Prolonged extracellular low sodium concentrations and subsequent their rapid correction modulate nitric oxide production dependent on NFAT5 in microglia.

Hyponatremia is the most common clinical electrolyte disorder. Chronic hyponatremia has been recently reported to be associated with falls, fracture, osteoporosis, neurocognitive impairment, and mental manifestations. In the treatment of chronic hyponatremia, overly rapid correction of hyponatremia can cause osmotic demyelination syndrome (ODS), a central demyelinating disease that is also associated with neurological morbidity and mortality. Using a rat model, we have previously shown that microglia play a critical role in the pathogenesis of ODS. However, the direct effect of rapid correction of hyponatremia on microglia is unknown. Furthermore, the effect of chronic hyponatremia on microglia remains elusive. Using microglial cell lines BV-2 and 6-3, we show here that low extracellular sodium concentrations (36 mmol/L decrease; LS) suppress Nos2 mRNA expression and nitric oxide (NO) production of microglia. On rapid correction of low sodium concentrations, NO production was significantly increased in both cells, suggesting that acute correction of hyponatremia partly directly contributes to increased Nos2 mRNA expression and NO release in ODS pathophysiology. LS also suppressed expression and nuclear translocation of nuclear factor of activated T cells-5 (NFAT5), a transcription factor that regulates the expression of genes involved in osmotic stress. Furthermore, overexpression of NFAT5 significantly increased Nos2 mRNA expression and NO production in BV-2 cells. Expressions of Nos2 and Nfat5 mRNA were also modulated in microglia isolated from cerebral cortex in chronic hyponatremia model mice. These data indicate that LS modulates microglial NO production dependent on NFAT5 and suggest that microglia contribute to hyponatremia-induced neuronal dysfunctions.

Nuclear factor of activated T-cells 5 is indispensable for a balanced adaptive transcriptional response of lung endothelial cells to hypoxia.

AIMS: Chronic hypoxia causes detrimental structural alterations in the lung, which may cause pulmonary hypertension and are partially mediated by the endothelium. While its relevance for the development of hypoxia-associated lung diseases is well known, determinants controlling the initial adaptation of the lung endothelium to hypoxia remain largely unexplored. METHODS AND RESULTS: We revealed that hypoxia activates the transcription factor nuclear factor of activated T-cells 5 (NFAT5) and studied its regulatory function in murine lung endothelial cells (MLECs). EC-specific knockout of Nfat5 (Nfat5(EC)-/-) in mice exposed to normobaric hypoxia (10% O2) for 21 days promoted vascular fibrosis and aggravated the increase in pulmonary right ventricular systolic pressure as well as right ventricular dysfunction as compared with control mice. Microarray- and single-cell RNA-sequencing-based analyses revealed an impaired growth factor-, energy-, and protein-metabolism-associated gene expression in Nfat5-deficient MLEC after exposure to hypoxia for 7 days. Specifically, loss of NFAT5 boosted the expression and release of platelet-derived growth factor B (Pdgfb)-a hypoxia-inducible factor 1 alpha (HIF1α)-regulated driver of vascular smooth muscle cell (VSMC) growth-in capillary MLEC of hypoxia-exposed Nfat5(EC)-/- mice, which was accompanied by intensified VSMC coverage of distal pulmonary arteries. CONCLUSION: Collectively, our study shows that early and transient subpopulation-specific responses of MLEC to hypoxia may determine the degree of organ dysfunction in later stages. In this context, NFAT5 acts as a protective transcription factor required to rapidly adjust the endothelial transcriptome to cope with hypoxia. Specifically, NFAT5 restricts HIF1α-mediated Pdgfb expression and consequently limits muscularization and resistance of the pulmonary vasculature.

Transcriptional upregulation of the myo-inositol biosynthesis pathway is enhanced by NFAT5 in hyperosmotically stressed tilapia cells.

Euryhaline fish experience variable osmotic environments requiring physiological adjustments to tolerate elevated salinity. Mozambique tilapia (Oreochromis mossambicus) possess one of the highest salinity tolerance limits of any fish. In tilapia and other euryhaline fish species, the myo-inositol biosynthesis (MIB) pathway enzymes, myo-inositol phosphate synthase (MIPS) and inositol monophosphatase 1 (IMPA1.1), are among the most upregulated mRNAs and proteins indicating the high importance of this pathway for hyperosmotic (HO) stress tolerance. These abundance changes must be precluded by HO perception and signaling mechanism activation to regulate the expression of MIPS and IMPA1.1 genes. In previous work using a O. mossambicus cell line (OmB), a reoccurring osmosensitive enhancer element (OSRE1) in both MIPS and IMPA1.1 was shown to transcriptionally upregulate these enzymes in response to HO stress. The OSRE1 core consensus (5'-GGAAA-3') matches the core binding sequence of the predominant mammalian HO response transcription factor, nuclear factor of activated T-cells (NFAT5). HO-challenged OmB cells showed an increase in NFAT5 mRNA suggesting NFAT5 may contribute to MIB pathway regulation in euryhaline fish. Ectopic expression of wild-type NFAT5 induced an IMPA1.1 promoter-driven reporter by 5.1-fold (P < 0.01). Moreover, expression of dominant negative NFAT5 in HO media resulted in a 47% suppression of the reporter signal (P < 0.005). Furthermore, reductions of IMPA1.1 (37-49%) and MIPS (6-37%) mRNA abundance were observed in HO-challenged NFAT5 knockout cells relative to control cells. Collectively, these multiple lines of experimental evidence establish NFAT5 as a tilapia transcription factor contributing to HO-induced activation of the MIB pathway.NEW & NOTEWORTHY In our study, we use a multi-pronged synthetic biology approach to demonstrate that the fish homolog of the predominant mammalian osmotic stress transcription factor nuclear factor of activated T-cells (NFAT5) also contributes to the activation of hyperosmolality inducible genes in cells of extremely euryhaline fish. However, in addition to NFAT5 the presence of other strong osmotically inducible signaling mechanisms is required for full activation of osmoregulated tilapia genes.

Heart-specific NFAT5 knockout suppresses type I interferon signaling and aggravates coxsackievirus-induced myocarditis.

Nuclear factor of activated T cells 5 (NFAT5) is an osmosensitive transcription factor that is well-studied in renal but rarely explored in cardiac diseases. Although the association of Coxsackievirus B3 (CVB3) with viral myocarditis is well-established, the role of NFAT5 in this disease remains largely unexplored. Previous research has demonstrated that NFAT5 restricts CVB3 replication yet is susceptible to cleavage by CVB3 proteases. Using an inducible cardiac-specific Nfat5-knockout mouse model, we uncovered that NFAT5-deficiency exacerbates cardiac pathology, worsens cardiac function, elevates viral load, and reduces survival rates. RNA-seq analysis of CVB3-infected mouse hearts revealed the significant impact of NFAT5-deficiency on gene pathways associated with cytokine signaling and inflammation. Subsequent in vitro and in vivo investigation validated the disruption of the cytokine signaling pathway in response to CVB3 infection, evidenced by reduced expression of key cytokines such as interferon ß1 (IFNß1), C-X-C motif chemokine ligand 10 (CXCL10), interleukin 6 (IL6), among others. Furthermore, NFAT5-deficiency hindered the formation of stress granules, leading to a reduction of important stress granule components, including plakophilin-2, a pivotal protein within the intercalated disc, thereby impacting cardiomyocyte structure and function. These findings unveil a novel mechanism by which NFAT5 inhibits CVB3 replication and pathogenesis through the promotion of antiviral type I interferon signaling and the formation of cytoplasmic stress granules, collectively identifying NFAT5 as a new cardio protective protein.

LncRNA AP000842.3 Triggers the Malignant Progression of Prostate Cancer by Regulating Cuproptosis Related Gene NFAT5.

OBJECTIVES: Prostate cancer (PRAD) is a highly malignant disease with poor prognosis, and its development is regulated by a complex network of genes and signaling pathways. LncRNAs and miRNAs have significant regulatory roles in PRAD through the ceRNA network. Cuproptosis is a unique form of programmed cell death that is involved in various signaling pathways and biological processes related to tumor development. Nuclear factor of activated T cells 5 (NFAT5), a transcription factor that activates T cells, has been implicated in cuproptosis. However, the regulatory mechanism by which NFAT5 is involved in the ceRNA network in PRAD remains unclear. METHODS: Through bioinformatics analysis, we found the ceRNA axis that regulates cuproptosis. By performing ROS assay and copper ion concentration assay, we demonstrated that inhibiting NFAT5 can increase the sensitivity of PRAD to cuproptosis inducers. By using luciferase assay, we discovered that AP000842.3 acts as the ceRNA of miR-206 to regulate the expression of NFAT5. RESULTS: In this study, we found that lncRNA AP000842.3, as a ceRNA of miR-206, was involved in the regulation of levels of the transcription factor NFAT5 associated with cuproptosis in PRAD. First, knocking down NFAT5 can increase the sensitivity of PRAD to cuproptosis inducers. Meanwhile, changes in the expression of AP000842.3 and miR-206 could affect the proliferation of PRAD by regulating NFAT5. Mechanistically, AP000842.3 acts as the ceRNA of miR-206 to regulate the expression of NFAT5. In addition, the effects of lncRNA AP000842.3 on malignant progression of PRAD and NFAT5 were partially dependent on miR-206. CONCLUSION: Taken together, our study reveals a key ceRNA regulatory network in PRAD and can be regarded as a new potential target for PRAD diagnosis and treatment.

Differentiation and immunosuppressive function of CD19+CD24hiCD27+ regulatory B cells are regulated through the miR-29a-3p/NFAT5 pathway.

BACKGROUND: Regulatory B cells (Bregs) is an indispensable element in inducing immune tolerance after liver transplantation. As one of the microRNAs (miRNAs), miR-29a-3p also inhibits translation by degrading the target mRNA, and yet the relationship between Bregs and miR-29a-3p has not yet been fully explored. This study aimed to investigate the impact of miR-29a-3p on the regulation of differentiation and immunosuppressive functions of memory Bregs (mBregs) and ultimately provide potentially effective therapies in inducing immune tolerance after liver transplantation. METHODS: Flow cytometry was employed to determine the levels of Bregs in peripheral blood mononuclear cells. TaqMan low-density array miRNA assays were used to identify the expression of different miRNAs, electroporation transfection was used to induce miR-29a-3p overexpression and knockdown, and dual luciferase reporter assay was used to verify the target gene of miR-29a-3p. RESULTS: In patients experiencing acute rejection after liver transplantation, the proportions and immunosuppressive function of mBregs in the circulating blood were significantly impaired. miR-29a-3p was found to be a regulator of mBregs differentiation. Inhibition of miR-29a-3p, which targeted nuclear factor of activated T cells 5 (NFAT5), resulted in a conspicuous boost in the differentiation and immunosuppressive function of mBregs. The inhibition of miR-29a-3p in CD19+ B cells was capable of raising the expression levels of NFAT5, thereby promoting B cells to differentiate into mBregs. In addition, the observed enhancement of differentiation and immunosuppressive function of mBregs upon miR-29a-3p inhibition was abolished by the knockdown of NFAT5 in B cells. CONCLUSIONS: miR-29a-3p was found to be a crucial regulator for mBregs differentiation and immunosuppressive function. Silencing miR-29a-3p could be a potentially effective therapeutic strategy for inducing immune tolerance after liver transplantation.

Association between skin lymphangiogenesis parameters and arterial hypertension status in patients: An observational study.

BACKGROUND: Recent studies have indicated that the skin lymphatic system and interstitium may play a role in the pathophysiology of arterial hypertension (AH). OBJECTIVES: We aimed to determine whether the set of pathway parameters described previously in rodents would allow for the distinction between hypertensive and normotensive patients. MATERIAL AND METHODS: Molecular and histopathological parameters from the skin and blood of patients with AH (AH group, n = 53), resistant AH (RAH group, n = 32) and control (C group, n = 45) were used, and a statistical multivariate bootstrap methodology combining partial least squares-discriminant analysis (PLS-DA) and selectivity ratio (SR) were applied. RESULTS: The C vs RAH model presented the best prediction performance (AUC test = 0.90) and had a sensitivity and specificity of 73.68% and 83.33%, respectively. However, the parameters selected for the C vs AH group model were the most important for the pathway described in the rodent model, i.e., greater density of the skin lymphatic vessels (D2-40 expression) and greater number of macrophages (CD68 expression), higher expression of the messenger ribonucleic acid (mRNA) of nuclear factor of activated T cells 5 (NFAT5), vascular endothelial growth factor C (VEGFC) and podoplanin (PDPN) in the skin, greater concentration of hyaluronic acid (HA) in the skin, and lower serum concentration of VEGF-C. CONCLUSIONS: Our study suggests that the NFAT5/VEGF-C/lymphangiogenesis pathway, previously described in rodent studies, may also be present in human HA. Further experiments are needed to confirm our findings.

TonEBP/NFAT5 expression is associated with cisplatin resistance and migration in macrophage-induced A549 cells.

BACKGROUND: Macrophages promote angiogenesis, metastasis, and drug resistance in several cancers. Similarly, TonEBP/NFAT5 induces metastasis in renal carcinoma and colon cancer cells. However, the role of this transcription factor and that of macrophages in lung cancer cells remains unclear. Therefore, this study investigated the effects of macrophages and TonEBP/NFAT5 expression on cisplatin resistance and migration in A549 lung adenocarcinoma cells. RESULTS: A549 cells were cultured alone or indirectly co-cultured with THP-1-derived macrophages using a transwell culture chamber. Cisplatin-induced cell death was markedly decreased and migration increased in co-cultured A549 cells. Macrophage-conditioned media (CM) showed a similar effect on drug resistance and migration. Cisplatin-induced apoptosis, DNA fragmentation, and cleaved apoptotic proteins PARP and caspase-3 were markedly reduced in macrophage CM-induced A549 cells. Here, ERK, p38, JNK, and NF-κB activities were increased by macrophage CM. Furthermore, the proteins involved in cisplatin resistance and cancer cell migration were identified using specific inhibitors of each protein. ERK and NF-κB inhibition considerably reduced cisplatin resistance. The increase in macrophage CM-induced migration was partially reduced by treatment with ERK, JNK, and NF-κB inhibitors. TonEBP/NFAT5 expression was increased by macrophages, resulting in increased cisplatin resistance, cell migration, and invasion. Moreover, RNAi-mediated knockdown of TonEBP/NFAT5 reduced cisplatin resistance, migration, and invasion in macrophage CM-induced A549 cells. CONCLUSIONS: These findings demonstrate that paracrine factors secreted from macrophages can change A549 cells, resulting in the induction of drug resistance against cisplatin and migration. In addition, the TonEBP/NFAT5 ratio, increased by macrophages, is an important regulator of the malignant transformation of cells.

A comparative analysis of TonEBP conditional knockout mouse models reveals inter-dependency between compartments of the intervertebral disc.

Interactions between notochord and sclerotome are required for normal embryonic spine patterning, but whether the postnatal derivatives of these tissues also require interactions for postnatal intervertebral disc (IVD) growth and maintenance is less established. We report here the comparative analysis of four conditional knockout mice deficient for TonEBP, a transcription factor known to allow cells to adapt to changes in extracellular osmotic pressure, in specific compartments of the IVD. We show that TonEBP deletion in nucleus pulposus (NP) cells does not affect their survival or aggrecan expression, but promoted cell proliferation in the NP and in adjacent vertebral growth plates (GPs). In cartilage end plates/GPs, TonEBP deletion induced cell death, but also structural alterations in the adjacent NP cells and vertebral bodies. Embryonic or postnatal TonEBP loss generated similar IVD changes. In addition to demonstrating the requirement of TonEBP in the different compartments of the IVD, this comparative analysis uncovers the in vivo interdependency of the different IVD compartments during the growth of the postnatal IVD-vertebral units.

SNHG14 Elevates NFAT5 Expression Through Sequestering miR-375-3p to Promote MPP + -Induced Neuronal Apoptosis, Inflammation, and Oxidative Stress in Parkinson's Disease.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the loss of dopaminergic neurons. LncRNA small nucleolar RNA host gene 14 (SNHG14) was found to promote neuron injury in PD. Here, we investigated the mechanisms of SNHG14 in PD process. In vivo or in vitro PD model was established by using 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced mice or 1-methyl-4-phenylpyridinium (MPP +)-stimulated SK-N-SH cells. The expression of genes and proteins was measured by qRT-PCR and Western blot. In vitro assays were conducted using ELISA, CCK-8, colony formation, EdU, flow cytometry, and Western blot assays, respectively. The oxidative stress was evaluated by determining the production of superoxide dismutase (SOD) and malondialdehyde (MDA). The direct interactions between miR-375-3p and NFAT5 (Nuclear factor of activated T-cells 5) or SNHG14 was verified using dual-luciferase reporter and RNA immunoprecipitation (RIP) assays. SNHG14 and NFAT5 were elevated, while miR-375-3p was decreased in MPTP-mediated PD mouse model and MPP + -induced SK-N-SH cells. Knockdown of SNHG14 or NFAT5, or overexpression of miR-375-3p reversed MPP + -induced neuronal apoptosis, inflammation, and oxidative stress. Mechanistically, SNHG14 directly bound to miR-375, which targeted NFAT5. Inhibition of miR-375-3p abolished the inhibitory activity of SNHG14 knockdown on MPP + -evoked neuronal damage. Besides that, NFAT5 up-regulation counteracted the effects of miR-375-3p on MPP + -mediated neuronal damage. SNHG14 contributed to MPP + -induced neuronal injury by miR-375/NFAT5 axis, suggesting a new insight into the pathogenesis of PD.

Inhibition of NFAT5-Dependent Astrocyte Swelling Alleviates Neuropathic Pain.

Astrocyte swelling is implicated in various neurological disorders. However, whether astrocyte swelling contributes to neuropathic pain remains elusive. This study elucidates the pivotal role of the nuclear factor of activated T-cells 5 (NFAT5) emerges as a master regulator of astrocyte swelling in the spinal dorsal horn (SDH) during neuropathic pain. Despite the ubiquitous expression of NFAT5 protein in SDH cell types, it selectively induces swelling specifically in astrocytes, not in microglia. Mechanistically, NFAT5 directly controls the expression of the water channel aquaporin-4 (AQP4), a key regulator exclusive to astrocytes. Additionally, aurora kinase B (AURKB) orchestrates NFAT5 phosphorylation, enhancing its protein stability and nuclear translocation, thereby regulating AQP4 expression. The findings establish NFAT5 as a crucial regulator for neuropathic pain through the modulation of astrocyte swelling. The AURKB-NFAT5-AQP4 pathway in astrocytes emerges as a potential therapeutic target to combat neuropathic pain.

Extracellular sodium regulates fibroblast growth factor 23 (FGF23) formation.

The bone-derived hormone fibroblast growth factor-23 (FGF23) has recently received much attention due to its association with chronic kidney disease and cardiovascular disease progression. Extracellular sodium concentration ([Na+]) plays a significant role in bone metabolism. Hyponatremia (lower serum [Na+]) has recently been shown to be independently associated with FGF23 levels in patients with chronic systolic heart failure. However, nothing is known about the direct impact of [Na+] on FGF23 production. Here, we show that an elevated [Na+] (+20 mM) suppressed FGF23 formation, whereas low [Na+] (-20 mM) increased FGF23 synthesis in the osteoblast-like cell lines UMR-106 and MC3T3-E1. Similar bidirectional changes in FGF23 abundance were observed when osmolality was altered by mannitol but not by urea, suggesting a role of tonicity in FGF23 formation. Moreover, these changes in FGF23 were inversely proportional to the expression of NFAT5 (nuclear factor of activated T cells-5), a transcription factor responsible for tonicity-mediated cellular adaptations. Furthermore, arginine vasopressin, which is often responsible for hyponatremia, did not affect FGF23 production. Next, we performed a comprehensive and unbiased RNA-seq analysis of UMR-106 cells exposed to low versus high [Na+], which revealed several novel genes involved in cellular adaptation to altered tonicity. Additional analysis of cells with Crisp-Cas9-mediated NFAT5 deletion indicated that NFAT5 controls numerous genes associated with FGF23 synthesis, thereby confirming its role in [Na+]-mediated FGF23 regulation. In line with these in vitro observations, we found that hyponatremia patients have higher FGF23 levels. Our results suggest that [Na+] is a critical regulator of FGF23 synthesis.

USF2 promotes autophagy and proliferation in chronic lymphocytic leukemia by inhibiting STUB1-induced NFAT5 ubiquitination.

Chronic lymphocytic leukemia (CLL) mainly affects the health of older adults and is difficult to cure. Upstream stimulatory factor 2 (USF2) has been implicated in several diseases and conditions including cancers. However, the effect of USF2 on CLL has not been elucidated. To investigate the effect of USP2 on proliferation and autophagy of CLL, and to explore the underlying mechanism. The mRNA of USF2 and STIP1 homology and U-Box containing protein 1 (STUB1) was analyzed using qRT-PCR. Western blots were used to evaluate the expression level of USF2, LC3II, Beclin-1, P62, STUB1, and NFAT5. The cell proliferation was evaluated using CCK-8 and EdU assays. The cell apoptosis was evaluated using flow cytometry. Indirect fluorescent assay (IFA) was performed to analyze LC3 signal. Nuclear factor of activated T-cells 5 (NFAT5) ubiquitination was detected using immunoprecipitation (IP) assay. The CLL progression was evaluated in xenotransplantation model of nude mice. USF2 was highly expressed in CLL tissues and cell lines. USF2 knockdown suppressed the cell viability and EdU incorporation, while promoting cell apoptosis. Meanwhile, USF2 knockdown reduced the level of LC3II and Beclin-1, but increased P62, illustrating USF2 knockdown inhibiting autophagy. USF2 induced NFAT5 ubiquitination and promoted NFAT5 protein level via repressing STUB1. The downregulation of USF2 weakened CLL progression in xenotransplantation model of nude mice. CLL survival and autophagy was dependent on highly expressed USF2 which promoted the expression and ubiquitination of NFAT5 through inhibiting the transcription of STUB1, which makes USF2 a promising therapeutic candidate for CLL treatment.

Direct ionic stress sensing and mitigation by the transcription factor NFAT5.

Homeostatic control of intracellular ionic strength is essential for protein, organelle and genome function, yet mechanisms that sense and enable adaptation to ionic stress remain poorly understood in animals. We find that the transcription factor NFAT5 directly senses solution ionic strength using a C-terminal intrinsically disordered region. Both in intact cells and in a purified system, NFAT5 forms dynamic, reversible biomolecular condensates in response to increasing ionic strength. This self-associative property, conserved from insects to mammals, allows NFAT5 to accumulate in the nucleus and activate genes that restore cellular ion content. Mutations that reduce condensation or those that promote aggregation both reduce NFAT5 activity, highlighting the importance of optimally tuned associative interactions. Remarkably, human NFAT5 alone is sufficient to reconstitute a mammalian transcriptional response to ionic or hypertonic stress in yeast. Thus NFAT5 is both the sensor and effector of a cell-autonomous ionic stress response pathway in animal cells.

Sodium accumulation in the skin is associated with higher density of skin lymphatic vessels in patients with arterial hypertension.

PURPOSE: Recent studies, conducted mainly on the rodent model, have demonstrated that regulatory pathway in the skin provided by glycosaminoglycans, nuclear factor of activated T cells 5 (NFAT5), vascular endothelial growth factor C (VEGF-C) and process of lymphangiogenesis may play an important role in extrarenal regulation of sodium (Na+) balance, body water volume, and blood pressure. We aimed to investigate the concentrations and relations among the main factors of this pathway in human skin to confirm that this regulatory axis also exists in humans. PATIENTS AND METHODS: Skin specimens from patients diagnosed with arterial hypertension and from control group were histologically and molecularly examined. RESULTS: The primary hypertensive and control groups did not differ in Na+ â€‹concentrations in the skin. However, the patients with hypertension and higher skin Na+ concentration had significantly greater density of skin lymphatic vessels. Higher skin Na+concentration was associated with higher skin water content. In turn, skin water content correlated with factors associated with lymphangiogenesis, i.e. NFAT5, VEGF-C, and podoplanin (PDPN) mRNA expression in the skin. The strong mutual pairwise correlations of the expressions of NFAT5, VEGF-C, vascular endothelial growth factor D (VEGF-D) and PDPN mRNA were noted in the skin in all of the studied groups. CONCLUSIONS: Our study confirms that skin interstitium and the lymphatic system may be important players in the pathophysiology of arterial hypertension in humans. Based on the results of our study and existing literature in this field, we propose the hypothetical model which might explain the phenomenon of salt-sensitivity.

Inflammation of Dry Eye Syndrome: A Cellular Study of the Epithelial and Macrophagic Involvement of NFAT5 and RAGE.

Dry eye inflammation is a key step in a vicious circle and needs to be better understood in order to break it. The goals of this work were to, first, characterize alarmins and cytokines released by ocular surface cells in the hyperosmolar context and, second, study the role of NFAT5 in this process. Finally, we studied the potential action of these alarmins in ocular surface epithelial cells and macrophages via RAGE pathways. HCE and WKD cell lines were cultured in a NaCl-hyperosmolar medium and the expression of alarmins (S100A4, S100A8, S100A9, and HMGB1), cytokines (IL6, IL8, TNFα, and MCP1), and NFAT5 were assessed using RT-qPCR, ELISA and multiplex, Western blot, immunofluorescence, and luciferase assays. In selected experiments, an inhibitor of RAGE (RAP) or NFAT5 siRNAs were added before the hyperosmolar stimulations. HCE and WKD cells or macrophages were treated with recombinant proteins of alarmins (with or without RAP) and analyzed for cytokine expression and chemotaxis, respectively. Hyperosmolarity induced epithelial cell inflammation depending on cell type. NFAT5, but not RAGE or alarmins, participated in triggering epithelial inflammation. Furthermore, the release of alarmins induced macrophage migration through RAGE. These in vitro results suggest that NFAT5 and RAGE have a role in dry eye inflammation.

2-methoxyestradiol restrains non-small cell lung cancer tumorigenesis through regulating circ_0010235/miR-34a-5p/NFAT5 axis.

BACKGROUND: Non-small cell lung cancer (NSCLC) is one of the most prevalent and severe malignant tumors in the world and its molecular mechanism is still unclear. In recent years, increasing evidence indicates the significant roles of circRNAs in NSCLC. It has been determined that 2-methoxyestradiol (2-MeOE2) exerts antitumor roles in many cancers. However, the molecular mechanism of 2-MeOE2 in regulating the development of lung cancer needs further elucidation. METHODS: The expression levels of circ_0010235, microRNA-34a-5p (miR-34a-5p), and nuclear factor of activated T cells 5 (NFAT5) were detected by quantitative real-time polymerase chain reaction (qRT-PCR). Cell proliferation, apoptosis and invasion were detected by cell counting kit-8 (CCK-8), 5-ethynyl-2'-deoxyuridine (EdU) assay, flow cytometry and transwell assays, respectively. The interaction between miR-34a-5p and circ_0010235 or NFAT5 was predicted by bioinformatic software and confirmed by dual-luciferase reporter assay. RESULTS: Our data showed 2-MeOE2 hindered cell proliferation, invasion and induced apoptosis in NSCLC, which could be reversed by upregulation of circ_0010235 and NFAT5 or miR-34a-5p knockdown. Circ_0010235 and NFAT5 expression levels were increased, and miR-34a-5p expression level was decreased in NSCLC tissues and cells. In addition, 2-MeOE2 treatment suppressed the expression of circ_0010235 and NFAT5 while promoted the expression of miR-34a-5p. Furthermore, circ_0010235 functioned as a molecular sponge of miR-34a-5p to regulate NFAT5 expression. Knockdown of circ_0010235 or 2-MeOE2 treatment constrained tumor growth in vivo, and circ_0010235 depletion enhanced the inhibitory effect of 2-MeOE2 on tumor growth in vivo. CONCLUSION: These findings demonstrated that 2-MeOE2 retarded NSCLC progression by modulating the circ_0010235/miR-34a-5p/NFAT5 axis, thus providing a new perspective for 2-MeOE2 treatment in NSCLC.

NFAT5 Restricts Bovine Herpesvirus 1 Productive Infection in MDBK Cell Cultures.

Bovine herpesvirus 1 (BoHV-1), an important bovine viral pathogen, causes severe disease in the upper respiratory tract and reproductive system. Tonicity-responsive enhancer-binding protein (TonEBP), also known as nuclear factor of activated T cells 5 (NFAT5), is a pleiotropic stress protein involved in a range of cellular processes. In this study, we showed that the knockdown of NFAT5 by siRNA increased BoHV-1 productive infection and overexpression of NFAT5 via plasmid transfection decreased virus production in bovine kidney (MDBK) cells. Virus productive infection at later stages significantly increased transcription of NFAT5 but not appreciably alter measurable NFAT5 protein levels. Virus infection relocalized NFAT5 protein and decreased the cytosol accumulation. Importantly, we found a subset of NFAT5 resides in mitochondria, and virus infection led to the depletion of mitochondrial NFAT5. In addition to full-length NFAT5, another two isoforms with distinct molecular weights were exclusively detected in the nucleus, where the accumulation was differentially affected following virus infection. In addition, virus infection differentially altered mRNA levels of PGK1, SMIT, and BGT-1, the canonical downstream targets regulated by NFAT5. Taken together, NFAT5 is a potential host factor that restricts BoHV-1 productive infection, and virus infection hijacks NFAT5 signaling transduction by relocalization of NFAT5 molecules in cytoplasm, nucleus, and mitochondria, as well as altered expression of its downstream targets. IMPORTANCE Accumulating studies have revealed that NFAT5 regulates disease development due to infection of numerous viruses, underlying the importance of the host factor in virus pathogenesis. Here, we report that NFAT5 has capacity to restrict BoHV-1 productive infection in vitro. And virus productive infection at later stages may alter NFAT5 signaling pathway as observed by relocalization of NFAT5 protein, reduced accumulation of NFAT5 in cytosol, and differential expression of NFAT5 downstream targets. Importantly, for the first time, we found that a subset of NFAT5 resides in mitochondria, implying that NFAT5 may regulate mitochondrial functions, which will extend our knowledge on NFAT5 biological activities. Moreover, we found two NFAT5 isoforms with distinct molecular weights were exclusively detected in the nucleus, where the accumulation was differentially affected following virus infection, representing a novel regulation mechanism on NFAT5 function in response to BoHV-1infection.

Study on the role of histone epigenetic modification in replication of hepatitis B virus.

Hepatitis B virus (HBV) infection is a global health problem and lacks effective therapies in clinic. This study attempted to investigate the role of histone deacetylase 3 (HDAC3) in HBV replication. Cells were treated with 1.3 folds of HBV genome. The expression patterns of HDAC3, miR-29a-3p, and nuclear factor of activated T-cells 5 (NFAT5) in cells were determined by real-time quantitative polymerase chain reaction and Western blot analysis. HBV replication was assessed by measurements of HBV DNA, HBV RNA, hepatitis B surface antigen, and hepatitis B E antigen. After chromatin immunoprecipitation and RNA pull-down assays to testify gene interactions, rescue experiments and animal experiments were performed to assess the role of miR-29a-3p/NFAT5 in HBV replication and the role of HDAC3 in vivo. HDAC3 level was decreased by pHBV1.3 plasmid in a concentration-dependent manner. HDAC3 overexpression can inhibit HBV replication, which was neutralized by miR-29a-3p overexpression or NFAT5 downregulation. Mechanically, HDAC3 overexpression reduced the enrichment of histone 3 lysine 9 acetylation on the miR-29a-3p promoter to inhibit miR-29a-3p expression and then promote NFAT5 transcription. In vivo, HDAC3 restrained HBV replication through the miR-29a-3p/NFAT5 axis. Overall, HDAC3 downregulation was associated with HBV replication and HDAC3 overexpression inhibited HBV replication through H3K9ac/miR-29a-3p/NFAT5.

A novel molecular mechanism mediated by circCCDC134 regulates non-small cell lung cancer progression.

BACKGROUND: Circular RNA (circRNA) plays a crucial role in non-small cell lung cancer (NSCLC) progression. However, the role of circCCDC134 in NSCLC is still largely unknown. METHODS: Quantitative real-time PCR was utilized for measuring circCCDC134, microRNA (miR)-625-5p and nuclear factor of activated T cell 5 (NFAT5) expression. Cell functions were evaluated by colony formation, EdU, transwell, and wound healing assays and flow cytometry. Glucose consumption, lactate production, and ATP level were determined to analyze cell glycolysis. Western blot analysis was used to detect protein expression. Animal experiments were performed to assess the effect of circCCDC134 on NSCLC tumor growth. RNA interaction was evaluated by dual-luciferase reporter assay and RIP assay. Exosomes were isolated from the serum of NSCLC patients and healthy normal controls. RESULTS: Highly expressed circCCDC134 was found in NSCLC tissues and cells, as well as in the serum exosomes of NSCLC patients. Downregulated circCCDC134 restrained NSCLC cell growth, metastasis and glycolysis. CircCCDC134 sponged miR-625-5p to regulate NFAT5. MiR-625-5p inhibitor abolished the regulation of circCCDC134 knockdown on NSCLC progression, and NFAT5 overexpression eliminated the effects of miR-625-5p on NSCLC cell behaviors. CircCCDC134 knockdown also inhibited NSCLC tumor growth. CONCLUSION: Our study revealed that circCCDC134 was involved in regulating NSCLC progression through the miR-625-5p/NFAT5 pathway, confirming that circCCDC134 might function as the diagnostic and therapeutic target for NSCLC.