Transcription Factor NFAT5 Research Articles

Analgesia linked to Nav1.7 loss of function requires µ- and δ-opioid receptors

Background: Functional deletion of the Scn9a (sodium voltage-gated channel alpha subunit 9) gene encoding sodium channel Nav1.7 makes humans and mice pain-free. Opioid signalling contributes to this analgesic state. We have used pharmacological and genetic approaches to identify the opioid receptors involved in this form of analgesia. We also examined the regulation of proenkephalin expression by the transcription factor Nfat5 that binds upstream of the Penk gene. Methods: We used specific µ-, δ- and κ-opioid receptor antagonists alone or in combination to examine which opioid receptors were necessary for Nav1.7 loss-associated analgesia in mouse behavioural assays of thermal pain. We also used µ- and δ-opioid receptor null mutant mice alone and in combination in behavioural assays to examine the role of these receptors in Nav1.7 knockouts pain free phenotype. Finally, we examined the levels of Penk mRNA in Nfat5-null mutant mice, as this transcription factor binds to consensus sequences upstream of the Penk gene. Results: The pharmacological block or deletion of both µ- and δ-opioid receptors was required to abolish Nav1.7-null opioid-related analgesia. κ-opioid receptor antagonists were without effect. Enkephalins encoded by the Penk gene are upregulated in Nav1.7 nulls. Deleting Nfat5, a transcription factor with binding motifs upstream of Penk, induces the same level of enkephalin mRNA expression as found in Nav1 .7 nulls, but without consequent analgesia. These data confirm that a combination of events linked to Scn9a gene loss is required for analgesia. Higher levels of endogenous enkephalins, potentiated opioid receptors, diminished electrical excitability and loss of neurotransmitter release together contribute to the analgesic phenotype found in Nav1.7-null mouse and human mutants. Conclusions: These observations help explain the failure of Nav1.7 channel blockers alone to produce analgesia and suggest new routes for analgesic drug development.

Analgesia linked to Nav1.7 loss of function requires µ- and δ-opioid receptors.

Background: Functional deletion of the Scn9a (sodium voltage-gated channel alpha subunit 9) gene encoding sodium channel Nav1.7 makes humans and mice pain-free. Opioid signalling contributes to this analgesic state. We have used pharmacological and genetic approaches to identify the opioid receptors involved in this form of analgesia. We also examined the regulation of proenkephalin expression by the transcription factor Nfat5 that binds upstream of the Penk gene. Methods: We used specific µ-, δ- and κ-opioid receptor antagonists alone or in combination to examine which opioid receptors were necessary for Nav1.7 loss-associated analgesia in mouse behavioural assays of thermal pain. We also used µ- and δ-opioid receptor null mutant mice alone and in combination in behavioural assays to examine the role of these receptors in Nav1.7 knockouts pain free phenotype. Finally, we examined the levels of Penk mRNA in Nfat5-null mutant mice, as this transcription factor binds to consensus sequences upstream of the Penk gene. Results: The pharmacological block or deletion of both µ- and δ-opioid receptors was required to abolish Nav1.7-null opioid-related analgesia. κ-opioid receptor antagonists were without effect. Enkephalins encoded by the Penk gene are upregulated in Nav1.7 nulls. Deleting Nfat5, a transcription factor with binding motifs upstream of Penk, induces the same level of enkephalin mRNA expression as found in Nav1 .7 nulls, but without consequent analgesia. These data confirm that a combination of events linked to Scn9a gene loss is required for analgesia. Higher levels of endogenous enkephalins, potentiated opioid receptors, diminished electrical excitability and loss of neurotransmitter release together contribute to the analgesic phenotype found in Nav1.7-null mouse and human mutants. Conclusions: These observations help explain the failure of Nav1.7 channel blockers alone to produce analgesia and suggest new routes for analgesic drug development.

Pro-inflammatory AGE-RAGE signaling is activated during arousal from hibernation in ground squirrel adipose.

BackgroundInflammation is generally suppressed during hibernation, but select tissues (e.g. lung) have been shown to activate both antioxidant and pro-inflammatory pathways, particularly during arousal from torpor when breathing rates increase and oxidative metabolism fueling the rewarming process produces more reactive oxygen species. Brown and white adipose tissues are now understood to be major hubs for the regulation of immune and inflammatory responses, yet how these potentially damaging processes are regulated by fat tissues during hibernation has hardly been studied. The advanced glycation end-product receptor (RAGE) can induce pro-inflammatory responses when bound by AGEs (which are glycated and oxidized proteins, lipids, or nucleic acids) or damage associated molecular pattern molecules (DAMPs, which are released from dying cells).MethodsSince gene expression and protein synthesis are largely suppressed during torpor, increases in AGE-RAGE pathway proteins relative to a euthermic control could suggest some role for these pro-inflammatory mediators during hibernation. This study determined how the pro-inflammatory AGE-RAGE signaling pathway is regulated at six major time points of the torpor-arousal cycle in brown and white adipose from a model hibernator, Ictidomys tridecemlineatus. Immunoblotting, RT-qPCR, and a competitive ELISA were used to assess the relative gene expression and protein levels of key regulators of the AGE-RAGE pathway during a hibernation bout.ResultsThe results of this study revealed that RAGE is upregulated as animals arouse from torpor in both types of fat, but AGE and DAMP levels either remain unchanged or decrease. Downstream of the AGE-RAGE cascade, nfat5 was more highly expressed during arousal in brown adipose.DiscussionAn increase in RAGE protein levels and elevated mRNA levels of the downstream transcription factor nfat5 during arousal suggest the pro-inflammatory response is upregulated in adipose tissue of the hibernating ground squirrel. It is unlikely that this cascade is activated by AGEs or DAMPs. This research sheds light on how a fat-but-fit organism with highly regulated metabolism may control the pro-inflammatory AGE-RAGE pathway, a signaling cascade that is often dysregulated in other obese organisms.

NFAT5-Regulated Macrophage Polarization Supports the Proinflammatory Function of Macrophages and T Lymphocytes.

Macrophages are exquisite sensors of tissue homeostasis that can rapidly switch between pro- and anti-inflammatory or regulatory modes to respond to perturbations in their microenvironment. This functional plasticity involves a precise orchestration of gene expression patterns whose transcriptional regulators have not been fully characterized. We had previously identified the transcription factor NFAT5 as an activator of TLR-induced responses, and in this study we explore its contribution to macrophage functions in different polarization settings. We found that both in classically and alternatively polarized macrophages, NFAT5 enhanced functions associated with a proinflammatory profile such as bactericidal capacity and the ability to promote Th1 polarization over Th2 responses. In this regard, NFAT5 upregulated the Th1-stimulatory cytokine IL-12 in classically activated macrophages, whereas in alternatively polarized ones it enhanced the expression of the pro-Th1 mediators Fizz-1 and arginase 1, indicating that it could promote proinflammatory readiness by regulating independent genes in differently polarized macrophages. Finally, adoptive transfer assays in vivo revealed a reduced antitumor capacity in NFAT5-deficient macrophages against syngeneic Lewis lung carcinoma and ID8 ovarian carcinoma cells, a defect that in the ID8 model was associated with a reduced accumulation of effector CD8 T cells at the tumor site. Altogether, detailed analysis of the effect of NFAT5 in pro- and anti-inflammatory macrophages uncovered its ability to regulate distinct genes under both polarization modes and revealed its predominant role in promoting proinflammatory macrophage functions.

Peptide affinity analysis of proteins that bind to an unstructured region containing the transactivating domain of the osmoprotective transcription factor NFAT5.

NFAT5 is a transcription factor originally identified because it is activated by hypertonicity and that activation increases expression of genes that protect against the adverse effects of the hypertonicity. However, its targets also include genes not obviously related to tonicity. The transactivating domain of NFAT5 is contained in its COOH-terminal region, which is predicted to be unstructured. Unstructured regions are common in transcription factors particularly in transactivating domains where they can bind co-regulatory proteins essential to their function. To identify potential binding partners of NFAT5 from either cytoplasmic or nuclear HEK293 cell extracts, we used peptide affinity chromatography followed by mass spectrometry. Peptide aptamer-baits consisted of overlapping 20 amino acid peptides within the predicted COOH-terminal unstructured region of NFAT5. We identify a total of 351 unique protein preys that associate with at least one COOH-terminal peptide bait from NFAT5 in either cytoplasmic or nuclear extracts from cells incubated at various tonicities (NaCl varied). In addition to finding many proteins already known to associate with NFAT5, we found many new ones whose function suggest novel aspects of NFAT5 regulation, interaction, and function. Relatively few of the proteins pulled down by peptide baits from NFAT5 are generally involved in transcription, and most, therefore, are likely to be specifically related to the regulation of NFAT5 or its function. The novel associated proteins are involved with cancer, effects of hypertonicity on chromatin, development, splicing of mRNA, transcription, and vesicle trafficking.

Context-dependent regulation of Th17-associated genes and IFNγ expression by the transcription factor NFAT5.

Stress-activated transcription factors influence T-cell function in different physiopathologic contexts. NFAT5, a relative of nuclear factor κB and the calcineurin-activated NFATc transcription factors, protects mammalian cells from hyperosmotic stress caused by the elevation of extracellular sodium levels. In T cells exposed to hypernatremia, NFAT5 not only induces osmoprotective gene products but also cytokines and immune receptors, which raises the question of whether this factor could regulate other T-cell functions in osmostress-independent contexts. Here we have used mice with a conditional deletion of Nfat5 in mature T lymphocytes to explore osmostress-dependent and -independent functions of this factor. In vitro experiments with CD4 T cells stimulated in hyperosmotic medium showed that NFAT5 enhanced the expression of IL-2 and the Th17-associated gene products RORγt and IL-23R. By contrast, NFAT5-deficient CD4 T cells activated in vivo by anti-CD3 antibody exhibited a different activation profile and were skewed towards enhanced interferon γ (IFNγ) and IL-17 expression and attenuated Treg responses. Using a model of experimental colitis, we observed that mice lacking NFAT5 in T cells exhibited exacerbated intestinal colitis and enhanced expression of IFNγ in draining lymph nodes and colon. These results show that NFAT5 can modulate different T-cell responses depending on stress conditions and stimulatory context.

Peptide affinity analysis of proteins that bind to an unstructured NH2-terminal region of the osmoprotective transcription factor NFAT5.

NFAT5 is an osmoregulated transcription factor that particularly increases expression of genes involved in protection against hypertonicity. Transcription factors often contain unstructured regions that bind co-regulatory proteins that are crucial for their function. The NH2-terminal region of NFAT5 contains regions predicted to be intrinsically disordered. We used peptide aptamer-based affinity chromatography coupled with mass spectrometry to identify protein preys pulled down by one or more overlapping 20 amino acid peptide baits within a predicted NH2-terminal unstructured region of NFAT5. We identify a total of 467 unique protein preys that associate with at least one NH2-terminal peptide bait from NFAT5 in either cytoplasmic or nuclear extracts from HEK293 cells treated with elevated, normal, or reduced NaCl concentrations. Different sets of proteins are pulled down from nuclear vs. cytoplasmic extracts. We used GeneCards to ascertain known functions of the protein preys. The protein preys include many that were previously known, but also many novel ones. Consideration of the novel ones suggests many aspects of NFAT5 regulation, interaction and function that were not previously appreciated, for example, hypertonicity inhibits NFAT5 by sumoylating it and the NFAT5 protein preys include components of the CHTOP complex that desumoylate proteins, an action that should contribute to activation of NFAT5.

How do kinases contribute to tonicity-dependent regulation of the transcription factor NFAT5?

NFAT5 plays a critical role in maintaining the renal functions. Its dis-regulation in the kidney leads to or is associated with certain renal diseases or disorders, most notably the urinary concentration defect. Hypertonicity, which the kidney medulla is normally exposed to, activates NFAT5 through phosphorylation of a signaling molecule or NFAT5 itself. Hypotonicity inhibits NFAT5 through a similar mechanism. More than a dozen of protein and lipid kinases have been identified to contribute to tonicity-dependent regulation of NFAT5. Hypertonicity activates NFAT5 by increasing its nuclear localization and transactivating activity in the early phase and protein abundance in the late phase. The known mechanism for inhibition of NFAT5 by hypotonicity is a decrease of nuclear NFAT5. The present article reviews the effect of each kinase on NFAT5 nuclear localization, transactivation and protein abundance, and the relationship among these kinases, if known. Cyclosporine A and tacrolimus suppress immune reactions by inhibiting the phosphatase calcineurin-dependent activation of NFAT1. It is hoped that this review would stimulate the interest to seek explanations from the NFAT5 regulatory pathways for certain clinical presentations and to explore novel therapeutic approaches based on the pathways. On the basic science front, this review raises two interesting questions. The first one is how these kinases can specifically signal to NFAT5 in the context of hypertonicity or hypotonicity, because they also regulate other cellular activities and even opposite activities in some cases. The second one is why these many kinases, some of which might have redundant functions, are needed to regulate NFAT5 activity. This review reiterates the concept of signaling through cooperation. Cells need these kinases working in a coordinated way to provide the signaling specificity that is lacking in the individual one. Redundancy in regulation of NFAT5 is a critical strategy for cells to maintain robustness against hypertonic or hypotonic stress.

Regulation of tonicity-dependent activation of NFAT5 by mitogen-activated protein kinases

Various tissues, most notably the kidney medulla, are normally exposed to a hypertonic environment. The transcription factor NFAT5 is essential for these tissues to survive and function properly by activating expression of the osmoprotective genes and genes that are unique for the functions of these tissues. Moreover, hypertonicity causes or is associated with certain diseases and disorders such as diabetes mellitus and inflammation. NFAT5 plays an important role in pathogenesis of these diseases and disorders. Hypertonicity activates NFAT5 through phosphorylation of signaling molecules or NFAT5 itself. More than a dozen of kinases have been identified. This mini-review will focus on mitogen-activated protein kinases (MAPKs), which include p38, ERK and JNK families, because they are the most studied kinases in regulation of NFAT5. p38alpha, ERK1/2 and some of their upstream kinases have been demonstrated to contribute to tonicity-dependent activation of NFAT5 by increasing its transactivating activity without significantly affecting its nuclear accumulation. However, many important questions remain. For example, hypotonicity, which inhibits NFAT5 activity, also activates p38 and ERK1/2, raising a question concerning how p38 and ERK1/2 relay the hypertonic signal to NFAT5. The present mini-review calls for attention to MAPKs isoforms, duration and strength of activation, cellular localization and interaction with specific scaffolds and other signaling molecules, when they are studied for their roles in regulation of NFAT5. Further, whether MAPKs regulate NFAT5 in the tissues remains to be addressed.

NFAT5 Expression in Circulating PBMCs is a Potential Biomarker of Whole‐body Dehydration and Stress

BackgroundOsmotic stress‐specific transcription factor NFAT5 (Nuclear Factor of Activated T cells) and heat stress‐specific factor HSF1 induce transcription of HSP70 and other protective proteins. Our previous work suggests NFAT5 as a novel cellular marker of whole‐body dehydration. We aimed to further characterize NFAT5, HSF1, and chaperone responses to progressive stress (dehydration, exercise, and heat) and recovery in humans.MethodsHealthy males (n=36) completed an overnight (16h) fluid deprivation (‐2.0±0.9% body mass) followed by 2h of cycling (146±2 bpm; 38°C, 40%RH; ‐4.2±0.9% total body mass). Subjects then partially rehydrated (1h; +1.90±0.57% body mass) and returned 24h later after continued ad libitum rehydration. We measured cellular HSF1, NFAT5, and HSP70 in PBMCs (flow cytometry) and plasma eHSP70 (ELISA).ResultsPBMC NFAT5 expression increased post overnight fluid deprivation (20.96%; p=0.056) and post exercise (additional 3.35%; p=0.026), and decreased with partial rehydration (‐4.78%; p=0.072). Cellular and plasma HSP70 responded to stress and recovery with different patterns, and without detectable changes in cellular HSF1.ConclusionPBMC NFAT5 expression responded to dehydration. Cellular and plasma HSP70 responded to stress differently and may thus have different function. Elucidating immune cell molecular chaperone responses to stress exposure and recovery will aid in defining the effect of stress on immune cell function.

NH4Cl Treatment Prevents Tissue Calcification in Klotho Deficiency.

Klotho, a cofactor in suppressing 1,25(OH)2D3 formation, is a powerful regulator of mineral metabolism. Klotho-hypomorphic mice (kl/kl) exhibit excessive plasma 1,25(OH)2D3, Ca(2+), and phosphate concentrations, severe tissue calcification, volume depletion with hyperaldosteronism, and early death. Calcification is paralleled by overexpression of osteoinductive transcription factor Runx2/Cbfa1, Alpl, and senescence-associated molecules Tgfb1, Pai-1, p21, and Glb1. Here, we show that NH4Cl treatment in drinking water (0.28 M) prevented soft tissue and vascular calcification and increased the life span of kl/kl mice >12-fold in males and >4-fold in females without significantly affecting extracellular pH or plasma concentrations of 1,25(OH)2D3, Ca(2+), and phosphate. NH4Cl treatment significantly decreased plasma aldosterone and antidiuretic hormone concentrations and reversed the increase of Runx2/Cbfa1, Alpl, Tgfb1, Pai-1, p21, and Glb1 expression in aorta of kl/kl mice. Similarly, in primary human aortic smooth muscle cells (HAoSMCs), NH4Cl treatment reduced phosphate-induced mRNA expression of RUNX2/CBFA1, ALPL, and senescence-associated molecules. In both kl/kl mice and phosphate-treated HAoSMCs, levels of osmosensitive transcription factor NFAT5 and NFAT5-downstream mediator SOX9 were higher than in controls and decreased after NH4Cl treatment. Overexpression of NFAT5 in HAoSMCs mimicked the effect of phosphate and abrogated the effect of NH4Cl on SOX9, RUNX2/CBFA1, and ALPL mRNA expression. TGFB1 treatment of HAoSMCs upregulated NFAT5 expression and prevented the decrease of phosphate-induced NFAT5 expression after NH4Cl treatment. In conclusion, NH4Cl treatment prevents tissue calcification, reduces vascular senescence, and extends survival of klotho-hypomorphic mice. The effects of NH4Cl on vascular osteoinduction involve decrease of TGFB1 and inhibition of NFAT5-dependent osteochondrogenic signaling.

Inhibition of Phosphate-Induced Vascular Smooth Muscle Cell Osteo-/Chondrogenic Signaling and Calcification by Bafilomycin A1 and Methylamine

Background/Aims: Excessive phosphate concentrations trigger vascular calcification, an active process promoted by osteoinduction of vascular smooth muscle cells (VSMCs) with increased expression and activity of transcription factor RUNX2 (Core-binding factor α1, CBFA1), alkaline phosphatase (ALPL), TGFß1, transcription factor NFAT5, and NFAT5-sensitive transcription factor SOX9. The osteoinductive signaling and vascular calcification of hyperphosphatemic klotho-hypomorphic mice could be reversed by treatment with NH₄Cl, effects involving decrease of TGFß1 and inhibition of NFAT5-dependent osteoinductive signaling. Known effects of NH₄Cl include alkalinization of acidic cellular compartments. The present study explored whether osteo-/chondrogenic signaling could be influenced by alkalinization of acidic cellular compartments following inhibition of the vacuolar H⁺ ATPase with bafilomycin A1 or following dissipation of the pH gradient across the membranes of acidic cellular compartments with methylamine. Methods: Primary human aortic smooth muscle cells (HAoSMCs) were treated with high phosphate to trigger osteo-/chondrogenic signaling and calcification in the absence or presence of bafilomycin A1 or methylamine. Calcium content was determined using a QuantiChrom Calcium assay, ALP activity by a colorimetric assay and transcript levels by quantitative RT-PCR. Results: High phosphate increased significantly the calcium deposition, CBFA1 and ALPL mRNA expression as well as alkaline phosphatase activity in HAoSMCs, all effects ameliorated by both, bafilomycin A1 and methylamine. High phosphate further significantly up-regulated the mRNA levels of TGFB1, NFAT5 and SOX9, effects significantly blunted by additional treatment with bafilomycin A1 or methylamine. Treatment of HAoSMCs with human TGFß1 protein or high phosphate up-regulated NFAT5, SOX9, CBFA1 and ALPL mRNA expression to similarly high levels which could not be further increased by combined treatment with high phosphate and TGFß1. Bafilomycin A1 failed to reverse the osteo-/chondrogenic signaling triggered by high phosphate together with TGFß1. Conclusions: Inhibition of the vacuolar H⁺ ATPase or dissipation of the pH gradient across the membranes of acidic cellular compartments both disrupt osteo-/chondrogenic signaling and calcium deposition in VSMCs, observations supporting the hypothesis that vascular calcification requires acidic cellular compartments.

High-Salt Enhances the Inflammatory Response by Retina Pigment Epithelium Cells following Lipopolysaccharide Stimulation.

High-salt has been shown to play a role in the pathogenesis of autoimmune disease. In this study, we investigated the effect of high-salt on the production of inflammatory mediators by ARPE-19 cells and the possible mechanisms involved. ARPE-19 cells were cultured with LPS in DMEM to which extra NaCl had been added (20 mM and 40 mM). NaCl had no influence on the apoptosis and proliferation of ARPE-19. Addition of 40 mM NaCl significantly induced IL-6 and MCP-1 production but had no effect on IL-8 secretion. High mannitol, as an osmotic stress control, did not affect the secretion of inflammatory mediators by ARPE-19 cells indicating that the effect was not mediated by osmolarity. Coculture of ARPE-19 cells with NaCl resulted in significant increases in the phosphorylation of p38 MAPK, Akt, and NF-κB and an upregulation of the transcription factors NFAT5 and SGK1. High-salt significantly promotes IL-6 and MCP-1 production by ARPE-19 cells and is associated with activation of the p38 MAPK, Akt, and NF-κB pathway and NFAT-SGK1 pathways.

Abstract 37: Secretion of von Willebrand Factor by Endothelial Cells Links Salt to Hypercoagulability and Thrombosis

Hypercoagulability increases the risk of thrombi that cause cardiovascular events. Dehydration and hypernatremia are often accompanied by thrombosis, but the mechanisms are not clear. Von Willebrand Factor is secreted by endothelium, affecting aggregation of platelets and promoting activation of the coagulation cascade and formation of thrombi. Here we show that in culture of primary Human Umbilical Vein Endothelia Cells, elevating medium osmolality to 320-380 mosmol/kg by adding NaCl reversibly increases both vWF mRNA and vWF secretion. The high NaCl increases expression of tonicity regulated transcription factor NFAT5 and its binding to promoter of vWF gene, suggesting that vWF upregulation is caused by hypertonic signaling. To elevate NaCl in vivo, we modeled mild dehydration, subjecting mice to water restriction (WR) for 9 days by feeding them with gel food containing 30% of water. Such WR elevates blood sodium from 145.1±0.5 to 150.2±1.3 mmol/l and activates hypertonic signaling as evidenced from increased expression of NFAT5 in tissues. WR increased vWF mRNA in liver and lung and raised vWF protein in blood. Immunostaining of liver revealed increased production of vWF protein by endothelium and increased number of microthrombi inside capillaries. WR also increased blood level of D-dimer, a fibrinogen degradation product indicative of ongoing coagulation and thrombolysis. We conclude that elevation of extracellular sodium within the physiological range raises expression and secretion of von Willebrand Factor sufficiently to increase coagulability of blood and risk of thrombosis. The results suggest that hydration and salt intake are modifiable factors that affect coagulability and thrombosis through high salt-dependent secretion of vWF from endothelial cells.

14-3-3-β and -{varepsilon} contribute to activation of the osmoprotective transcription factor NFAT5 by increasing its protein abundance and its transactivating activity.

Having previously found that high NaCl causes rapid exit of 14‐3‐3 isoforms from the nucleus, we used siRNA‐mediated knockdown to test whether 14‐3‐3s contribute to the high NaCl‐induced increase in the activity of the osmoprotective transcription factor NFAT5. We find that, when NaCl is elevated, knockdown of 14‐3‐3‐β and/or 14‐3‐3‐ε decreases NFAT5 transcriptional activity, as assayed both by luciferase reporter and by the mRNA abundance of the NFAT5 target genes aldose reductase and the sodium‐ and chloride‐dependent betaine transporter, BGT1. Knockdown of other 14‐3‐3 isoforms does not significantly affect NFAT5 activity. 14‐3‐3‐β and/or 14‐3‐3‐ε do not act by affecting the nuclear localization of NFAT5, but by at least two other mechanisms: (1) 14‐3‐3‐β and 14‐3‐3‐ε increase protein abundance of NFAT5 and (2) they increase NFAT5 transactivating activity. When NaCl is elevated, knockdown of 14‐3‐3‐β and/or 14‐3‐3‐ε reduces the protein abundance of NFAT5, as measured by Western blot, without affecting the level of NFAT5 mRNA, and the knockdown also decreases NFAT5 transactivating activity, as measured by luciferase reporter. The 14‐3‐3s increase NFAT5 protein, not by increasing its translation, but by decreasing the rate at which it is degraded, as measured by cycloheximide chase. It is not clear at this point whether the 14‐3‐3s affect NFAT5 directly or indirectly through their effects on other proteins that signal activation of NFAT5.

Sfmbt2 10th intron-hosted miR-466(a/e)-3p are important epigenetic regulators of Nfat5 signaling, osmoregulation and urine concentration in mice

Sfmbt2-hosted miR-466a-3p and its close relatives are often among the most significantly up-regulated or down-regulated miRNAs in responses to numerous deleterious environmental stimuli. The exact roles of these miRNAs in cellular stress responses, however, are not clear. Here we showed that many Sfmbt2-hosted miRNAs were highly hypertonic stress responsive in vitro and in vivo. In renal medulla, water deprivation induced alterations in the expression of miR-466(a/b/c/e/p)-3p in a pattern similar to that of miR-200b-3p, a known regulator of osmoresponsive transcription factor Nfat5. Remarkably, exposure of mIMCD3 cells to an arginine vasopressin analog time-dependently down-regulated the expression of miR-466(a/b/c/e/p)-3p and miR-200b-3p, which provides a novel regulatory mechanism for these osmoresponsive miRNAs. In cultured mIMCD3 cells we further demonstrated that miR-466a-3p and miR-466g were capable of targeting Nfat5 by interacting with its 3′UTR. In transgenic mice overexpressing miR-466a-3p, significant down-regulation of Nfat5 and many other osmoregulation-related genes was observed in both the renal cortex and medulla. Moreover, sustained transgenic over-expression of miR-466a-3p was found to be associated with polydipsia, polyuria and disturbed ion homeostasis and kidney morphology. Since the mature sequence of miR-466a-3p is completely equivalent to that of miR-466e-3p and that the seed sequence of miR-466a-3p is completely equivalent to that of miR-297(a/b/c)-3p, miR-466d-3p, miR-467g and miR-669d-3p, and that miR-466a-3p differs from miR-466(b/c/p)-3p only in a 5′ nucleotide, we propose that miR-466a-3p and many of its close relatives are important epigenetic regulators of renal Nfat5 signaling, osmoregulation and urine concentration in mice.

Differential DNA Methylation Regions in Cytokine and Transcription Factor Genomic Loci Associate with Childhood Physical Aggression

BackgroundAnimal and human studies suggest that inflammation is associated with behavioral disorders including aggression. We have recently shown that physical aggression of boys during childhood is strongly associated with reduced plasma levels of cytokines IL-1α, IL-4, IL-6, IL-8 and IL-10, later in early adulthood. This study tests the hypothesis that there is an association between differential DNA methylation regions in cytokine genes in T cells and monocytes DNA in adult subjects and a trajectory of physical aggression from childhood to adolescence.Methodology/Principal FindingsWe compared the methylation profiles of the entire genomic loci encompassing the IL-1α, IL-6, IL-4, IL-10 and IL-8 and three of their regulatory transcription factors (TF) NFkB1, NFAT5 and STAT6 genes in adult males on a chronic physical aggression trajectory (CPA) and males with the same background who followed a normal physical aggression trajectory (control group) from childhood to adolescence. We used the method of methylated DNA immunoprecipitation with comprehensive cytokine gene loci and TF loci microarray hybridization, statistical analysis and false discovery rate correction. We found differentially methylated regions to associate with CPA in both the cytokine loci as well as in their transcription factors loci analyzed. Some of these differentially methylated regions were located in known regulatory regions whereas others, to our knowledge, were previously unknown as regulatory areas. However, using the ENCODE database, we were able to identify key regulatory elements in many of these regions that indicate that they might be involved in the regulation of cytokine expression.ConclusionsWe provide here the first evidence for an association between differential DNA methylation in cytokines and their regulators in T cells and monocytes and male physical aggression.

High NaCl-induced inhibition of PTG contributes to activation of NFAT5 through attenuation of the negative effect of SHP-1

Activation of the transcription factor NFAT5 by high NaCl involves changes in phosphorylation. By siRNA screening, we previously found that protein targeting to glycogen (PTG), a regulatory subunit of protein phosphatase1 (PP1), contributes to regulation of high NaCl-induced NFAT5 transcriptional activity. The present study addresses the mechanism involved. We find that high NaCl-induced inhibition of PTG elevates NFAT5 activity by increasing NFAT5 transactivating activity, protein abundance, and nuclear localization. PTG acts via a catalytic subunit PP1γ. PTG associates physically with PP1γ, and NaCl reduces both this association and remaining PTG-associated PP1γ activity. High NaCl-induced phosphorylation of p38, ERK, and SHP-1 contributes to activation of NFAT5. Knockdown of PTG does not affect phosphorylation of p38 or ERK. However, PTG and PP1γ bind to SHP-1, and knockdown of either PTG or PP1γ increases high NaCl-induced phosphorylation of SHP-1-S591, which inhibits SHP-1. Mutation of SHP-1-S591 to alanine, which cannot be phosphorylated, increases inhibition of NFAT5 by SHP-1. Thus high NaCl reduces the stimulatory effect of PTG and PP1γ on SHP-1, which in turn reduces the inhibitory effect of SHP-1 on NFAT5. Our findings add to the known functions of PTG, which was previously recognized only for its glycogenic activity.

Circulating and intracellular HSP72 responds acutely to cumulative exercise stress, heat stress, dehydration, and rehydration and may be mediated by collective effects of multiple stress‐specific transcription factors

We have previously shown that protective protein HSP72 responds acutely and chronically during 11d of daily exercise‐heat stress. In this study (n=6, men), we aimed to characterize acute HSP72 and transcription factor responses to 2d of sequential stress and rest periods. In each of 4 repeat visits, participants: 1) passively dehydrated overnight, 2) exercised for 2h with no fluid in a hot and humid environmental chamber, 3) rested and rehydrated for 1h, 4) maximally exercised for 0.5h, and 5) returned after 24h of at‐home recovery and rehydration. We tracked rectal temperature, hydration status, circulating HSP72, and intracellular (flow cytometry) expression of HSP72 and cellular stress‐specific transcription factor NFAT5. We found that circulating and intracellular HSP72 significantly correlated (p<0.05) with physiological manifestations of heat, exercise, and dehydration stresses and recovery/rehydration periods. NFAT5 expression correlated (p<0.05) with measures of hydration status suggesting for the first time that this cellular osmotic stress‐induced transcription factor may reflect whole body hydration status, and contribute to general protective responses during aggregate stress.

Salt, skeletons, and suicide. Focus on “Hyperosmotic stress regulates the distribution and stability of myocardin-related transcription factor, a key modulator of the cytoskeleton”

high NaCl and other sources of hypertonicity are cellular stresses that span evolution. A protective response common to most organisms is cellular accumulation of compatible organic osmolytes, driven by increased transcription of transporters and synthesizing enzymes. In mammalian cells the