[Show abstract][Hide abstract] ABSTRACT: The kinase mammalian target of rapamycin (mTOR) is a central regulator of cell growth and proliferation that integrates inputs from growth factor receptors, nutrient availability, intracellular ATP (adenosine 5'-triphosphate), and a variety of stressors. Since early works in the mid-1990s uncovered the role of mTOR in stimulating protein translation, this kinase has emerged as a rather multifaceted regulator of numerous processes. Whereas mTOR is generally activated by growth- and proliferation-stimulating signals, its activity can be reduced and even suppressed when cells are exposed to a variety of stress conditions. However, cells can also adapt to stress while maintaining their growth capacity and mTOR function. Despite knowledge accumulated on how stress represses mTOR, less is known about mTOR influencing stress responses. In this review, we discuss the capability of mTOR, in particular mTOR complex 1 (mTORC1), to activate stress-responsive transcription factors, and we outline open questions for future investigation.
[Show abstract][Hide abstract] ABSTRACT: The Rel-like transcription factors nuclear factor kappa B (NF-κB) and the calcineurin-dependent nuclear factor of activated T cells (NFATc) control specific points of thymocyte maturation. Thymocytes also express a distinct member of the Rel family, the calcineurin-independent, osmostress response regulator NFAT5. Here we show that IKKβ regulates the expression of NFAT5 in thymocytes, which in turn contributes to the survival of T-cell receptor αβ thymocytes and the transition from the β-selection checkpoint to the double-positive stage in an osmostress-independent manner. NFAT5-deficient thymocytes had normal expression and proximal signaling of the pre-T-cell receptor but exhibited a partial defect in β-chain allelic exclusion and increased apoptosis. Further analysis showed that NFAT5 regulated the expression of the prosurvival factors A1 and Bcl2 and attenuated the proapoptotic p53/Noxa axis. These findings position NFAT5 as a target of the IKKβ/NF-κB pathway in thymocytes and as a downstream effector of the prosurvival role of the pre-T-cell receptor.
Proceedings of the National Academy of Sciences 09/2013; · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Toll-like receptors (TLRs) engage networks of transcriptional regulators to induce genes essential for antimicrobial immunity. We report that NFAT5, previously characterized as an osmostress responsive factor, regulates the expression of multiple TLR-induced genes in macrophages independently of osmotic stress. NFAT5 was essential for the induction of the key antimicrobial gene Nos2 (inducible nitric oxide synthase [iNOS]) in response to low and high doses of TLR agonists but is required for Tnf and Il6 mainly under mild stimulatory conditions, indicating that NFAT5 could regulate specific gene patterns depending on pathogen burden intensity. NFAT5 exhibited two modes of association with target genes, as it was constitutively bound to Tnf and other genes regardless of TLR stimulation, whereas its recruitment to Nos2 or Il6 required TLR activation. Further analysis revealed that TLR-induced recruitment of NFAT5 to Nos2 was dependent on inhibitor of κB kinase (IKK) β activity and de novo protein synthesis, and was sensitive to histone deacetylases. In vivo, NFAT5 was necessary for effective immunity against Leishmania major, a parasite whose clearance requires TLRs and iNOS expression in macrophages. These findings identify NFAT5 as a novel regulator of mammalian anti-pathogen responses.
Journal of Experimental Medicine 02/2012; 209(2):379-93. · 13.21 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Although stress can suppress growth and proliferation, cells can induce adaptive responses that allow them to maintain these functions under stress. While numerous studies have focused on the inhibitory effects of stress on cell growth, less is known on how growth-promoting pathways influence stress responses. We have approached this question by analyzing the effect of mammalian target of rapamycin (mTOR), a central growth controller, on the osmotic stress response. Our results showed that mammalian cells exposed to moderate hypertonicity maintained active mTOR, which was required to sustain their cell size and proliferative capacity. Moreover, mTOR regulated the induction of diverse osmostress response genes, including targets of the tonicity-responsive transcription factor NFAT5 as well as NFAT5-independent genes. Genes sensitive to mTOR-included regulators of stress responses, growth and proliferation. Among them, we identified REDD1 and REDD2, which had been previously characterized as mTOR inhibitors in other stress contexts. We observed that mTOR facilitated transcription-permissive conditions for several osmoresponsive genes by enhancing histone H4 acetylation and the recruitment of RNA polymerase II. Altogether, these results reveal a previously unappreciated role of mTOR in regulating transcriptional mechanisms that control gene expression during cellular stress responses.
Nucleic Acids Research 01/2012; 40(10):4368-84. · 8.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Immune cells rely on the transcription factor NFAT5 to adapt to hypertonic stress. The hypertonicity-dependent role of NFAT5 in T cells in vivo remains unclear because mouse models of NFAT5 deficiency have produced substantially different T cell phenotypes. In this study, we analyzed the T cell compartment in NFAT5-null and T cell-specific NFAT5 knockout mice. We found that NFAT5-null mice had constitutive, pronounced hypernatremia and suffered a severe immunodeficiency, with T cell lymphopenia, altered CD8 naive/memory homeostasis, and inability to reject allogeneic tumors. By contrast, T cell-specific NFAT5 knockout mice had normal plasma tonicity, rejected allogeneic tumors, and exhibited only a mild, low-penetrance memory bias in CD8 cells. Notably, when T cells from these mice were cultured ex vivo in hypernatremic media, they exhibited features found in NFAT5-null mice, with pronounced naive/memory imbalance and impaired homeostatic survival in response to IL-7, as well as a severe inhibition of their mitogen-induced proliferation. By analyzing surface receptors whose expression might be affected in NFAT5-deficient cells, we identified CD24 as a novel NFAT5 target induced by hypertonicity both in vitro and in vivo, and required to sustain T cell expansion under osmostress. NFAT5 bound to the Cd24 promoter in response to hypertonicity facilitated the local derepression of chromatin and enhanced the expression of CD24 mRNA and protein. Altogether, our results indicate that the systemic hypernatremia of NFAT5-null mice is a major contributor to their immunodeficiency, and highlight the role of NFAT5 and CD24 in the homeostasis of T cells under osmostress in vivo.
The Journal of Immunology 10/2010; 185(11):6624-35. · 5.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Nuclear factor of activated T cells 5 (NFAT5) is a member of the Rel family of transcription factors and is an essential inducer of osmoprotective gene products in mammalian cells. Its activation by hypertonicity requires p38 mitogen-activated protein kinase (MAPK) signaling and other pathways. A study now elucidates a signaling cascade regulated by the guanine nucleotide exchange factor Brx that leads to the activation of p38alpha MAPK and the induction of nfat5 messenger RNA in response to osmotic stress in lymphocytes and renal medullary cells. Brx-deficient lymphocytes showed impaired responses to hypertonicity, and brx(+/-) mice exhibited immune defects similar to those of nfat5-deficient mice. These findings support a major role for Brx in regulating the osmoprotective function of NFAT5 in different cell types.
[Show abstract][Hide abstract] ABSTRACT: Hypertonicity can perturb cellular functions, induce DNA damage-like responses and inhibit proliferation. The transcription factor NFAT5 induces osmoprotective gene products that allow cells to adapt to sustained hypertonic conditions. Although it is known that NFAT5-deficient lymphocytes and renal medullary cells have reduced proliferative capacity and viability under hypertonic stress, less is understood about the contribution of this factor to DNA damage responses and cell cycle regulation.
We have generated conditional knockout mice to obtain NFAT5(-/-) T lymphocytes, which we used as a model of proliferating cells to study NFAT5-dependent responses. We show that hypertonicity triggered an early, NFAT5-independent, genotoxic stress-like response with induction of p53, p21 and GADD45, downregulation of cyclins, and cell cycle arrest. This was followed by an NFAT5-dependent adaptive phase in wild-type cells, which induced an osmoprotective gene expression program, downregulated stress markers, resumed cyclin expression and proliferation, and displayed enhanced NFAT5 transcriptional activity in S and G2/M. In contrast, NFAT5(-/-) cells failed to induce osmoprotective genes and exhibited poorer viability. Although surviving NFAT5(-/-) cells downregulated genotoxic stress markers, they underwent cell cycle arrest in G1/S and G2/M, which was associated with reduced expression of cyclins E1, A2 and B1. We also show that pathologic hypertonicity levels, as occurring in plasma of patients and animal models of osmoregulatory disorders, inhibited the induction of cyclins and aurora B kinase in response to T cell receptor stimulation in fresh NFAT5(-/-) lymphocytes.
We conclude that NFAT5 facilitates cell proliferation under hypertonic conditions by inducing an osmoadaptive response that enables cells to express fundamental regulators needed for cell cycle progression.
PLoS ONE 02/2009; 4(4):e5245. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The transcription factor NFAT5 is a major inducer of osmoprotective genes and is required to maintain the proliferative capacity of cells exposed to hypertonic stress. In response to hypertonicity, NFAT5 translocates to the nucleus, binds to regulatory regions of osmoprotective genes and activates their transcription. Besides stimulus-specific regulatory mechanisms, the activity of transcription factors in cycling cells is also regulated by the passage through mitosis, when most transcriptional processes are downregulated. It was not known whether mitosis could be a point of control for NFAT5.
Using confocal microscopy we observed that NFAT5 was excluded from chromatin during mitosis in both isotonic and hypertonic conditions. Analysis of NFAT5 deletions showed that exclusion was mediated by the carboxy-terminal domain (CTD). NFAT5 mutants lacking this domain showed constitutive binding to mitotic chromatin independent of tonicity, which caused them to localize in the nucleus and remain bound to chromatin in the subsequent interphase without hypertonic stimulation. We analyzed the contribution of the CTD, DNA binding, and nuclear import and export signals to the subcellular localization of this factor. Our results indicated that cytoplasmic localization of NFAT5 in isotonic conditions required both the exclusion from mitotic DNA and active nuclear export in interphase. Finally, we identified several regions within the CTD of NFAT5, some of them overlapping with transactivation domains, which were separately capable of causing its exclusion from mitotic chromatin.
Our results reveal a multipart mechanism regulating the subcellular localization of NFAT5. The transactivating module of NFAT5 switches its function from an stimulus-specific activator of transcription in interphase to an stimulus-independent repressor of binding to DNA in mitosis. This mechanism, together with export signals acting in interphase, resets the cytoplasmic localization of NFAT5 and prevents its nuclear accumulation and association with DNA in the absence of hypertonic stress.
PLoS ONE 01/2009; 4(9):e7036. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The transcription factor NFAT5/TonEBP regulates the response of mammalian cells to hypertonicity. However, little is known about the physiopathologic tonicity thresholds that trigger its transcriptional activity in primary cells. Wilkins et al. recently developed a transgenic mouse carrying a luciferase reporter (9xNFAT-Luc) driven by a cluster of NFAT sites, that was activated by calcineurin-dependent NFATc proteins. Since the NFAT site of this reporter was very similar to an optimal NFAT5 site, we tested whether this reporter could detect the activation of NFAT5 in transgenic cells.
The 9xNFAT-Luc reporter was activated by hypertonicity in an NFAT5-dependent manner in different types of non-transformed transgenic cells: lymphocytes, macrophages and fibroblasts. Activation of this reporter by the phorbol ester PMA plus ionomycin was independent of NFAT5 and mediated by NFATc proteins. Transcriptional activation of NFAT5 in T lymphocytes was detected at hypertonic conditions of 360-380 mOsm/kg (isotonic conditions being 300 mOsm/kg) and strongly induced at 400 mOsm/kg. Such levels have been recorded in plasma in patients with osmoregulatory disorders and in mice deficient in aquaporins and vasopressin receptor. The hypertonicity threshold required to activate NFAT5 was higher in bone marrow-derived macrophages (430 mOsm/kg) and embryonic fibroblasts (480 mOsm/kg). Activation of the 9xNFAT-Luc reporter by hypertonicity in lymphocytes was insensitive to the ERK inhibitor PD98059, partially inhibited by the PI3-kinase inhibitor wortmannin (0.5 microM) and the PKA inhibitor H89, and substantially downregulated by p38 inhibitors (SB203580 and SB202190) and by inhibition of PI3-kinase-related kinases with 25 microM LY294002. Sensitivity of the reporter to FK506 varied among cell types and was greater in primary T cells than in fibroblasts and macrophages.
Our results indicate that NFAT5 is a sensitive responder to pathologic increases in extracellular tonicity in T lymphocytes. Activation of NFAT5 by hypertonicity in lymphocytes was mediated by a combination of signaling pathways that differed from those required in other cell types. We propose that the 9xNFAT-Luc transgenic mouse model might be useful to study the physiopathological regulation of both NFAT5 and NFATc factors in primary cells.
[Show abstract][Hide abstract] ABSTRACT: Stress, be it from environmental factors or intrinsic to the cell as result of growth and metabolism, can be harmful to cells. Mammalian cells have developed numerous mechanisms to respond to diverse forms of stress. These mechanisms combine signaling cascades and activation of gene expression programs to orchestrate an adaptive response that will allow the cell to survive and resume its normal functioning. In this review we will focus on the transcription factor NFAT5, a fundamental regulator of the response to osmotic stress in mammalian cells. Identified in 1999, NFAT5 is the latest addition to the Rel family, which comprises the NF-kappaB and NFATc proteins. Though in some of its structural and functional features NFAT5 is a hybrid between these two major groups of Rel proteins, it has unique characteristics that make it stand on its own as a third type of Rel transcription factor. Since its discovery, NFAT5 has been studied mostly in the context of the hypertonicity stress response. The advent of mouse models deficient in NFAT5 and other recent advances have confirmed a fundamental osmoprotective role for this factor in mammals, but also revealed features that suggest it may have a wider range of functions.
[Show abstract][Hide abstract] ABSTRACT: Tumor necrosis factor (TNF) is a pro-inflammatory cytokine that plays an important role in a variety of infectious and autoimmune disorders. Its transcription is regulated in a stimulus- and cell-type-specific manner via the recruitment of distinct DNA/activator complexes forming secondary structures or enhanceosomes. NFATp, a member of the nuclear factor of activated T cells (NFAT) family of transcription factors, plays a critical role in TNF gene regulation under a variety of conditions. In this study, we show that NFAT5, the most recently described NFAT family member, binds to the TNF promoter in a manner distinct from other NFAT proteins and is a key mediator in the activation of TNF gene transcription during hypertonic stress alone.
Nucleic Acids Research 02/2005; 33(12):3845-54. · 8.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The transcription factor NFAT5/TonEBP, a member of the NFAT/Rel family of transcription factors, has been implicated in diverse cellular responses, including the response to osmotic stress, integrin-dependent cell migration, T cell activation, and the Ras pathway in Drosophila. To clarify the in vivo role of NFAT5, we generated NFAT5-null mice. Homozygous mutants were genetically underrepresented after embryonic day 14.5. Surviving mice manifested a progressive and profound atrophy of the kidney medulla with impaired activation of several osmoprotective genes, including those encoding aldose reductase, Na+/Cl--coupled betaine/gamma-aminobutyric acid transporter, and the Na+/myo-inositol cotransporter. The aldose reductase gene is controlled by a tonicity-responsive enhancer, which was refractory to hypertonic stress in fibroblasts lacking NFAT5, establishing this enhancer as a direct transcriptional target of NFAT5. Our findings demonstrate a central role for NFAT5 as a tonicity-responsive transcription factor required for kidney homeostasis and function.
Proceedings of the National Academy of Sciences 03/2004; 101(8):2392-7. · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The transcription factor NFAT5/TonEBP, a member of the NFAT/Rel family of transcription factors, has been implicated in diverse
cellular responses, including the response to osmotic stress, integrin-dependent cell migration, T cell activation, and the
Ras pathway in Drosophila. To clarify the in vivo role of NFAT5, we generated NFAT5-null mice. Homozygous mutants were genetically underrepresented after embryonic day 14.5.
Surviving mice manifested a progressive and profound atrophy of the kidney medulla with impaired activation of several osmoprotective
genes, including those encoding aldose reductase, Na+/Cl–-coupled betaine/γ-aminobutyric acid transporter, and the Na+/myo-inositol cotransporter. The aldose reductase gene is controlled by a tonicity-responsive enhancer, which was refractory to hypertonic stress in fibroblasts lacking NFAT5,
establishing this enhancer as a direct transcriptional target of NFAT5. Our findings demonstrate a central role for NFAT5
as a tonicity-responsive transcription factor required for kidney homeostasis and function.
Proceedings of the National Academy of Sciences 02/2004; 101(8):2392-2397. · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Integrins, receptors for extracellular matrix ligands, are critical regulators of the invasive phenotype. Specifically, the alpha(6)beta(4) integrin has been linked with epithelial cell motility, cellular survival and carcinoma invasion, hallmarks of metastatic tumours. Previous studies have also shown that antagonists of the NFAT (nuclear factor of activated T-cells) family of transcription factors exhibit strong anti-tumour-promoting activity. This suggests that NFAT may function in tumour metastasis. Here, we investigate the involvement of NFAT in promoting carcinoma invasion downstream of the alpha(6)beta(4) integrin. We provide evidence that both NFAT1, and the recently identified NFAT5 isoform, are expressed in invasive human ductal breast carcinomas and participate in promoting carcinoma invasion using cell lines derived from human breast and colon carcinomas. NFAT1 and NFAT5 activity correlates with the expression of the alpha(6)beta(4) integrin. In addition, the transcriptional activity of NFAT5 is induced by alpha(6)beta(4) clustering in the presence of chemo-attractants, resulting in enhanced cell migration. These observations show that NFATs are targets of alpha(6)beta(4) integrin signalling and are involved in promoting carcinoma invasion, highlighting a novel function for this family of transcription factors in human cancer.
[Show abstract][Hide abstract] ABSTRACT: Tonicity-responsive enhancer binding protein (TonEBP), also known as NFAT5, is a unique member of the NFAT family of transcription factors that regulates gene expression induced by osmotic stress in mammalian cells. Unlike monomeric members of the NFAT family, TonEBP exists as a homodimer and binds asymmetric TonE DNA sites; furthermore, the affinity of TonEBP for DNA is much lower than that of other NFAT proteins. How TonEBP recognizes the TonE site and regulates the activation of hypertonicity response genes has not been clear. Here we show that TonEBP adopts a NF-kappaB-like structure upon binding to DNA, providing a direct structural link between the NFAT and NF-kappaB family of transcription factors. We also show that TonEBP completely encircles its DNA target and present biochemical evidence that the DNA encirclement may lead to increased kinetic stability of the TonEBP-DNA complex. Thus, the list of proteins that bind DNA by encirclement is now expanded to include sequence-specific transcription factors.
[Show abstract][Hide abstract] ABSTRACT: The transcription factor NFAT5/TonEBP is evolutionarily the oldest member of the NFAT/Rel family of transcription factors. We show that NFAT5 is uniquely related to NF-kappaB and is the only member of the Rel/NFAT family to be activated by osmotic stress. Like Rel/NF-kappaB proteins but unlike the calcium-regulated NFAT proteins, NFAT5 is constitutively dimeric, and dimerization is essential for DNA binding and transcriptional activity. Using dominant-negative proteins that inhibit NFAT5 dimerization, we show that NFAT5 regulates expression of the TNFalpha and lymphotoxin-beta genes in osmotically stressed T cells. Chromatin immunoprecipitation experiments confirm that NFAT5 binds to the TNFalpha promoter in vivo. We suggest that NFAT5 participates in specific aspects of host defense by upregulating TNF family genes and other target genes in T cells.
[Show abstract][Hide abstract] ABSTRACT: Combinatorial regulation is a powerful mechanism that enables tight control of gene expression, via integration of multiple signaling pathways that induce different transcription factors required for enhanceosome assembly. The four calcium-regulated transcription factors of the NFAT family act synergistically with AP-1 (Fos/Jun) proteins on composite DNA elements which contain adjacent NFAT and AP-1 binding sites, where they form highly stable ternary complexes to regulate the expression of diverse inducible genes. Concomitant induction of NFAT and AP-1 requires concerted activation of two different signaling pathways: calcium/calcineurin, which promotes NFAT dephosphorylation, nuclear translocation and activation; and protein kinase C (PKC)/Ras, which promotes the synthesis, phosphorylation and activation of members of the Fos and Jun families of transcription factors. A fifth member of the NFAT family, NFAT5, controls the cellular response to osmotic stress, by a mechanism that requires dimer formation and is independent of calcineurin or of interaction with AP-1. Pharmacological interference with theNFAT:AP-1 interaction may be useful in selective manipulation of the immune response. Balanced activation of NFAT and AP-1 is known to be required for productive immune responses, but the role of NFAT:AP-1 interactions in other cell types and biological processes remains to be understood.
[Show abstract][Hide abstract] ABSTRACT: The flow of information from calcium-mobilizing receptors to nuclear factor of activated T cells (NFAT)-dependent genes is critically dependent on interaction between the phosphatase calcineurin and the transcription factor NFAT. A high-affinity calcineurin-binding peptide was selected from combinatorial peptide libraries based on the calcineurin docking motif of NFAT. This peptide potently inhibited NFAT activation and NFAT-dependent expression of endogenous cytokine genes in T cells, without affecting the expression of other cytokines that require calcineurin but not NFAT. Substitution of the optimized peptide sequence into the natural calcineurin docking site increased the calcineurin responsiveness of NFAT. Compounds that interfere selectively with the calcineurin-NFAT interaction without affecting calcineurin phosphatase activity may be useful as therapeutic agents that are less toxic than current drugs.
[Show abstract][Hide abstract] ABSTRACT: NFAT transcription factors are related to NF-kappaB/Rel proteins and form cooperative complexes with Fos and Jun on DNA. We have identified an NFAT-related protein, NFAT5, which differs from the conventional NFAT proteins NFAT1-4 in its structure, DNA binding, and regulation. NFAT5 contains a NFAT-like Rel homology domain, conserves the DNA contact residues of NFAT1-4, and binds DNA sequences similar to those found in the regulatory regions of well-characterized NFAT-dependent genes. However, it lacks the majority of Fos/Jun contact residues and does not bind cooperatively with Fos and Jun to DNA. Unlike NFAT1-4, whose nuclear import is tightly regulated by calcineurin-mediated dephosphorylation, NFAT5 is a constitutively nuclear phosphoprotein regardless of calcineurin activation. These features suggest that unlike the conventional NFAT proteins, NFAT1-4, which activate gene transcription by integrating inputs from calcium/calcineurin and protein kinase C/mitogen-activated protein kinase signaling pathways, NFAT5 participates in as-yet-unidentified signaling pathways in diverse immune and nonimmune cells.
Proceedings of the National Academy of Sciences 07/1999; 96(13):7214-9. · 9.81 Impact Factor