdown in RA synoviocytes and HUVECs inhibited their proliferation/survival and impeded angiogenic processes in HUVECs. Mice with NF-AT5 haploinsufficiency (NF-AT5(+/-)) developed a very limited degree of synovial proliferation, as seen on histologic analysis, and decreased angiogenesis, and they exhibited a nearly complete suppression of experimentally induced arthritis. CONCLUSION: NF-AT5 regulates synovial proliferation and angiogenesis in chronic arthritis.

Distinct cellular pathways for resistance to urea stress and hypertonic stress.

During antidiuresis with elevated vasopressin, urea accumulates in the renal medulla to very high concentrations, imposing considerable cellular stress. How local cells cope with urea stress is relevant to the whole kidney because the renal medulla is the major site of residence for the renal stem cells. Previous studies showed that renal cells were incapable of preconditioning in moderate urea concentrations to enhance resistance to urea stress. Instead, preconditioning in moderately high salinity (moderate hypertonicity) has been shown to promote resistance to urea stress due to the induction of the molecular chaperone heat shock protein 70 (Hsp70), which is mediated by the transcription factor tonicity-responsive enhancer binding protein (TonEBP). Here we report that cell lines derived from the kidney and fibroblasts display enhanced resistance to urea stress after pretreatment in moderate, nonstressful concentrations of urea. Using TonEBP knockdown and immunoblot analyses, we demonstrate that TonEBP and Hsp70 are dispensable for the increased resistance to urea stress. These data suggest that cells in the renal medulla are capable of overcoming urea stress by activating distinct cellular pathways.

TonEBP stimulates multiple cellular pathways for adaptation to hypertonic stress: organic osmolyte-dependent and -independent pathways.

TonEBP (tonicity-responsive enhancer binding protein) is a transcription factor that promotes cellular accumulation of organic osmolytes in the hypertonic renal medulla by stimulating expression of its target genes. Genetically modified animals with deficient TonEBP activity in the kidney suffer from severe medullary atrophy in association with cell death, demonstrating that TonEBP is essential for the survival of the renal medullary cells. Using both TonEBP knockout cells and RNA interference of TonEBP, we found that TonEBP promoted cellular adaptation to hypertonic stress. Microarray analyses revealed that the genetic response to hypertonicity was dominated by TonEBP in that expression of totally different sets of genes was increased by hypertonicity in those cells with TonEBP vs. those without TonEBP activity. Of over 100 potentially new TonEBP-regulated genes, we selected seven for further analyses and found that their expressions were all dependent on TonEBP. RNA interference experiments showed that some of these genes, asporin, insulin-like growth factor-binding protein-5 and -7, and an extracellular lysophospholipase D, plus heat shock protein 70, a known TonEBP target gene, contributed to the adaptation to hypertonicity without promoting organic osmolyte accumulation. We conclude that TonEBP stimulates multiple cellular pathways for adaptation to hypertonic stress in addition to organic osmolyte accumulation.

Renal ischemia-reperfusion injury causes intercalated cell-specific disruption of occludin in the collecting duct.

Renal ischemic events open tight junctions and disrupt epithelial polarity. The purpose of this study was to examine the effects of ischemia-reperfusion (IR) injury on expression and distribution of the tight junction proteins, occludin and ZO-1, in the rat kidney. IR injury was induced by clamping both renal pedicles for 30 min and animals were killed at 6 h after the reperfusion. IR injury decreased blood bicarbonate level, but did not persistently alter pH, Na(+), K(+), or Cl(-). In control kidneys, occludin immunoreactivity was intense in the tight junctions in the thick ascending limb, distal convoluted tubule, and collecting duct, moderate in the thin limbs of the loop of Henle, and was not detected in the proximal tubule, glomerulus, and blood vessels. ZO-1 was expressed in the same sites in which occludin was expressed, and additionally was also expressed in the proximal tubule, glomerulus, and vascular endothelial cells. IR kidneys exhibited damaged renal tubular epithelial cells in both proximal tubule and collecting duct segments in the outer medulla. In the collecting duct, the response of intercalated cells and principal cells differed. Following IR injury, intercalated cells, but not principal cells, lost their normal epithelial polarity and were frequently extruded into the tubule lumen. Occludin, instead of being localized to tight junctions, was localized diffusely in the cytoplasm in intercalated cells of IR kidneys. Principal cells, in contrast, were not detectably affected and neither occludin nor ZO-1 expression were altered in response to IR injury. The normal localization of ZO-1 expression to tight junction sites in both the proximal tubule and collecting duct was altered in response to IR, and, instead, ZO-1 expression was present diffusely in the cytoplasm. IR injury did not alter detectably either occludin or ZO-1 localization to the tight junction of the thick ascending limb cells. The abundance of total occludin protein by immunoblot analysis was not changed with IR injury. These results demonstrate that renal IR injury causes tight junction disruptions in both the proximal tubule and the collecting duct, and that altered distribution of the tight junction protein, occludin, may play a critical role in the collecting duct dysfunction which IR induces.

Urea promotes TonEBP expression and cellular adaptation in extreme hypertonicity.

The transcriptional activator TonEBP is a central regulator of osmolality in the renal medulla and whole body water homeostasis. In order to understand the regulation of TonEBP in the renal medulla, we examined MDCK cells, a kidney-derived epithelial cell line, under conditions mimicking the renal medulla. Moderate changes in ambient tonicity, which was tolerated without prior adaptation, displayed lasting effects on TonEBP in bidirectional manner-stimulated by hypertonicity and inhibited by hypotonicity. TonEBP expression was further enhanced by extreme hypertonicity observed in the inner medullae of antidiuretic animals. Urea stimulated TonEBP expression and promoted cellular proliferation under the conditions of extreme hypertonicity. On the other hand, the TonEBP activity was negatively modulated under these conditions presumably to temper the highly abundant TonEBP. We conclude that urea is critical to the cellular adaptation to extreme hypertonicity and the high level of TonEBP expression in the inner medulla.

Hypertonicity stimulates PGE2 signaling in the renal medulla by promoting EP3 and EP4 receptor expression.

Hypertonicity in the renal medulla stimulates local cyclooxygenase 2 expression, leading to abundant PGE(2) production. Here we found that mRNA expression by the PGE(2)-activated G-protein-coupled receptors, EP3 and EP4 in the renal medulla was decreased by furosemide treatment, a procedure that reduces medullary hypertonicity. When HepG2 cells were cultured in hypertonic conditions by addition of salt or sorbitol, EP3 expression was induced. A specific EP3 agonist inhibited cAMP production, indicating receptor functionality, and this led to a substantial increase in cell survival in hypertonic media. Survival was independent of the SLC5A3 inositol transporter and aldose reductase expression, suggesting that EP3 promoted cell survival under hypertonic conditions independent of cellular organic osmolyte accumulation. Reduced cAMP production did not contribute to increased survival. EP4 expression was stimulated by hypertonicity in MDCK and HepG2 cells, which was associated with increased cAMP production in response to an EP4 agonist. Our study shows that local hypertonicity promotes PGE(2) signaling in the renal medulla by stimulating cognate receptor and cyclooxygenase 2 expression that likely regulates local hemodynamics and tubular transport.Kidney International (2009) 75, 278-284. doi:10.1038/ki.2008.498.

Interstitial tonicity controls TonEBP expression in the renal medulla.

Cells in the hyperosmotic kidney medulla, express a transcriptional activator termed tonicity responsive enhancer binding protein (TonEBP). Genes targeted by TonEBP protect kidney cells from the deleterious effects of hyperosmolality by inducing the expression of organic osmolytes and molecular chaperones, and other genes that mediate urine concentration such as aquaporin-2 and urea transporters. We tested here the effect of hypertonicity and hyperosmotic salt in the renal medullary interstitium on the expression TonEBP. When massive water diuresis was induced in rats the medullary sodium concentrations did not change, neither did TonEBP expression. In these animals the medullary tonicity was unchanged despite the production of dilute urine. On the other hand, treatment with the loop diurectic furosemide resulted in a dose-dependent decrease in the medullary sodium concentration causing a reduction in interstitial tonicity. Here, TonEBP expression was blunted in the outer and inner medulla which was due, in part, to decreased mRNA abundance. As expected, the expression of TonEBP target genes in the renal medulla also decreased in response to furosemide. Hence TonEBP expression in the renal medulla is stimulated by interstitial hypertonicity.

Osmotically-induced genes are controlled by the transcription factor TonEBP in cultured cardiomyocytes.

Changes in cardiac osmolarity occur in myocardial infarction. Osmoregulatory mechanisms may, therefore, play a crucial role in cardiomyocyte survival. Tonicity-responsive enhancer binding protein (TonEBP) is a key transcription factor participating in the adaptation of cells to increases in tonicity. However, it is unknown whether cardiac TonEBP is activated by tonicity. Hypertonicity activated transcriptional activity of TonEBP, increased the amounts of both TonEBP mRNA and protein, and induced both the mRNA and protein of TonEBP target genes (aldose reductase and heat shock protein-70). Hypotonicity decreased the amount of TonEBP protein indicating bidirectional osmoregulation of this transcription factor. Adenoviral expression of a dominant negative TonEBP suppressed the hypertonicity-dependent increase of aldose reductase protein. These results indicated that TonEBP controls osmoregulatory mechanisms in cardiomyocytes.

Transcriptional activator TonE-binding protein in cellular protection and differentiation.

The TonE-binding protein (TonEBP) is a transcriptional activator in the Rel family that includes NFkappaB and NFAT. TonEBP is critical for the development and function of the renal medulla, which is a major regulator of water homeostasis. TonEBP is also implicated in diabetic nephropathy and inflammation. Established methods for biochemical and histochemical detection and functional analysis of TonEBP, including identification of novel TonEBP target genes, are described for those who are interested in investigating function and regulation of TonEBP.

RGS2 promotes formation of neurites by stimulating microtubule polymerization.

Regulator of G-protein signaling (RGS) proteins interact with alpha subunits of heterotrimeric G-proteins via the RGS domain and attenuate their activity by accelerating GTPase activity. RGS2, a member of the RGS family, regulates synaptic development via hereto unknown mechanism. In this study, we found that RGS2 directly interacted with tubulin via a short region at the N-terminus: amino acids 41-60. RGS2 enhanced microtubule polymerization in vitro, and the tubulin binding region was necessary and sufficient for this activity. In Vero cells, polymerization of microtubule was stimulated when peptides containing the tubulin binding region were microinjected. Immunocytochemical analysis showed that endogenous RGS2 was localized at the termini of neurites in differentiated PC12 cells. Over-expression of RGS2 enhanced the nerve growth factor-induced neurite outgrowth in PC12 cells, while specific knock-down of endogenous RGS2 suppressed the neurite outgrowth. These findings demonstrate that RGS2 contributes to the neuronal cell differentiation via regulation of microtubule dynamics.

TonEBP/NFAT5 stimulates transcription of HSP70 in response to hypertonicity.

While hyperosmolality of the kidney medulla is essential for urinary concentration, it imposes a great deal of stress. Cells in the renal medulla adapt to the stress of hypertonicity (hyperosmotic salt) by accumulating organic osmolytes. Tonicity-responsive enhancer (TonE) binding protein (TonEBP) (or NFAT5) stimulates transcription of transporters and a synthetic enzyme for the cellular accumulation of organic osmolytes. We found that dominant-negative TonEBP reduced expression of HSP70 as well as the transporters and enzyme. Near the major histocompatibility complex class III locus, there are three HSP70 genes named HSP70-1, HSP70-2, and HSC70t. While HSP70-1 and HSP70-2 were heat inducible, only HSP70-2 was induced by hypertonicity. In the 5' flanking region of the HSP70-2 gene, there are three sites for TonEBP binding. In cells transfected with a reporter plasmid containing this region, expression of luciferase was markedly stimulated in response to hypertonicity. Coexpression of the dominant-negative TonEBP reduced the luciferase expression. Mutating all three sites in the reporter plasmid led to a complete loss of induction by hypertonicity. Thus, TonEBP rather than heat shock factor stimulates transcription of the HSP70-2 gene in response to hypertonicity. We conclude that TonEBP is a master regulator of the renal medulla for cellular protection against high osmolality via organic osmolytes and molecular chaperones.

TonEBP is inhibited by RNA helicase A via interaction involving the E'F loop.

TonEBP [TonE (tonicity-responsive enhancer)-binding protein] is a transcriptional activator of the Rel family like NF-kappaB (nuclear factor kappaB) and NFAT (nuclear factor of activated T-cells). TonEBP plays a key role in the protection of cells in the kidney medulla from the deleterious effects of hyperosmolality. This is achieved by enhancing expression of HSP70 (heat-shock protein 70) and other genes whose products drive cellular accumulation of organic osmolytes. TonEBP is stimulated by ambient hypertonicity via multiple pathways that regulate nuclear translocation and transactivation. In the present paper, we report that TonEBP is associated in vivo with RHA (RNA helicase A). The N- and C-termini of RHA bound the E'F loop of the DNA-binding domain of TonEBP. The interaction was not affected by DNA binding or dimerization of TonEBP. Overexpression of RHA inhibited the activity of TonEBP; however, catalytic activity of RHA was dispensable for the inhibition. When the ambient tonicity was raised, the TonEBP-RHA interaction decreased, suggesting that dissociation of RHA is a pathway to stimulate TonEBP. We conclude that the E'F loop of TonEBP interacts with RHA like NFAT and NF-kappaB interact with AP1 (activator protein 1) and the high-mobility group protein HMG-I(Y) respectively. While RHA interacts with and stimulates other transcription factors such as CREB (cAMP-response-element-binding protein), NF-kappaB and mineralocorticoid receptor, it inhibits TonEBP.

Transcription factor NFAT5 promotes macrophage survival in rheumatoid arthritis.

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.