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Patrick G. Hogan - One of the best experts on this subject based on the ideXlab platform.
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balanced interactions of calcineurin with akap79 regulate ca2 calcineurin NFAT signaling
Nature Structural & Molecular Biology, 2012Co-Authors: Matthew D Pink, Patrick G. Hogan, Jonathan G Murphy, Alexander Stein, Mark L DellacquaAbstract:In hippocampal neurons, the scaffold protein AKAP79 recruits the phosphatase calcineurin to L-type Ca(2+) channels and couples Ca(2+) influx to activation of calcineurin and of its substrate, the transcription factor NFAT. Here we show that an IAIIIT anchoring site in human AKAP79 binds the same surface of calcineurin as the PxIxIT recognition peptide of NFAT, albeit more strongly. A modest decrease in calcineurin-AKAP affinity due to an altered anchoring sequence is compatible with NFAT activation, whereas a further decrease impairs activation. Counterintuitively, increasing calcineurin-AKAP affinity increases recruitment of calcineurin to the scaffold but impairs NFAT activation; this is probably due to both slower release of active calcineurin from the scaffold and sequestration of active calcineurin by 'decoy' AKAP sites. We propose that calcineurin-AKAP79 scaffolding promotes NFAT signaling by balancing strong recruitment of calcineurin with its efficient release to communicate with NFAT.
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dephosphorylation of the nuclear factor of activated t cells NFAT transcription factor is regulated by an rna protein scaffold complex
Proceedings of the National Academy of Sciences of the United States of America, 2011Co-Authors: Sonia Sharma, Patrick G. Hogan, Valentina A. Schmidt, Gregory M Findlay, Hozefa S Bandukwala, Shalini Oberdoerffer, Beate Baust, Zhigang Li, David B. SacksAbstract:Nuclear factor of activated T cells (NFAT) proteins are Ca2+-regulated transcription factors that control gene expression in many cell types. NFAT proteins are heavily phosphorylated and reside in the cytoplasm of resting cells; when cells are stimulated by a rise in intracellular Ca2+, NFAT proteins are dephosphorylated by the Ca2+/calmodulin-dependent phosphatase calcineurin and translocate to the nucleus to activate target gene expression. Here we show that phosphorylated NFAT1 is present in a large cytoplasmic RNA-protein scaffold complex that contains a long intergenic noncoding RNA (lincRNA), NRON [noncoding (RNA) repressor of NFAT]; a scaffold protein, IQ motif containing GTPase activating protein (IQGAP); and three NFAT kinases, casein kinase 1, glycogen synthase kinase 3, and dual specificity tyrosine phosphorylation regulated kinase. Combined knockdown of NRON and IQGAP1 increased NFAT dephosphorylation and nuclear import exclusively after stimulation, without affecting the rate of NFAT rephosphorylation and nuclear export; and both NRON-depleted T cells and T cells from IQGAP1-deficient mice showed increased production of NFAT-dependent cytokines. Our results provide evidence that a complex of lincRNA and protein forms a scaffold for a latent transcription factor and its regulatory kinases, and support an emerging consensus that lincRNAs that bind transcriptional regulators have a similar scaffold function.
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a genome wide drosophila rnai screen identifies dyrk family kinases as regulators of NFAT
Nature, 2006Co-Authors: Yousang Gwack, Sonia Sharma, Julie Nardone, Heidi Okamura, Bogdan Tanasa, Alina Iuga, Sonal Srikanth, Diana Bolton, Stefan Feske, Patrick G. HoganAbstract:Down's syndrome is caused by an extra chromosome; somehow a 1.5-fold increase in the dosage of a gene or genes on chromosome 21 causes the wide-reaching effects associated with the condition. A study using ‘knockout’ mice now identifies two genes as candidates for involvement. A 1.5-fold increase in dosage of DSCR1 and DYRK1a destabilizes the regulation of signalling pathways involving the NFAT transcription factor. The discovery follows the surprise finding that NFATc1-4 and calcineurin mutant mice demonstrate nearly all the characteristics of Down's syndrome. In an unrelated paper, a genome-wide RNAi screen reveals conserved regulators of NFAT in Drosophila. NFAT is a purely vertebrate transcription factor, but this work breaks new ground by using Drosophila cells to study the function of a protein artificially introduced from a mammalian species. Pathways regulating the subcellular localization of NFAT proteins are strongly conserved across species and this new approach can identify new regulators of a transcription factor normally expressed in vertebrates. Precise regulation of the NFAT (nuclear factor of activated T cells) family of transcription factors (NFAT1–4) is essential for vertebrate development and function1. In resting cells, NFAT proteins are heavily phosphorylated and reside in the cytoplasm; in cells exposed to stimuli that raise intracellular free Ca2+ levels, they are dephosphorylated by the calmodulin-dependent phosphatase calcineurin and translocate to the nucleus1. NFAT dephosphorylation by calcineurin is countered by distinct NFAT kinases, among them casein kinase 1 (CK1) and glycogen synthase kinase 3 (GSK3)1,2,3,4,5. Here we have used a genome-wide RNA interference (RNAi) screen in Drosophila6,7 to identify additional regulators of the signalling pathway leading from Ca2+–calcineurin to NFAT. This screen was successful because the pathways regulating NFAT subcellular localization (Ca2+ influx, Ca2+–calmodulin–calcineurin signalling and NFAT kinases) are conserved across species8,9, even though Ca2+-regulated NFAT proteins are not themselves represented in invertebrates. Using the screen, we have identified DYRKs (dual-specificity tyrosine-phosphorylation regulated kinases) as novel regulators of NFAT. DYRK1A and DYRK2 counter calcineurin-mediated dephosphorylation of NFAT1 by directly phosphorylating the conserved serine-proline repeat 3 (SP-3) motif of the NFAT regulatory domain, thus priming further phosphorylation of the SP-2 and serine-rich region 1 (SRR-1) motifs by GSK3 and CK1, respectively. Thus, genetic screening in Drosophila can be successfully applied to cross evolutionary boundaries and identify new regulators of a transcription factor that is expressed only in vertebrates.
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inhibition of the calcineurin NFAT interaction by small organic molecules reflects binding at an allosteric site
Journal of Biological Chemistry, 2005Co-Authors: Sunghyun Kang, Anjana Rao, Patrick G. HoganAbstract:Transcriptional signaling from the Ca(2+)-calmodulin-activated phosphatase calcineurin to its substrate NFAT (nuclear factor of activated T cells, also termed NFATc) is critically dependent on a protein-protein docking interaction between calcineurin and the PXIXIT motif in NFAT. Several inhibitors of NFAT-calcineurin association (INCA compounds) prevent binding of NFAT or the peptide ligand PVIVIT to calcineurin. Here we show that the binding site on calcineurin for INCA1, INCA2, and INCA6 is centered on cysteine 266 of calcineurin Aalpha and does not coincide with the core PXIXIT-binding site. Although ample evidence indicates that INCA1 and INCA2 react covalently with cysteine 266, covalent derivatization alone is not sufficient for maximal inhibition of the calcineurin-PVIVIT interaction, because the maleimide INCA12 reacts with the same site and produces only very modest inhibition. Thus, inhibition arises through an allosteric change affecting the PXIXIT docking site, which may be assisted by covalent binding but depends on other specific features of the ligand. The spatial arrangement of the binding sites for PVIVIT and INCA makes it probable that the change in conformation involves the beta11-beta12 loop of calcineurin. The finding that an allosteric site controls NFAT binding opens new alternatives for inhibition of calcineurin-NFAT signaling.
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transcriptional regulation by calcium calcineurin and NFAT
Genes & Development, 2003Co-Authors: Patrick G. Hogan, Lin Chen, Julie Nardone, Anjana RaoAbstract:The NFAT family of transcription factors encompasses five proteins evolutionarily related to the Rel/NF B family (Chytil and Verdine 1996; Graef et al. 2001b). The primordial family member is NFAT5, the only NFATrelated protein represented in the Drosophila genome. NFAT5 is identical to TonEBP (tonicity element binding protein), a transcription factor crucial for cellular responses to hypertonic stress (Lopez-Rodriguez et al. 1999; Miyakawa et al. 1999). We focus here on the remaining four NFAT proteins (NFAT1–NFAT4, also known as NFATc1–c4; see Table 1), referring to them collectively as NFAT. The distinguishing feature of NFAT is its regulation by Ca and the Ca/calmodulin-dependent serine phosphatase calcineurin. NFAT proteins are phosphorylated and reside in the cytoplasm in resting cells; upon stimulation, they are dephosphorylated by calcineurin, translocate to the nucleus, and become transcriptionally active, thus providing a direct link between intracellular Ca signaling and gene expression. NFAT activity is further modulated by additional inputs from diverse signaling pathways, which affect NFAT kinases and nuclear partner proteins. In the first part of this review, we describe the influence of these multiple inputs on the nuclear–cytoplasmic distribution and transcriptional function of NFAT. Recent structural data emphasize the remarkable versatility of NFAT binding to DNA. At composite NFAT:AP-1 elements found in the regulatory regions of many target genes, NFAT proteins bind cooperatively with an unrelated transcription factor, AP-1 (Fos–Jun; Chen et al. 1998). At DNA elements that resemble NF B sites, NFAT proteins bind DNA as dimers (Giffin et al. 2003; Jin et al. 2003). In the second section of this review, we describe these two modes of DNA binding by NFAT. NFAT also acts synergistically with transcription factors other than Fos and Jun, but the structural basis for synergy remains unknown. Drawing on published structures, we discuss the potential cooperation of NFAT with other classes of DNA-binding proteins. It is clear that NFAT activates transcription of a large number of genes during an effective immune response (Rao et al. 1997; Kiani et al. 2000; Serfling et al. 2000; Macian et al. 2001). In the third part of this review, we present information obtained from these studies, highlighting experimental and bioinformatics approaches to identifying NFAT target genes. We discuss the finding that NFAT and NFAT–Fos–Jun complexes activate distinct subsets of target genes in lymphocytes (Macian et al. 2002). We also describe a novel aspect of gene regulation by NFAT, in which this transcription factor participates in an early phase of chromatin remodeling that occurs at specific genetic loci in differentiating T cells (Avni et al. 2002). There is evidence that NFAT regulates cell differentiation programs in cell types other than immune cells (Crabtree and Olson 2002; Horsley and Pavlath 2002; Graef et al. 2003; Hill-Eubanks et al. 2003). In the last section of this review, we select three differentiation programs—fiber-type specification in differentiated skeletal muscle, cardiac valve development, and osteoclast differentiation—for detailed consideration. We evaluate the evidence for NFAT involvement, point out novel cellular and molecular mechanisms that might regulate this familiar transcription factor, and discuss how NFAT exerts its biological effects. Because the phenotypes of NFAT knockout mice have been reviewed elsewhere (Crabtree and Olson 2002; Horsley and Pavlath 2002), we refer to them only as necessary to illustrate specific points.
Anjana Rao - One of the best experts on this subject based on the ideXlab platform.
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Manipulating Immune Responses Review with Immunosuppressive Agents that Target NFAT iting a remarkable gain of function relative to the drugs themselves (Schreiber and Crabtree, 1992). In the immune system, calcineurin and NFAT, the ma- jor targets of CsA
2020Co-Authors: Alexander Kiani, Anjana Rao, Jose AramburuAbstract:, 1999). this normally protective system overreacts and directs The main drawback of these immunosuppressive regiits forces at the organism itself. The clinical syndrome mens, however, is that they produce major toxic side of septic shock results from overactive host responses effects and therefore are rarely employed except in serito gram-negative bacterial infections and can be fatal ous clinical situations. because of an overwhelming production of proinflamOur thesis in this review is that since NFAT is a valimatory cytokines. Transplantation of solid organs is dated target for two clinically important immunosupconfounded by the immunologic problem of graft rejecpressive drugs, interference with NFAT regulation might tion, while graft-versus-host-disease is one of the major be expected to yield additional avenues for immunosupcomplications of transplantation with allogeneic bone pression. Moreover, since NFAT is only one of many marrow or peripheral blood stem cells, severely limiting calcineurin substrates, compounds that interfere selecthe application of these potentially life-saving therapeutively with NFAT regulation might be expected to have tic options. Other examples of inappropriate immune fewer side effects than CsA and FK506. Unfortunately, responses include allergic asthma and chronic autoimthis simple premise is complicated by the existence of mune diseases such as rheumatoid arthritis. A major multiple NFAT proteins, our incomplete knowledge of challenge for clinicians is how to suppress deleterious their regulation and function in immune and nonimmune immune responses in these diverse clinical settings. cells, and our lack of information about the mechanisms The fungal metabolites cyclosporin A (CsA) and tacrolunderlying the toxic effects of CsA and FK506. imus (FK506) are among the most potent immunosupHere we review our current understanding of these pressive drugs available today. CsA revolutionized the points. We consider what features of NFAT regulation field of organ transplantation following its discovery in might be exploited to develop selective inhibitors of 1976 (Borel et al., 1976); it was approved for clinical use NFAT, whether such drugs would be as immunosupin 1983. Tacrolimus was described soon after (Kino et pressive as CsA or FK506, whether they would lack the al., 1987) and introduced into clinical trials in 1989. Detoxic effects of CsA and FK506, and whether they might spite their lack of structural similarity, the modes of have unexpected side effects in immune and nonimmune action of these two drugs are very similar: at the low organs. An informed understanding of these issues might concentrations relevant to their immunosuppressive efspur the development of better, less toxic immunosupfects, they inhibit the calcium-dependent serine/threopressive agents that target the NFAT pathway. nine phosphatase calcineurin and its substrate, the transcription factor NFAT (Schreiber and Crabtree, 1992). The drugs do not interact directly with calcineurin but What Features of NFAT Regulation Might Be Exploited rather form complexes with intracellular receptors known to Develop Selective Inhibitors of NFAT? as immunophilins. The immunophilin receptors for cyclo- The NFAT family, consisting of the "classical" members sporin A are the cyclophilins, while those for FK506 are NFAT1 (p, c2), NFAT2 (c, c1), NFAT3 (c4), and NFAT4 the FK506-binding proteins (FKBPs). Both classes of (x, c3), has recently been extended by a fifth member, immunophilin receptors are widely expressed, abundant NFAT5/TonEBP ( , 1997a, 1997b). For NFAT4, the results were less consistent: in one study, mutation of two residues be envisioned to explain this result; neither has yet been tested experimentally. Calcineurin might be needed as in the serine-rich region of NFAT4 resulted in nuclear localization (Chow et al., 1997), while in another report, an integral part of the NFAT transcriptional complex to maintain NFAT in an active and dephosphorylated form, deletion of the entire serine-rich region rendered the protein hypersensitive to calcium ionophore, a result particularly if DNA-bound NFAT present in active transcriptional complexes is still capable of being inactiinterpreted by the authors as the effect of deleting the docking site for CK1␣, a proposed NFAT kinase (Zhu vated by rephosphorylation. Alternatively, the DNAbound, transcriptionally active fraction of NFAT might et al., 1998; see below). The latter study located the phosphoserine residues responsible for nuclear import not be accessible to calcineurin or nuclear kinases; if so, the ongoing requirement for calcineurin indicates in the first SP motif of NFAT4 (Zhu et al., 1998). While, overall, the results implicate both the serine-rich region that this fraction of NFAT would be in continuous equilibrium with free nuclear NFAT. These possibilities are not and the SP motifs in regulating nuclear import, the specific discrepancies are puzzling given the high degree mutually exclusive: the detailed structure of NFAT-containing transcriptional complexes is likely to vary deof sequence conservation in the NFAT regulatory domain and the fact that all four NFATs are regulated by pending on the regulatory region involved, and hence the accessibility of NFAT to modifying enzymes could calcineurin. These discrepancies will need to be resolved before pharmacological strategies targeting debe different in each case. Paradoxically, it has been suggested that calcineurin phosphorylation-regulated nuclear import can be devised. activity is not necessary for transcription mediated by NFAT2 and NFAT4 that have been artificially localized to DNA Binding and Transcriptional Activity Have Undesired Side Effects? Functions of NFAT in Nonimmune Cells This is an area in dire need of more specific information. There is much evidence for NFAT expression and funcAs discussed in previous sections, the contribution of tion in cells outside the immune system. NFAT2-defi-NFAT to CsA and FK506 toxicity is not known. However, cient mice die in utero because of defects in the morpho- family member may be useful include allergic asthma they lack the toxicity of CsA and FK506, and whether (NFAT2) and cardiac hypertrophy (NFAT3). The developthey have unexpected side expects of their own. Indeed, ment of such specific inhibitors will require a greater a transgenic mouse has been generated that expresses appreciation of shared versus unique regulatory mechaa "dominant-negative" NFAT, whose effectiveness is at nisms, and shared versus unique functions, of individual least partly due to inhibition of NFAT-calcineurin binding NFAT proteins within and outside the immune system
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transcriptional complexes formed by NFAT dimers regulate the induction of t cell tolerance
Journal of Experimental Medicine, 2009Co-Authors: Noemi Sotonieves, Anjana Rao, Irene Puga, Brian T Abe, Sanmay Bandyopadhyay, Ian Baine, Fernando MacianAbstract:In T cells, anergy can be induced after T cell receptor engagement in the absence of costimulation. Under these conditions, the expression of a specific set of anergy-associated genes is activated. Several lines of evidence suggest that nuclear factor of activated T cells (NFAT) proteins may regulate the expression of many of those genes; however, the nature of the complexes responsible for the induction of this new program of gene expression is unknown. Here, we show that transcriptional complexes formed by NFAT homodimers are directly responsible for the activation of at least two anergy-inducing genes, Grail and Caspase3. Our data shows that Grail expression is activated by direct binding of NFAT dimers to the Grail promoter at two different sites. Consequently, a mutant NFAT protein with impaired ability to dimerize is not able to induce an unresponsive state in T cells. Our results not only identify a new biological function for NFAT dimers but also reveal the different nature of NFAT-containing complexes that induce anergy versus those that are activated during a productive immune response. These data also establish a basis for the design of immunomodulatory strategies that specifically target each type of complex.
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inhibition of the calcineurin NFAT interaction by small organic molecules reflects binding at an allosteric site
Journal of Biological Chemistry, 2005Co-Authors: Sunghyun Kang, Anjana Rao, Patrick G. HoganAbstract:Transcriptional signaling from the Ca(2+)-calmodulin-activated phosphatase calcineurin to its substrate NFAT (nuclear factor of activated T cells, also termed NFATc) is critically dependent on a protein-protein docking interaction between calcineurin and the PXIXIT motif in NFAT. Several inhibitors of NFAT-calcineurin association (INCA compounds) prevent binding of NFAT or the peptide ligand PVIVIT to calcineurin. Here we show that the binding site on calcineurin for INCA1, INCA2, and INCA6 is centered on cysteine 266 of calcineurin Aalpha and does not coincide with the core PXIXIT-binding site. Although ample evidence indicates that INCA1 and INCA2 react covalently with cysteine 266, covalent derivatization alone is not sufficient for maximal inhibition of the calcineurin-PVIVIT interaction, because the maleimide INCA12 reacts with the same site and produces only very modest inhibition. Thus, inhibition arises through an allosteric change affecting the PXIXIT docking site, which may be assisted by covalent binding but depends on other specific features of the ligand. The spatial arrangement of the binding sites for PVIVIT and INCA makes it probable that the change in conformation involves the beta11-beta12 loop of calcineurin. The finding that an allosteric site controls NFAT binding opens new alternatives for inhibition of calcineurin-NFAT signaling.
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activation and deactivation of gene expression by ca2 calcineurin NFAT mediated signaling
Molecules and Cells, 2004Co-Authors: Anjana RaoAbstract:Ca2+/calcineurin-NFAT-mediated signaling pathways are involved in diverse cellular reactions by regulating gene expression either positively or negatively. The transcriptional activity of NFAT proteins can be either activating or deactivating depending on which binding partners are involved. Interaction of NFAT with AP-1 turns on the genes involved in active immune responses, while NFAT without cooperative binding of AP-1 turns on a T cell anergy program and blocks T cell activation and proliferation. In addition, interaction of NFAT with histone deacetylase (HDAC) proteins induces gene silencing. In this review we focus on the dual function, activator or deactivator, of NFAT and the binding partners that determine the role of NFAT in gene expression.
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transcriptional regulation by calcium calcineurin and NFAT
Genes & Development, 2003Co-Authors: Patrick G. Hogan, Lin Chen, Julie Nardone, Anjana RaoAbstract:The NFAT family of transcription factors encompasses five proteins evolutionarily related to the Rel/NF B family (Chytil and Verdine 1996; Graef et al. 2001b). The primordial family member is NFAT5, the only NFATrelated protein represented in the Drosophila genome. NFAT5 is identical to TonEBP (tonicity element binding protein), a transcription factor crucial for cellular responses to hypertonic stress (Lopez-Rodriguez et al. 1999; Miyakawa et al. 1999). We focus here on the remaining four NFAT proteins (NFAT1–NFAT4, also known as NFATc1–c4; see Table 1), referring to them collectively as NFAT. The distinguishing feature of NFAT is its regulation by Ca and the Ca/calmodulin-dependent serine phosphatase calcineurin. NFAT proteins are phosphorylated and reside in the cytoplasm in resting cells; upon stimulation, they are dephosphorylated by calcineurin, translocate to the nucleus, and become transcriptionally active, thus providing a direct link between intracellular Ca signaling and gene expression. NFAT activity is further modulated by additional inputs from diverse signaling pathways, which affect NFAT kinases and nuclear partner proteins. In the first part of this review, we describe the influence of these multiple inputs on the nuclear–cytoplasmic distribution and transcriptional function of NFAT. Recent structural data emphasize the remarkable versatility of NFAT binding to DNA. At composite NFAT:AP-1 elements found in the regulatory regions of many target genes, NFAT proteins bind cooperatively with an unrelated transcription factor, AP-1 (Fos–Jun; Chen et al. 1998). At DNA elements that resemble NF B sites, NFAT proteins bind DNA as dimers (Giffin et al. 2003; Jin et al. 2003). In the second section of this review, we describe these two modes of DNA binding by NFAT. NFAT also acts synergistically with transcription factors other than Fos and Jun, but the structural basis for synergy remains unknown. Drawing on published structures, we discuss the potential cooperation of NFAT with other classes of DNA-binding proteins. It is clear that NFAT activates transcription of a large number of genes during an effective immune response (Rao et al. 1997; Kiani et al. 2000; Serfling et al. 2000; Macian et al. 2001). In the third part of this review, we present information obtained from these studies, highlighting experimental and bioinformatics approaches to identifying NFAT target genes. We discuss the finding that NFAT and NFAT–Fos–Jun complexes activate distinct subsets of target genes in lymphocytes (Macian et al. 2002). We also describe a novel aspect of gene regulation by NFAT, in which this transcription factor participates in an early phase of chromatin remodeling that occurs at specific genetic loci in differentiating T cells (Avni et al. 2002). There is evidence that NFAT regulates cell differentiation programs in cell types other than immune cells (Crabtree and Olson 2002; Horsley and Pavlath 2002; Graef et al. 2003; Hill-Eubanks et al. 2003). In the last section of this review, we select three differentiation programs—fiber-type specification in differentiated skeletal muscle, cardiac valve development, and osteoclast differentiation—for detailed consideration. We evaluate the evidence for NFAT involvement, point out novel cellular and molecular mechanisms that might regulate this familiar transcription factor, and discuss how NFAT exerts its biological effects. Because the phenotypes of NFAT knockout mice have been reviewed elsewhere (Crabtree and Olson 2002; Horsley and Pavlath 2002), we refer to them only as necessary to illustrate specific points.
Jeffery D. Molkentin - One of the best experts on this subject based on the ideXlab platform.
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isoform and tissue specific regulation of the ca2 sensitive transcription factor NFAT in cardiac myocytes and heart failure
American Journal of Physiology-heart and Circulatory Physiology, 2010Co-Authors: Andreas Rinne, Kathrin Banach, Nidhi Kapur, Donald M. Bers, Steven M. Pogwizd, Jeffery D. Molkentin, Lothar A BlatterAbstract:Nuclear factors of activated T cells (NFATs) are Ca2+-sensitive transcription factors that have been implicated in hypertrophy, heart failure (HF), and arrhythmias. Cytosolic NFAT is activated by dephosphorylation by the Ca2+-sensitive phosphatase calcineurin, resulting in translocation to the nucleus, which is opposed by kinase activity, rephosphorylation, and nuclear export. Four different NFAT isoforms are expressed in the heart. The activation and regulation of NFAT in adult cardiac myocytes, which may depend on the NFAT isoform and cell type, are not fully understood. This study compared basal localization, import, and export of NFATc1 and NFATc3 in adult atrial and ventricular myocytes to identify isoform- and tissue-specific regulatory mechanisms of NFAT activation under physiological conditions and in HF. NFAT-green fluorescent protein fusion proteins and NFAT immunocytochemistry were used to analyze NFAT regulation in adult cat and rabbit myocytes. NFATc1 displayed basal nuclear localization in atrial and ventricular myocytes, an effect that was attenuated by reducing intracellular Ca2+ concentration and inhibiting calcineurin, and enhanced by the inhibition of nuclear export. In contrast, NFATc3 was localized to the cytoplasm but could be driven to the nucleus by angiotensin II and endothelin-1 stimulation in atrial, but not ventricular, cells. Inhibition of nuclear export (by leptomycin B) facilitated nuclear localization in both cell types. Ventricular myocytes from HF rabbits showed increased basal nuclear localization of endogenous NFATc3 and reduced responsiveness of NFAT translocation to phenylephrine stimulation. In control myocytes, Ca2+ overload, leading to spontaneous Ca2+ waves, induced substantial translocation of NFATc3 to the nucleus. We conclude that the activation of NFAT in adult cardiomyocytes is isoform and tissue specific and is tightly controlled by nuclear export. NFAT is activated in myocytes from HF animals and may be secondary to Ca2+ overload.
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calcineurin NFAT coupling participates in pathological but not physiological cardiac hypertrophy
Circulation Research, 2004Co-Authors: Benjamin J Wilkins, Yanshan Dai, Orlando F Bueno, Stephanie A Parsons, David M Plank, Fred Jones, Thomas R Kimball, Jeffery D. MolkentinAbstract:Calcineurin (PP2B) is a calcium/calmodulin-activated, serine-threonine phosphatase that transmits signals to the nucleus through the dephosphorylation and translocation of nuclear factor of activated T cell (NFAT) transcription factors. Whereas calcineurin-NFAT signaling has been implicated in regulating the hypertrophic growth of the myocardium, considerable controversy persists as to its role in maintaining versus initiating hypertrophy, its role in pathological versus physiological hypertrophy, and its role in heart failure. To address these issues, NFAT-luciferase reporter transgenic mice were generated and characterized. These mice showed robust and calcineurin-specific activation in the heart that was inhibited with cyclosporin A. In the adult heart, NFAT-luciferase activity was upregulated in a delayed, but sustained manner throughout eight weeks of pathological cardiac hypertrophy induced by pressure-overload, or more dramatically following myocardial infarction-induced heart failure. In contrast, physiological hypertrophy as produced in two separate models of exercise training failed to show significant calcineurin-NFAT coupling in the heart at multiple time points, despite measurable increases in heart to body weight ratios. Moreover, stimulation of hypertrophy with growth hormone-insulin-like growth factor-1 (GH-IGF-1) failed to activate calcineurin-NFAT signaling in the heart or in culture, despite hypertrophy, activation of Akt, and activation of p70 S6K. Calcineurin Abeta gene-targeted mice also showed a normal hypertrophic response after GH-IGF-1 infusion. Lastly, exercise- or GH-IGF-1-induced cardiac growth failed to show induction of hypertrophic marker gene expression compared with pressure-overloaded animals. Although a direct cause-and-effect relationship between NFAT-luciferase activity and pathological hypertrophy was not proven here, our results support the hypothesis that separable signaling pathways regulate pathological versus physiological hypertrophic growth of the myocardium, with calcineurin-NFAT potentially serving a regulatory role that is more specialized for maladaptive hypertrophy and heart failure.
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requirement of transcription factor NFAT in developing atrial myocardium
Journal of Cell Biology, 2003Co-Authors: William Schubert, Teddy T C Yang, Michael P. Lisanti, Mercedes Rincon, Stephen M. Factor, Jeffery D. Molkentin, Xiaoyong Yang, Chiwing ChowAbstract:Nuclear factor of activated T cell (NFAT) is a ubiquitous regulator involved in multiple biological processes. Here, we demonstrate that NFAT is temporally required in the developing atrial myocardium between embryonic day 14 and P0 (birth). Inhibition of NFAT activity by conditional expression of dominant-negative NFAT causes thinning of the atrial myocardium. The thin myocardium exhibits severe sarcomere disorganization and reduced expression of cardiac troponin-I (cTnI) and cardiac troponin-T (cTnT). Promoter analysis indicates that NFAT binds to and regulates transcription of the cTnI and the cTnT genes. Thus, regulation of cytoskeletal protein gene expression by NFAT may be important for the structural architecture of the developing atrial myocardium.
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inhibition of calcineurin NFAT hypertrophy signaling by cgmp dependent protein kinase type i in cardiac myocytes
Proceedings of the National Academy of Sciences of the United States of America, 2002Co-Authors: Beate Fiedler, Jeffery D. Molkentin, Suzanne M Lohmann, Albert Smolenski, Stephan Linnemuller, Burkert Pieske, Frank Schroder, Helmut Drexler, Kai C WollertAbstract:Recent investigation has focused on identifying signaling pathways that inhibit cardiac hypertrophy, a major risk factor for cardiovascular morbidity and mortality. In this context, nitric oxide (NO), signaling via cGMP and cGMP-dependent protein kinase type I (PKG I), has been recognized as a negative regulator of cardiac myocyte (CM) hypertrophy. However, the underlying mechanisms are poorly understood. Here, we show that PKG I inhibits CM hypertrophy by targeting the calcineurin-NFAT signaling pathway. Calcineurin, a Ca2+-dependent phosphatase, promotes hypertrophy in part by activating NFAT transcription factors which induce expression of hypertrophic genes, including brain natriuretic peptide (BNP). Activation of PKG I by NO/cGMP in CM suppressed NFAT transcriptional activity, BNP induction, and cell enlargement in response to alpha(1)-adrenoreceptor stimulation but not in response to adenoviral expression of a Ca2+-independent, constitutively active calcineurin mutant, thus demonstrating NO-cGMP-PKG I inhibition of calcineurin-NFAT signaling upstream of calcineurin. PKG I suppressed single L-type Ca2+-channel open probability, [Ca2+]i transient amplitude, and, most importantly, L-type Ca2+-channel current-induced NFAT activation, indicating that PKG I targets Ca2+-dependent steps upstream of calcineurin. Adenoviral expression of PKG I enhanced NO/cGMP inhibitory effects upstream of calcineurin, confirming that PKG I mediates NO/cGMP inhibition of calcineurin-NFAT signaling. In CM overexpressing PKG I, NO/cGMP also suppressed BNP induction and cell enlargement but not NFAT activation elicited by constitutively active calcineurin, which is consistent with additional, NFAT-independent inhibitory effect(s) of PKG I downstream of calcineurin. Inhibition of calcineurin-NFAT signaling by PKG I provides a framework for understanding how NO inhibits cardiac myocyte hypertrophy.
Laurie H Glimcher - One of the best experts on this subject based on the ideXlab platform.
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the nuclear factor of activated t cells NFAT transcription factor NFATp NFATc2 is a repressor of chondrogenesis
Journal of Experimental Medicine, 2000Co-Authors: Ann M Ranger, Louis C Gerstenfeld, Jinxi Wang, Ellen M Gravallese, Melvin J Glimcher, Laurie H GlimcherAbstract:Nuclear factor of activated T cells (NFAT) transcription factors regulate gene expression in lymphocytes and control cardiac valve formation. Here, we report that NFATp regulates chondrogenesis in the adult animal. In mice lacking NFATp, resident cells in the extraarticular connective tissues spontaneously differentiate to cartilage. These cartilage cells progressively differentiate and the tissue undergoes endochondral ossification, recapitulating the development of endochondral bone. Proliferation of already existing articular cartilage cells also occurs in some older animals. At both sites, neoplastic changes in the cartilage cells occur. Consistent with these data, NFATp expression is regulated in mesenchymal stem cells induced to differentiate along a chondrogenic pathway. Lack of NFATp in articular cartilage cells results in increased expression of cartilage markers, whereas overexpression of NFATp in cartilage cell lines extinguishes the cartilage phenotype. Thus, NFATp is a repressor of cartilage cell growth and differentiation and also has the properties of a tumor suppressor.
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a potential role for the nuclear factor of activated t cells family of transcriptional regulatory proteins in adipogenesis
Proceedings of the National Academy of Sciences of the United States of America, 1998Co-Authors: John Kim, John W Rooney, Bruce M Spiegelman, Laurie H GlimcherAbstract:NFAT (nuclear factor of activated T cells) is a family of transcription factors implicated in the control of cytokine and early immune response gene expression. Recent studies have pointed to a role for NFAT proteins in gene regulation outside of the immune system. Herein we demonstrate that NFAT proteins are present in 3T3-L1 adipocytes and, upon fat cell differentiation, bind to and transactivate the promoter of the adipocyte-specific gene aP2. Further, fat cell differentiation is inhibited by cyclosporin A, a drug shown to prevent NFAT nuclear localization and hence function. Thus, these data suggest a role for NFAT transcription factors in the regulation of the aP2 gene and in the process of adipocyte differentiation.
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novel NFAT sites that mediate activation of the interleukin 2 promoter in response to t cell receptor stimulation
Molecular and Cellular Biology, 1995Co-Authors: John W Rooney, Laurie H Glimcher, Yalin Sun, T HoeyAbstract:The transcription factors NFAT and AP-1 have been shown to be essential for inducible interleukin-2 (IL-2) expression in activated T cells. NFAT has been previously reported to bind to two sites in the IL-2 promoter: in association with AP-1 at the distal antigen response element at -280 and at -135. On the basis of DNase I footprinting with recombinant NFAT and AP-1 proteins, gel shift assays, and transfection experiments, we have identified three additional NFAT sites in the IL-2 promoter. Strikingly, all five NFAT sites are essential for the full induction of promoter activity in response to T-cell receptor stimulation. Four of the five NFAT sites are part of composite elements able to bind AP-1 in association with NFAT. These sites display a diverse range of cooperativity and interdependency on NFAT and AP-1 proteins for binding. One of the NFAT sites directly overlaps the CD28-responsive element. We present evidence that CD28 inducibility is conferred by the AP-1 component in NFAT-AP-1 composite elements. These findings provide further insight into the mechanisms involved in the regulation of the IL-2 promoter.
Anna C Hartwig - One of the best experts on this subject based on the ideXlab platform.
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NFAT calcineurin signaling promotes oligodendrocyte differentiation and myelination by transcription factor network tuning
Nature Communications, 2018Co-Authors: Matthias Weider, Laura Starost, Katharina Groll, Melanie Kuspert, Elisabeth Sock, Miriam Wedel, Franziska Frob, Christian Schmitt, Tina Baroti, Anna C HartwigAbstract:Oligodendrocytes produce myelin for rapid transmission and saltatory conduction of action potentials in the vertebrate central nervous system. Activation of the myelination program requires several transcription factors including Sox10, Olig2, and Nkx2.2. Functional interactions among them are poorly understood and important components of the regulatory network are still unknown. Here, we identify NFAT proteins as Sox10 targets and regulators of oligodendroglial differentiation in rodents and humans. Overall levels and nuclear fraction increase during differentiation. Inhibition of NFAT activity impedes oligodendrocyte differentiation in vitro and in vivo. On a molecular level, NFAT proteins cooperate with Sox10 to relieve reciprocal repression of Olig2 and Nkx2.2 as precondition for oligodendroglial differentiation and myelination. As NFAT activity depends on calcium-dependent activation of calcineurin signaling, regulatory network and oligodendroglial differentiation become sensitive to calcium signals. NFAT proteins are also detected in human oligodendrocytes, downregulated in active multiple sclerosis lesions and thus likely relevant in demyelinating disease. Oligodendrocyte differentiation is known to depend on transcription factors Sox10, Nkx2.2, and Olig2. Here, the authors show that NFAT/calcineurin signaling contributes to oligodendrocyte differentiation by relieving mutual repression of Nkx2.2 and Olig2.
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NFAT calcineurin signaling promotes oligodendrocyte differentiation and myelination by transcription factor network tuning
Nature Communications, 2018Co-Authors: Matthias Weider, Laura Starost, Katharina Groll, Melanie Kuspert, Elisabeth Sock, Miriam Wedel, Franziska Frob, Christian Schmitt, Tina Baroti, Anna C HartwigAbstract:Oligodendrocytes produce myelin for rapid transmission and saltatory conduction of action potentials in the vertebrate central nervous system. Activation of the myelination program requires several transcription factors including Sox10, Olig2, and Nkx2.2. Functional interactions among them are poorly understood and important components of the regulatory network are still unknown. Here, we identify NFAT proteins as Sox10 targets and regulators of oligodendroglial differentiation in rodents and humans. Overall levels and nuclear fraction increase during differentiation. Inhibition of NFAT activity impedes oligodendrocyte differentiation in vitro and in vivo. On a molecular level, NFAT proteins cooperate with Sox10 to relieve reciprocal repression of Olig2 and Nkx2.2 as precondition for oligodendroglial differentiation and myelination. As NFAT activity depends on calcium-dependent activation of calcineurin signaling, regulatory network and oligodendroglial differentiation become sensitive to calcium signals. NFAT proteins are also detected in human oligodendrocytes, downregulated in active multiple sclerosis lesions and thus likely relevant in demyelinating disease.