NFAT Pathway Research Articles

The NFATc1 transcription factor is widely expressed in white cells and translocates from the cytoplasm to the nucleus in a subset of human lymphomas

Stimulation of lymphoid cells via their surface receptors triggers signalling pathways that terminate in the nucleus, where they induce alterations in gene transcription. Nuclear factor of activated T cells (NFAT) transcription factors, involved in a major Ca2+-dependent signalling pathway, normally reside in the cytoplasm but re-locate to the nucleus when activation of the pathway (e.g. following ligation of antigen receptors) leads to their dephosphorylation. This study found that one member of the NFAT family (NFATc1/NFAT2) can be detected in routine biopsy samples, where it is seen in essentially all lymphoid cells, but is absent from the great majority of non-haematopoietic cells. An immunohistological evaluation of NFATc1 in almost 300 lymphomas showed that most neoplastic lymphoid cells also express NFATc1 as a cytoplasmic constituent, although it is absent in classical Hodgkin's disease and plasma cell proliferations. Of particular interest was the finding that NFATc1 was relocated to the nucleus in a minority of lymphoid neoplasms (usually diffuse large B-cell lymphomas or Burkitt lymphoma), presumably reflecting activation of the NFAT pathway. It would be of interest to correlate this feature with patterns of gene expression and also with prognosis, since it may identify a subset of human lymphoma that is distinct in its molecular and clinical features.

Distinct immune responses in TLR2, TLR4 or MyD88 deficient mice following Pseudomonas aeruginosa lung infection

The calcineurin-nuclear factor of activated T-cell (NFAT) signal transduction pathway regulates the expression of a plethora of genes in the myocardium. Cytosolic calcium overloading occurs in atrial fibrillation (AF), and this fulfills the condition needed for activation of this pathway. We therefore investigated the NFAT pathway in atrial tissue in a porcine model of AF. AF was induced in eight adult pigs by rapid atrial pacing. Investigations on the calcineurin and NFAT pathway were performed on transmural left atrial tissue obtained 6 weeks after implantation of the pacemaker (pacing for 4 weeks, and AF without pacing for 2 weeks). In the AF group, the left atrial dimension increased significantly (26 +/- 4 mm vs 31 +/- 4 mm, respectively, p < 0.05 [mean +/- SD]). Calcineurin enzyme activity increased significantly in pigs with AF (n = 8) when compared to control pigs (n = 6) [0.143 +/- 0.034 vs 0.038 +/- 0.063 mmol PO(4)(-) released, p < 0.01]. We found that both NFAT-c3 and NFAT-c4, the downstream effectors of calcineurin, increased significantly in the nuclei in AF tissue using immunoblotting. Translocation of NFAT-c3 and NFAT-c4 into the nuclei was also demonstrated in AF tissue microsections using immunohistochemistry. The electrophoresis mobility shift assay further demonstrated that nuclear extracts from AF tissue had a significantly larger binding capacity for NFAT-specific oligonucleotide probes. Our results demonstrate that calcineurin activity was increased in AF with subsequent NFAT-c3 and NFAT-c4 translocation into the nucleus. Activation of this signal transduction pathway may play an important role in the pathogenesis of AF.

Increased activity of the calcineurin-nuclear factor of activated T cells pathway in squirrel monkey B-Lymphoblasts identified by PowerBlot.

New World primate-derived cell lines were instrumental in identifying the primary factors causing glucocorticoid resistance in these primate species. Their use is expanding because it has been recognized that some of these cell lines exhibit differential sensitivity to retroviral infection. To enhance their utility as cell models, we have further characterized one of these cell lines, squirrel monkey-derived B-lymphoblast (SML) cells, using PowerBlot. PowerBlot is a high-throughput, proteomic screen designed to identify differentially expressed proteins. We compared proteins expressed in SML cells and in a human B-lymphoblast (HL) cell line. We found that, relative to HL cells, SML cells overexpress the calcineurin-activated transcription factor nuclear factor of activated T cells 1 (NFAT-1), which exists in a cyclosporine A (CsA)-sensitive dephosphorylated, constitutively active state. We show that there is increased binding of NFAT-1 to deoxyribonucleic acid and greater activity of an NFAT-sensitive human interleukin-2 (IL-2) promoter-luciferase reporter gene in SML compared with activity in HL cells. The increased NFAT activity does not likely result from calcium-dependent activation of calcineurin because cytosolic calcium levels were not different in SML and HL cells. Rather, SML cells express a truncated form of the catalytic subunit of calcineurin that we propose is responsible for the increased activity of the NFAT pathway. Thus, these novel findings first uncovered by a proteomic screen will enhance the value of these New World primate cell lines as "experiments of nature" to gain insight into mechanisms of NFAT activation.

Reduced expression of NFAT-associated genes in UCB versus adult CD4+T lymphocytes during primary stimulation

AbstractThe cellular and molecular mechanisms underlying the blunted allo-responsiveness of umbilical cord blood (UCB) T cells have not been fully elucidated. Protein expression of NFATc2 (nuclear factor of activated T cells c2), a critical transcription factor necessary for up-regulation of multiple cytokines known to amplify T-cell allogeneic responses, is reduced in UCB T cells. Affymetrix oligonucleotide microarrays were used to compare gene expression of primary purified CD4+UCB T cells to adult peripheral blood CD4+T cells (AB) at baseline, 6, and 16 hours of primary stimulation. NFAT-regulated genes exhibited lower expression in UCB CD4+T cells including the following: granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), interleukin 3 (IL-3), IL-4, IL-5, IL-13, IL-2 receptor α (IL-2Rα; CD25), CD40L, and macrophage inflammatory protein 1 α (MIP-1α). Transcription factors involved in the NFAT pathway including C/EBPβ, JunB, and Fosl1 (Fra-1), as well as Th1- and Th2-related transcription factors STAT4 (signal transducers and activators of transcription 4), T-bet, and c-maf showed reduced expression in UCB compared with AB during primary stimulation. Reduced cytokine, chemokine, and receptor expression was also found in UCB. Gene array data were confirmed using RNase protection assays, flow cytometry, and quantitative multiplexed cytokine measurements. Reduced global expression of NFAT-associated genes, as well as cytokines and chemokines, in UCB CD4+T cells may contribute to the decreased graft-versus-host disease (GVHD) observed after UCB transplantation. (Blood. 2003;102:4608-4617)

Morphine Negatively Regulates Interferon-γ Promoter Activity in Activated Murine T Cells through Two Distinct Cyclic AMP-dependent Pathways

To explore the mechanism by which morphine promotes the incidence of HIV infection, we evaluated the regulatory role of morphine on the interferon-gamma (IFN-gamma) promoter in activated T cells from wild type and mu-opioid receptor knockout mice. Our results show that morphine inhibited anti-CD3/CD28-stimulated IFN-gamma promoter activity in a dose-dependent manner. Chronic morphine treatment of T cells increased intracellular cAMP. To evaluate the role of cAMP in morphine's modulatory function, the effects of dibutyryl cyclic AMP and forskolin were investigated. Both dibutyryl cyclic AMP and forskolin treatment inhibited IFN-gamma promoter activity. Treatment with pertussis toxin, but not with a protein kinase A inhibitor, antagonized morphine's inhibitory effects. Morphine inhibited phosphorylation of ERK1/2 and p38 MAPK; in addition, morphine treatment in the presence of either ERK1/2 or p38 MAPK inhibitor (PD98059 or SB203580) resulted in an additive inhibition of IFN-gamma promoter activity. The transcription factor activator protein-1, NF-kappaB, and nuclear factor of activated T cells (NFAT) were negatively regulated by morphine. Overexpression of NF-kappaB p65 rescued the inhibitory effect of morphine on IFN-gamma promoter activity. However, only when NFATc1 was co-overexpressed with c-fos was the inhibitory effect of morphine on IFN-gamma promoter counteracted. The inhibitory effects of morphine were not observed in T cells obtained from mu-opioid receptor knockout mice, suggesting that morphine modulation of IFN-gamma promoter activity is mediated through the mu-opioid receptor. In summary, our data indicate that morphine modulation of IFN-gamma promoter activity is mediated through two distinct cAMP-dependent pathways, the NF-kappaB signaling pathway and the ERK1/2, p38 MAPK, AP-1/NFAT pathway.

Susceptibility to T cell-mediated injury in immune complex disease is linked to local activation of renin-angiotensin system: the role of NF-AT pathway.

FcR provides a critical link between ligands and effector cells in immune complex diseases. Emerging evidence reveals that angiotensin (Ang)II exerts a wide variety of cellular effects and contributes to the pathogenesis of inflammatory diseases. In anti-glomerular basement membrane Ab-induced glomerulonephritis (GN), we have previously noted that FcR-deficient mice (gamma(-/-)) surviving from lethal initial damage still developed mesangial proliferative GN, which was drastically prevented by an AngII type 1 receptor (AT1) blocker. We further examined the mechanisms by which renin-Ang system (RAS) participates in this immune disease. Using bone marrow chimeras between gamma(-/-) and AT1(-/-) mice, we found that glomerular injury in gamma(-/-) mice was associated with CD4(+) T cell infiltration depending on renal AT1-stimulation. Based on findings in cutaneous delayed-type hypersensitivity, we showed that AngII-activated renal resident cells are responsible for the recruitment of effector T cells. We next examined the chemotactic activity of AngII-stimulated mesangial cells, as potential mechanisms coupling RAS and cellular immunity. Chemotactic activity for T cells and Th1-associated chemokine (IFN-gamma-inducible protein-10 and macrophage-inflammatory protein 1alpha) expression was markedly reduced in mesangial cells from AT1(-/-) mice. Moreover, this activity was mainly through calcineurin-dependent NF-AT. Although IFN-gamma-inducible protein-10 was NF-kappaB-dependent, macrophage-inflammatory protein 1alpha was dominantly regulated by NF-AT. Furthermore, AT1-dependent NF-AT activation was observed in injured glomeruli by Southwestern histochemistry. In conclusion, our data indicate that local RAS activation, partly via the local NF-AT pathway, enhances the susceptibility to T cell-mediated injury in anti-glomerular basement membrane Ab-induced GN. This novel mechanism affords a rationale for the use of drugs interfering with RAS in immune renal diseases.

Regulation of IFN regulatory factor 4 expression in human T cell leukemia virus-I-transformed T cells.

IFN regulatory factor (IRF)-4 is a lymphoid/myeloid-restricted member of the IRF transcription factor family that plays an essential role in the homeostasis and function of mature lymphocytes. IRF-4 expression is tightly regulated in resting primary T cells and is transiently induced at the mRNA and protein levels after activation by Ag-mimetic stimuli such as TCR cross-linking or treatment with phorbol ester and calcium ionophore (PMA/ionomycin). However, IRF-4 is constitutively upregulated in human T cell leukemia virus type I (HTLV-I) infected T cells as a direct gene target for the HTLV-I Tax oncoprotein. In this study we demonstrate that chronic IRF-4 expression in HTLV-I-infected T lymphocytes is associated with a leukemic phenotype, and we examine the mechanisms by which continuous production of IRF-4 is achieved in HTLV-I-transformed T cells. IRF-4 expression in HTLV-1-infected cells is driven through activation of the NF-kappaB and NF-AT pathways, resulting in the binding of p50, p65, and c-Rel to the kappaB1 element and p50, c-Rel, and NF-ATp to the CD28RE element within the -617 to -209 region of the IRF-4 promoter. Furthermore, mutation of either the kappaB1 or CD28RE sites blocks Tax-mediated transactivation of the human IRF-4 promoter in T cells. These experiments constitute the first detailed analysis of human IRF-4 transcriptional regulation within the context of HTLV-I infection and transformation of CD4(+) T lymphocytes.

Vav-Rac1-mediated activation of the c-Jun N-terminal kinase/c-Jun/AP-1 pathway plays a major role in stimulation of the distal NFAT site in the interleukin-2 gene promoter.

Vav, a hematopoiesis-specific signaling protein, plays an important role in T-cell development and activation. Vav upregulates the expression of the interleukin-2 (IL-2) gene, primarily via activation of the distal NFAT site in the IL-2 gene promoter (NFAT-IL-2). However, since this site cooperatively binds NFAT and AP-1, the relative contribution of Vav to NFAT versus AP-1 activation has not been determined. Here, we studied the respective roles of the AP-1 and NFAT pathways in the T-cell receptor (TCR)-mediated, Vav-dependent activation of NFAT-IL-2. Although Vav stimulated the transcriptional activity of an NFAT-IL-2 reporter gene, it failed to stimulate the transcriptional or DNA-binding activities of an AP-1-independent NFAT site derived from the human gamma interferon gene promoter. Vav also did not stimulate detectable Ca(2+) mobilization and nuclear translocation of NFATc or NFATp. On the other hand, Vav induced the activation of Rac1 or Cdc42 and c-Jun N-terminal kinase (JNK), enhanced the transcriptional and DNA-binding activities of AP-1, and induced increased phosphorylation of c-Jun. Dominant-negative Vav and/or Rac1 mutants blocked the TCR-mediated stimulation of these events, demonstrating the physiological relevance of these effects. Vav also associated with Rac1 or Cdc42 in T cells, and anti-CD3 antibody stimulation enhanced this association. These findings indicate that a Rac1-dependent JNK/c-Jun/AP-1 pathway, rather than the Ca(2+)/NFAT pathway, plays the predominant role in NFAT-IL-2 activation by Vav.

Transcriptional activation of the interleukin-2 promoter by hepatitis C virus core protein.

Most patients infected with hepatitis C virus (HCV) become chronic carriers. Viruses that efficiently establish persistent infections must have effective ways of evading host defenses. In the case of HCV, little is known about how chronic infections are established or maintained. Besides hepatocytes, several reports suggest that HCV can infect T and B lymphocytes. Since T cells are essential for viral clearance, direct or indirect effects of HCV on T-cell function could influence the outcome of infection. Given that T-cell growth and differentiation require the cytokine interleukin 2 (IL-2), we asked whether HCV might modulate synthesis of IL-2. Portions of the HCV polyprotein were expressed in Jurkat cells under a variety of conditions. We found that the highly conserved HCV core protein, in combination with other stimuli, was able to dramatically activate transcription from the IL-2 promoter. The carboxy-terminal hydrophobic portion of the core protein was required for this activity. Activation was dependent on nuclear factor of activated T cells (NFAT), occurred in cells deficient in the tyrosine kinase p56(lck), and could be blocked by addition of cyclosporin A and by depletion of calcium. These results suggest that the HCV core protein can activate transcription of the IL-2 promoter through the NFAT pathway. This novel activity may have consequences for T-cell development and establishment of persistent infections.

Regulatory Defects in Cbl and Mitogen-Activated Protein Kinase (Extracellular Signal-Related Kinase) Pathways Cause Persistent Hyperexpression of CD40 Ligand in Human Lupus T Cells

To identify intrinsic defects in lupus, we studied short-term, CD4(+) T cell lines that were established from 16 lupus patients (active or inactive) and 15 normal subjects by stimulating once with anti-CD3, anti-CD28, and IL-2. After resting, the pure CD4(+) T cells were exposed to anergy-inducing stimulation with plate-bound anti-CD3 mAb in the absence of APC. Lupus T cells showed prolonged high level expression of CD40 ligand (CD40L, CD154) even in the face of anergy protocol, which shut down CD40L expression in normal T cells. The sustained CD40L expression in lupus T cells did not correlate with memory status or Th deviation, and was relatively independent of IL-2 or other autocrine or paracrine signals via CD28 or CTLA-4. Cyclosporin A could block CD40L expression by lupus T cells when added early during the anti-CD3 stimulation period, but only partially when added later, indicating that another mechanism regulates the prolonged hyperexpression of CD40L besides the Ca(2+) --> calcineurin-dependent NF-AT pathway. When exposed to the anergy protocol, lupus T cells, in marked contrast to normal T cells, did not phosphorylate Cbl/Cbl-b but continued to express strongly phosphorylated extracellular signal-regulated kinase (ERK); U0126, a specific inhibitor of mitogen-activated protein kinase kinase --> ERK, could block both the early and the prolonged hyperexpression of CD40L. Thus, pathways regulating the activities of Cbl and one particular mitogen-activated protein kinase, ERK, are involved in the prolonged hyperexpression of CD40L in lupus T cells.

Two distinct action mechanisms of immunophilin-ligand complexes for the blockade of T-cell activation.

The immunosuppressive effects of cyclosporin A (CsA) and FK506 are mediated through binding to immunophilins. Here we show that FK506-FKBP complex suppresses the activation of JNK and p38 pathways at a level upstream of mitogen-activated protein kinase (MAPK) kinase kinase (MAPKK-K) besides the calcineurin-NFAT pathway. A238L, a viral gene product that binds to immunophilin, also blocks activation of both pathways. In contrast, direct inhibitors of calcineurin, Cabin 1 and FR901725, suppress the activation of NFAT but not the JNK or p38 pathway. We further demonstrate that co-expression of a constitutively active NFAT and a constitutively active MEKK1 renders the interleukin-2 promoter in Jurkat T lymphocytes resistant to CsA and FK506, whereas Jurkat cells expressing a constitutively active NFAT alone are still sensitive to CsA or FK506. Therefore, CsA and FK506 exert their immunosuppressive effects through targeting both the calcineurin-dependent NFAT pathway and calcineurin-independent activation pathway for JNK and p38.

Myosin heavy chain isoform expression following reduced neuromuscular activity: potential regulatory mechanisms.

In this review, the adaptations in myosin heavy chain (MHC) isoform expression induced by chronic reductions in neuromuscular activity (including electrical activation and load bearing) of the intact neuromuscular unit are summarized and evaluated. Several different animal models and human clinical conditions of reduced neuromuscular activity are categorized based on the manner and extent to which they alter the levels of electrical activation and load bearing, resulting in three main categories of reduced activity. These are: 1) reduced activation and load bearing (including spinal cord injury, spinal cord transection, and limb immobilization with the muscle in a shortened position); 2) reduced loading (including spaceflight, hindlimb unloading, bed rest, and unilateral limb unloading); and 3) inactivity (including spinal cord isolation and blockage of motoneuron action potential conduction by tetrodotoxin). All of the models discussed resulted in increased expression of fast MHC isoforms at the protein and/or mRNA levels in slow and fast muscles (with the possible exception of unilateral limb unloading in humans). However, the specific fast MHC isoforms that are induced (usually the MHC-IIx isoform in slow muscle and the MHC-IIb isoform in fast muscle) and the degree and rate of adaptation are dependent upon the animal species and the specific model or condition that is being studied. Recent studies designed to elucidate the mechanisms by which electrical activation and load bearing alter expression of MHC isoforms at the cellular and genetic levels are also reviewed. Two main mechanisms have been proposed, the myogenin:MyoD and calcineurin:NF-AT pathways. Collectively, the data suggest that the regulation of MHC isoform expression involves a complex interaction of multiple control mechanisms including the myogenin:MyoD and calcineurin:NF-AT pathways; however, other intracellular signaling pathways are likely to contribute.

Inhibiting pathways instead of molecules

One of the medically important roles that the phosphatase calcineurin plays in regulating T cell responses is the dephospho-rylation of the transcription factor NFAT, which then can move into the nucleus and initiate transcription and cell activation. Current immunosuppressive drugs inhibit calcineurin, and therefore inhibit all of its functions, which may contribute to the known toxicity of the drugs. Aramburu et al . have made a more specific inhibitor of the NFAT activation pathway by identification of a peptide that binds with high affinity to the site on calcineurin to which NFAT binds, thereby inhibiting the association of the two proteins and specifically blocking only the NFAT pathway in the cell. This result demonstrates that interaction sites may make good targets for specifically inhibiting a particular signaling pathway and provide alternatives for the development of therapeutics. Aramburu, J., Yaffe, M.B., López-Rodríguez, C., Cantley, L.C., Hogan, P.G., and Rao, A. (1999) Affinity-driven peptide selection of an NFAT inhibitor more selective than cyclosporin A. Science 285 : 2129-2133. [Abstract] [Full Text]

The role of calcineurin in immune system responses

In this article I offer my own perspective on regulation of the immune system, which emphasizes some of the intracellular signaling events that underlie these biologic responses. Although this article highlights work that my collaborators and I have carried out, I would like to acknowledge at the outset the important contributions of many investigators in this area whose work I will be unable to discuss in detail. As stated elsewhere in this supplement, the immune system represents an example of remarkable complexity and involves the coordinated interaction of many cell types. On the macroscopic level, these responses are achieved through intercellular communication and the tightly controlled differentiation and expansion of specific subsets of lymphoid cells. However, these events are ultimately set in motion through the regulation of key intracellular signaling pathways, which determine the production and release of cytokines or other mediators. Early studies of lymphocyte signaling focused primarily on the initial receptor-mediated steps involving activation of tyrosine kinases and their substrates. 1 Although very informative about the effector coupling events, these studies were unable to pin-point the critical downstream steps that link stimulus recognition to the cellular response or developmental commitment. Nonetheless, the existence of these subsequent signaling events had been inferred through pharmacologic studies involving drugs that can modify a wide range of immune cell responses. During the past 7 years, my laboratory has focused its efforts on the biologic roles of calcineurin, a protein phosphatase that is regulated by Ca z+ through its association with the Ca2+-binding protein, calmodulin. In this respect it is unique among protein phosphatases in that its activity can