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Jose Aramburu - One of the best experts on this subject based on the ideXlab platform.
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Regulation of Inflammatory Functions of Macrophages and T Lymphocytes by NFAT5
Frontiers Media S.A., 2019Co-Authors: Jose Aramburu, Cristina López-rodríguezAbstract:The transcription factor NFAT5, also known as TonEBP, belongs to the family of Rel homology domain-containing factors, which comprises the NF-κB proteins and the calcineurin-dependent NFAT1 to NFAT4. NFAT5 shares several structural and functional features with other Rel-family factors, for instance it recognizes DNA elements with the same core sequence as those bound by NFAT1 to 4, and like NF-κB it responds to Toll-like receptors (TLR) and activates macrophage responses to microbial products. On the other hand, NFAT5 is quite unique among Rel-family factors as it can be activated by hyperosmotic stress caused by elevated concentrations of extracellular sodium ions. NFAT5 regulates specific genes but also others that are inducible by NF-κB and NFAT1 to 4. The ability of NFAT5 to do so in response to hypertonicity, microbial products, and inflammatory stimuli may extend the capabilities of immune cells to mount effective anti-pathogen responses in diverse microenvironment and signaling conditions. Recent studies identifying osmostress-dependent and -independent functions of NFAT5 have broadened our understanding of how NFAT5 may modulate immune function. In this review we focus on the role of NFAT5 in macrophages and T cells in different contexts, discussing findings from in vivo mouse models of NFAT5 deficiency and reviewing current knowledge on its mechanisms of regulation. Finally, we propose several questions for future research
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Context-dependent regulation of Th17-associated genes and IFNγ expression by the transcription factor NFAT5
Immunology and cell biology, 2016Co-Authors: Maria Alberdi, Cristina López-rodríguez, Marcos Iglesias, Sonia Tejedor, Ramón Merino, Jose AramburuAbstract: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.
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NFAT5 induction by the pre t cell receptor serves as a selective survival signal in t lymphocyte development
Proceedings of the National Academy of Sciences of the United States of America, 2013Co-Authors: Rosa Bergabolanos, Jose Aramburu, Maria Alberdi, Maria Buxade, Cristina LopezrodriguezAbstract: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.
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NFAT5 induction by the pre–T-cell receptor serves as a selective survival signal in T-lymphocyte development
Proceedings of the National Academy of Sciences of the United States of America, 2013Co-Authors: Rosa Berga-bolaños, Maria Alberdi, Jose Aramburu, Maria Buxade, Cristina López-rodríguezAbstract: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.
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Analysis of the transcriptional activity of endogenous NFAT5 in primary cells using transgenic NFAT-luciferase reporter mice
BMC molecular biology, 2008Co-Authors: Beatriz Morancho, Jeffery D. Molkentin, Cristina López-rodríguez, Jordi Minguillón, Jose AramburuAbstract:Background 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.
Anjana Rao - One of the best experts on this subject based on the ideXlab platform.
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hyperactivation of nuclear factor of activated t cells 1 nfat1 in t cells attenuates severity of murine autoimmune encephalomyelitis
Proceedings of the National Academy of Sciences of the United States of America, 2010Co-Authors: Srimoyee Ghosh, Yoshiteru Sasaki, Klaus Rajewsky, Anjana Rao, Sergei B Koralov, Irena Stevanovic, Mark S Sundrud, Martin MullerAbstract:Nuclear factor of activated T cells (NFAT) proteins are a group of Ca2+-regulated transcription factors residing in the cytoplasm of resting cells. Dephosphorylation by calcineurin results in nuclear translocation of NFAT and subsequent expression of target genes; rephosphorylation by kinases, including casein kinase 1 (CK1), restores NFAT to its latent state in the cytoplasm. We engineered a hyperactivable version of NFAT1 with increased affinity for calcineurin and decreased affinity for casein kinase 1. Mice expressing hyperactivable NFAT1 in their T-cell compartment exhibited a dramatically increased frequency of both IL-17– and IL-10–producing cells after differentiation under Th17 conditions—this was associated with direct binding of NFAT1 to distal regulatory regions of Il-17 and Il-10 gene loci in Th17 cells. Despite higher IL-17 production in culture, the mice were significantly less prone to myelin oligodendrocyte glycoprotein peptide-induced experimental autoimmune encephalomyelitis than controls, correlating with increased production of the immunomodulatory cytokine IL-10 and enhanced accumulation of regulatory T cells within the CNS. Thus, NFAT hyperactivation paradoxically leads to decreased susceptibility to experimental autoimmune encephalomyelitis, supporting previous observations linking defects in Ca2+/NFAT signaling to lymphoproliferation and autoimmune disease.
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a cd8 t cell intrinsic role for the calcineurin nfat pathway for tolerance induction in vivo
Blood, 2010Co-Authors: Anjana Rao, Thomas Fehr, Carrie L Lucas, Josef Kurtz, Takashi Onoe, Guiling Zhao, Timothy P Hogan, Casey Vallot, Megan SykesAbstract:Previous studies have indicated that blockade of signaling through the T-cell receptor (TCR)/calcineurin/nuclear factor of activated T cells (NFAT) pathway impairs transplantation tolerance induced with anti-CD154 antibody. By using an allogeneic bone marrow transplantation model, we examined the role of the TCR/calcineurin/NFAT pathway for tolerance induction with anti-CD154. Calcineurin blockade by cyclosporine A led to a failure of CD8 but not CD4 tolerance, and experiments in NFAT1−/− mice replicated this effect. Studies in thymectomized mice demonstrated that blockade of the calcineurin/NFAT pathway after bone marrow transplantation led to a failure of peripheral CD8 tolerance. Moreover, CD8 adoptive transfer studies demonstrated that NFAT1 is cell-intrinsically required for peripheral CD8 tolerance. NFAT1 deficiency did not impair CD8 T-cell up-regulation of PD1, which is required for CD8 tolerance in this model. NFAT1 has previously been shown to have a role in CD4 cells for anergy induction and for programming CD4 cells to become regulatory cells. By generating mice lacking NFAT1 in CD4 but not CD8 cells, we demonstrate that NFAT1 is neither required for CD4 tolerance induction nor for their regulatory function on CD8 T cells. Thus, our study reveals a CD8 T cell–intrinsic NFAT1 requirement for CD8 tolerance in vivo.
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Expression of Hyperactivable NFAT1 from the ROSA26 Locus Leads to Detrimental Effects during Embryonic Development.
Blood, 2007Co-Authors: Martin R. Mueller, Yoshiteru Sasaki, Sonia Sharma, Klaus Rajewsky, Srimoyee Ghosh, Curtis Gelinas, Edward L. Lamperti, Anjana RaoAbstract:NFAT is a family of highly phosphorylated proteins residing in the cytoplasm of resting cells. Upon dephosphorylation by calcineurin, NFAT translocates to the nucleus, where it orchestrates several developmental programs, including those of the immune, cardiovascular and central nervous systems. NFAT is rephosphorylated and inactivated by different kinases (CK1, GSK-3, DYRK). The major docking site for calcineurin is located at the N terminus of the NFAT regulatory domain and has the consensus sequence PxIxIT (SPRIEIT in NFAT1). Substitution of the SPRIEIT sequence with HPVIVIT increases the basal calcineurin sensitivity of the protein significantly. Similarly, mutation of the CK1 docking site from FSILF to ASILA leads to partial nuclear localization by decreasing rephosphorylation and nuclear export. To assess the impact of hyperactivable NFAT mutants on activation kinetics and signal responsiveness, we retrovirally transduced T cells from NFAT1−/− mice with contructs expressing wild type NFAT1, NFAT1-ASILA, NFAT1-HPVIVIT or NFAT1-ASILA-HPVIVIT. Analysis by western blotting and immunocytochemistry revealed, that the wild type protein was entirely localized in the cytoplasm and completely phosphorylated under resting conditions, whereas the hyperactivable mutants exhibited an increasing degree of nuclear translocation and dephosphorylation (ASILA 20%, HPVIVIT 30–40%, ASILA-HPVIVIT 50–70%). Upon stimulation with PMA and ionomycin, the hyperactivable mutants exhibited an incremental acceleration of nuclear translocation and delay of nuclear export as compared to the wild type protein. Furthermore, T cells expressing hyperactivable NFAT proteins exhibited significantly higher expression rates of different cytokines (IL-2, TNF-α, IFN-γ) upon stimulation with low doses of ionomycin documenting their hyperresponsiveness and biological activity. To provide a new tool for the analysis of the calcineurin-NFAT pathway in vivo and to assess the effect of modulating the affinity of signaling molecules for their upstream regulators, which are consistently kept at low to moderate affinity during evolution, we subsequently generated transgenic mice conditionally expressing different hyperactivable NFAT1 mutants from the ROSA26 locus (NFAT1-HPVIVIT, NFAT1-ASILA-HPVIVIT). Transgene expression in the T cell lineage was achieved by breeding the ROSA26 transgenic mice to CD4-Cre mice, which express the Cre recombinase under the control of the CD4 promoter. CD4 and CD8 T cells from these mice showed significant hyperactivability as assessed by accelerated nuclear translocation and delayed nuclear export of NFAT and substantially increased cytokine expression upon stimulation. Expression of the hyperactivable NFAT proteins early in the germline was achieved by breeding the ROSA26 mice to Cre-Del mice, which express the Cre recombinase under the control of the ACE promoter. While breeding of ROSA26-YFP control mice to Cre-Del mice resulted in transgene expression in all T and B cells, expression of hyperactivable NFAT proteins early in the germline resulted in incremental mosaicism in T and B cells (70% transgene expression in NFAT1-HPVIVIT and 20–30% transgene expression in NFAT1-ASILA-HPVIVIT). This data demonstrate that hyperactivable NFAT1 proteins result in a selective disadvantage for the expressing cells in embryonic development and provide a potential explanation why evolution chose to keep the respective docking sites at moderate affinity.
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NFAT5 binds to the tnf promoter distinctly from nfatp c 3 and 4 and activates tnf transcription during hypertonic stress alone
Nucleic Acids Research, 2005Co-Authors: Jonathan H Esensten, Anjana Rao, Cristina Lopezrodriguez, Alla V Tsytsykova, Filipa Ligeiro, Anne E GoldfeldAbstract: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.
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a conserved docking motif for ck1 binding controls the nuclear localization of nfat1
Molecular and Cellular Biology, 2004Co-Authors: Heidi Okamura, Carmen Garciarodriguez, Holly M Martinson, Jun Qin, David M Virshup, Anjana RaoAbstract:In resting cells, the NFAT1 transcription factor is kept inactive in the cytoplasm by phosphorylation on multiple serine residues. These phosphorylated residues are primarily contained within two types of serine-rich motifs, the SRR-1 and SP motifs, which are conserved within the NFAT family. Several different kinases have been proposed to regulate NFAT, but no single candidate displays the specificity required to fully phosphorylate both types of motifs; thus, the identity of the kinase that regulates NFAT activity remains unclear. Here we show that the NFAT1 serine motifs are regulated by distinct kinases that must coordinate to control NFAT1 activation. CK1 phosphorylates only the SRR-1 motif, the primary region required for NFAT1 nuclear import. CK1 exists with NFAT1 in a high-molecular-weight complex in resting T cells but dissociates upon activation. GSK3 does not phosphorylate the SRR-1 region but can target the NFAT1 SP-2 motif, and it synergizes with CK1 to regulate NFAT1 nuclear export. We identify a conserved docking site for CK1 in NFAT proteins and show that mutation of this site disrupts NFAT1-CK1 interaction and causes constitutive nuclear localization of NFAT1. The CK1 docking motif is present in proteins of the Wnt, Hedgehog, and circadian-rhythm pathways, which also integrate the activities of CK1 and GSK3.
Cristina López-rodríguez - One of the best experts on this subject based on the ideXlab platform.
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Regulation of Inflammatory Functions of Macrophages and T Lymphocytes by NFAT5
Frontiers Media S.A., 2019Co-Authors: Jose Aramburu, Cristina López-rodríguezAbstract:The transcription factor NFAT5, also known as TonEBP, belongs to the family of Rel homology domain-containing factors, which comprises the NF-κB proteins and the calcineurin-dependent NFAT1 to NFAT4. NFAT5 shares several structural and functional features with other Rel-family factors, for instance it recognizes DNA elements with the same core sequence as those bound by NFAT1 to 4, and like NF-κB it responds to Toll-like receptors (TLR) and activates macrophage responses to microbial products. On the other hand, NFAT5 is quite unique among Rel-family factors as it can be activated by hyperosmotic stress caused by elevated concentrations of extracellular sodium ions. NFAT5 regulates specific genes but also others that are inducible by NF-κB and NFAT1 to 4. The ability of NFAT5 to do so in response to hypertonicity, microbial products, and inflammatory stimuli may extend the capabilities of immune cells to mount effective anti-pathogen responses in diverse microenvironment and signaling conditions. Recent studies identifying osmostress-dependent and -independent functions of NFAT5 have broadened our understanding of how NFAT5 may modulate immune function. In this review we focus on the role of NFAT5 in macrophages and T cells in different contexts, discussing findings from in vivo mouse models of NFAT5 deficiency and reviewing current knowledge on its mechanisms of regulation. Finally, we propose several questions for future research
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Context-dependent regulation of Th17-associated genes and IFNγ expression by the transcription factor NFAT5
Immunology and cell biology, 2016Co-Authors: Maria Alberdi, Cristina López-rodríguez, Marcos Iglesias, Sonia Tejedor, Ramón Merino, Jose AramburuAbstract: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.
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NFAT5 induction by the pre–T-cell receptor serves as a selective survival signal in T-lymphocyte development
Proceedings of the National Academy of Sciences of the United States of America, 2013Co-Authors: Rosa Berga-bolaños, Maria Alberdi, Jose Aramburu, Maria Buxade, Cristina López-rodríguezAbstract: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.
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Analysis of the transcriptional activity of endogenous NFAT5 in primary cells using transgenic NFAT-luciferase reporter mice
BMC molecular biology, 2008Co-Authors: Beatriz Morancho, Jeffery D. Molkentin, Cristina López-rodríguez, Jordi Minguillón, Jose AramburuAbstract:Background 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.
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Analysis of the transcriptional activity of endogenous NFAT5 in primary cells using transgenic NFAT-luciferase reporter mice
BMC Molecular Biology, 2008Co-Authors: Beatriz Morancho, Jeffery D. Molkentin, Cristina López-rodríguez, Jordi Minguillón, Jose AramburuAbstract:Background 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. Results 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 μM) and the PKA inhibitor H89, and substantially downregulated by p38 inhibitors (SB203580 and SB202190) and by inhibition of PI3-kinase-related kinases with 25 μM LY294002. Sensitivity of the reporter to FK506 varied among cell types and was greater in primary T cells than in fibroblasts and macrophages. Conclusion 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.
Cristina Lopezrodriguez - One of the best experts on this subject based on the ideXlab platform.
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NFAT5 induction by the pre t cell receptor serves as a selective survival signal in t lymphocyte development
Proceedings of the National Academy of Sciences of the United States of America, 2013Co-Authors: Rosa Bergabolanos, Jose Aramburu, Maria Alberdi, Maria Buxade, Cristina LopezrodriguezAbstract: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.
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regulation of the hypertonic stress response and other cellular functions by the rel like transcription factor NFAT5
Biochemical Pharmacology, 2006Co-Authors: Jose Aramburu, Beatriz Morancho, Jordi Minguillón, Katherine Drewselger, Anais Estradagelonch, Veronica Santiago, Cristina LopezrodriguezAbstract: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.
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NFAT5 binds to the tnf promoter distinctly from nfatp c 3 and 4 and activates tnf transcription during hypertonic stress alone
Nucleic Acids Research, 2005Co-Authors: Jonathan H Esensten, Anjana Rao, Cristina Lopezrodriguez, Alla V Tsytsykova, Filipa Ligeiro, Anne E GoldfeldAbstract: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.
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loss of NFAT5 results in renal atrophy and lack of tonicity responsive gene expression
Proceedings of the National Academy of Sciences of the United States of America, 2004Co-Authors: Cristina Lopezrodriguez, Christopher L Antos, John M Shelton, James A Richardson, Tatiana Novobrantseva, Roderick T Bronson, Peter Igarashi, Eric N OlsonAbstract: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.
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loss of NFAT5 results in renal atrophy and lack of tonicity responsive gene expression
Proceedings of the National Academy of Sciences of the United States of America, 2004Co-Authors: Cristina Lopezrodriguez, Christopher L Antos, John M Shelton, James A Richardson, Tatiana Novobrantseva, Roderick T Bronson, Peter Igarashi, Fangming Lin, Anjana RaoAbstract: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.
Jeffery D. Molkentin - One of the best experts on this subject based on the ideXlab platform.
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Analysis of the transcriptional activity of endogenous NFAT5 in primary cells using transgenic NFAT-luciferase reporter mice
BMC molecular biology, 2008Co-Authors: Beatriz Morancho, Jeffery D. Molkentin, Cristina López-rodríguez, Jordi Minguillón, Jose AramburuAbstract:Background 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.
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Analysis of the transcriptional activity of endogenous NFAT5 in primary cells using transgenic NFAT-luciferase reporter mice
BMC Molecular Biology, 2008Co-Authors: Beatriz Morancho, Jeffery D. Molkentin, Cristina López-rodríguez, Jordi Minguillón, Jose AramburuAbstract:Background 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. Results 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 μM) and the PKA inhibitor H89, and substantially downregulated by p38 inhibitors (SB203580 and SB202190) and by inhibition of PI3-kinase-related kinases with 25 μM LY294002. Sensitivity of the reporter to FK506 varied among cell types and was greater in primary T cells than in fibroblasts and macrophages. Conclusion 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.
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Calcineurin and NFAT4 Induce Chondrogenesis
Journal of Biological Chemistry, 2002Co-Authors: Masuhiro Tomita, Jeffery D. Molkentin, Martina I. Reinhold, Michael C. NaskiAbstract:Abstract Nuclear factor of activated T-cells (NFAT) and calcineurin are essential regulators of immune cell and mesenchymal cell differentiation. Here we show that elevated intracellular calcium induces chondrogenesis through a calcineurin/NFAT signaling axis that activates bone morphogenetic protein (BMP) expression. The calcium ionophore, ionomycin, induced chondrogenesis through activation of calcineurin. The calcineurin substrate, NFAT4, also induced chondrogenesis and chondrocyte gene expression. Significantly, the BMP antagonist, noggin, or dominant negative BMP receptors blocked the effects of elevated intracellular calcium on chondrogenesis. This suggested that calcineurin/NFAT4 activates BMP expression. Consistent with this, BMP2 gene expression was increased by ionomycin and suppressed by the calcineurin inhibitor, cyclosporine A. Furthermore, activated NFAT4 induced BMP2 gene expression. These results have important implications for the effects of NFATs during development and adaptive responses.
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a calcineurin nfatc3 dependent pathway regulates skeletal muscle differentiation and slow myosin heavy chain expression
Molecular and Cellular Biology, 2000Co-Authors: Ulrike Delling, Jolana Tureckova, Leon J De Windt, Peter Rotwein, Jeffery D. MolkentinAbstract:Skeletal muscle cell differentiation is coordinated by endocrine, paracrine, and autocrine inductive factors that activate discrete intracellular signal transduction pathways, resulting in the modulation of transcription factor activity and the reprogramming of gene expression. During embryonic development, the MyoD family of basic helix-loop-helix transcription factors directly regulate myocyte cell specification and differentiation (reviewed in reference 33). The myogenic basic helix-loop-helix proteins operate in concert with other transcriptional regulators such as MEF2, serum response factor, and CBP/p300 to promote myocyte differentiation (17, 34, 44, 49, 60). In turn, these transcriptional regulators are themselves regulated by intracellular signaling pathways and phosphorylation cascades. In general, growth factors such as fibroblast growth factor and transforming growth factor β antagonize myocyte differentiation through signaling pathways involving ras, mitogen-activated protein kinase, and protein kinase C (14, 28, 41). Proliferation-inducing transduction pathways enhance AP-1 activity, increase Id expression, and directly attenuate the activity of the myogenic basic helix-loop-helix transcription factors through cell cycle-dependent mechanisms (20, 33, 48). In contrast, inductive factors such as insulin-like growth factor 1 (IGF-1) promote myocyte differentiation or hypertrophy (4, 38, 39, 43, 47, 55), partly through a transduction pathway involving phosphatidylinositol 3-kinase (24, 25, 38). Superimposed on the myocyte differentiation program are molecular pathways which regulate fiber type specificity. During development, maturing myofibers first express embryonic myosin, followed by neonatal myosin, followed again by various isoforms of fast myosin and then slow myosin (reviewed in reference 51). Less is known about the intracellular regulatory pathways that control fiber type specificity, although evidence has accumulated implicating a calcium-dependent pathway (10, 16). Calcium levels in resting fast fibers are reported to be 50 nM, while prolonged or chronic stimulation of fast fibers, associated with increased intracellular calcium levels, induces slow-fiber transformation (3, 8, 45, 50, 56, 58). Recent data have implicated calcineurin, a calcium-calmodulin-regulated serine/threonine phosphatase, in the control of IGF-1-dependent myocyte hypertrophy and fiber type specificity (10, 16, 38, 46). Calcineurin participates in the transduction of extracellular signals to the nucleus by targeting members of the NFAT family of transcription factors (reviewed in references 13 and 42). Calcineurin-directed dephosphorylation of NFAT factors unmasks their nuclear localization signal, resulting in nuclear translocation and gene activation. Five NFAT genes have thus far been identified, NFATc1 (NFATc or NFAT2), NFATc2 (NFATp or NFAT1), NFATc3 (NFAT4 or NFATx), NFATc4 (NFAT3), and NFAT5 (29, 42). Calcineurin-mediated signaling pathways contribute to T-cell activation, to the establishment of cardiac hypertrophy, and to the regulation of neuronal activity (13). Treatment of cultured human myoblasts with the calcineurin inhibitor cyclosporine A attenuates myogenic differentiation in culture and cyclosporine administration inhibits regeneration in response to acute injury in the mouse (1). Cyclosporine also prevents skeletal muscle hypertrophy in response to muscle overloading in vivo (16). More recently, myoblast cell lines expressing the local form of IGF-1 were shown to utilize a calcineurin-dependent pathway to promote hypertrophy in culture (39, 46). Lastly, cyclosporine induced fast-fiber-type switching in rodent skeletal muscle (10, 16), and transgenic mice expressing an activated calcineurin cDNA in skeletal muscle had increased numbers of slow fibers (39a). In the present study, we used four different model systems to examine the mechanisms whereby calcineurin and NFAT regulate myocyte differentiation and/or fiber type specificity. We demonstrate that calcineurin directly potentiates myocyte differentiation through a slow-fiber-type program in C2C12 myoblasts, Sol8 myoblasts, and MyoD-converted fibroblasts. Inhibition of calcineurin with a noncompetitive peptide inhibitor (cain) delays the differentiation of C2C12 and Sol8 cells and prevents MyoD-directed differentiation of 10T1/2 fibroblasts. Expression of activated calcineurin in C2C12 myoblasts overexpressing an inhibitory IGF binding protein overrides blocked differentiation. We demonstrate that NFATc3 is a specific target of calcineurin and translocates to the nucleus at the onset of myoblast differentiation, enhancing myogenesis.