NFAT Expression Research Articles

Reduced CTLA-4 protein and messenger RNA expression in umbilical cord blood T lymphocytes

A favorable incidence and severity of graft-vs-host disease is observed in patients transplanted with banked, unrelated, HLA-mismatched umbilical cord blood (UCB) grafts, while the incidence of malignant relapse remains low. CTLA-4 mediates negative T-cell signaling and may contribute to the development of allogeneic tolerance. In this study, we compared protein and mRNA expression of CTLA-4 in stimulated UCB and adult peripheral blood T cells. T cells were isolated from UCB and adult peripheral blood and stimulated with anti-CD3 and anti-CD28 monoclonal antibodies. Cells were immunostained and analyzed by flow cytometry for both surface and intracellular expression of CTLA-4 in the presence and absence of cyclosporin A, and kinetics of CTLA-4 expression compared. CTLA-4 mRNA expression was measured using quantitative real-time polymerase chain reaction. NFAT1 protein levels were measured by Western blot analysis. These studies demonstrate reduced surface and intracellular expression of CTLA-4 in stimulated UCB T cells compared to adult controls. Furthermore, reduced CTLA-4 protein expression in UCB T cells was noted to be in part transcriptionally regulated, as CTLA-4 mRNA levels also were significantly lower. Reduced CLTA-4 expression by UCB T cells followed the kinetics of delayed and reduced expression of the transcription factor NFAT1 by UCB T lymphocytes during primary stimulation. Moreover, cyclosporin A, which is known to modulate NFAT activation, reduced CTLA-4 protein expression in adult and UCB T cells. Reduced expression of the key regulatory proteins CTLA-4 and NFAT-1 may contribute to favorable UCB T lymphocyte allogeneic responses.

High-performance affinity beads for identifying anti-NF-κB drug receptors

will not work in cell lines that fail to express either calcineurin or NF-AT. These problems can be circumvented by cotransfection with NF-AT and/or calcineurin expression plasmids. Electroporation has proved to be an easy and effective means of transfecting Jurkat cells with plasmid DNA, whereas other cell lines may require a different means of transfection (liposomes, retrovirus, CaC12, etc.). This assay could be adapted to yield significant information about calcineurin activity in different human cell lines and provide a readout of H202-induced oxidative stress.

Human eosinophils constitutively express nuclear factor of activated T cells p and c

Background: Eosinophils are now known to produce a variety of proinflammatory cytokines, although the molecular factors that regulate their production are poorly understood. The expression of almost all of the cytokines produced by eosinophils, including the proallergic cytokine IL-4, is now known to be regulated at the level of transcription by members of the nuclear factor of activated T cells (NFAT) family of transcription factors. Objective: We sought to characterize the expression of different NFAT proteins in resting and activated eosinophils. Methods: Nuclear and whole cell extracts were obtained from both peripheral blood eosinophils and those obtained from bronchoalveolar lavage fluid of asthmatic subjects after endobronchial allergen challenge. NFAT expression was determined by using immunoprecipitation and Western blot analysis, DNA-binding assays, and RT-PCR analysis of eosinophil mRNA. Results: Both peripheral blood and bronchoalveolar lavage fluid eosinophils expressed NFATp and NFATc protein. Unlike activated T cells, which express multiple NFATc isoforms, eosinophils preferentially express the approximately 85-kd isoform. In addition, eosinophils were found to constitutively express NFATc mRNA. A brief incubation with the TH2 cytokines IL-4 and IL-5 was sufficient to induce the nuclear translocation of NFATc. Eosinophil nuclear extracts contain multiple factors that can specifically recognize the IL-4 promoter P1 NFAT site in DNA-binding assays, including NFATp. Conclusion: NFATp and NFATc can regulate the expression of cytokines and other genes in eosinophils but appear to be regulated by a novel signal transduction mechanism in these cells. (J Allergy Clin Immunol 2001;107:143-52.)

The calcineurin-NFAT pathway and muscle fiber-type gene expression.

To test for a role of the calcineurin-NFAT (nuclear factor of activated T cells) pathway in the regulation of fiber type-specific gene expression, slow and fast muscle-specific promoters were examined in C2C12 myotubes and in slow and fast muscle in the presence of calcineurin or NFAT2 expression plasmids. Overexpression of active calcineurin in myotubes induced both fast and slow muscle-specific promoters but not non-muscle-specific reporters. Overexpression of NFAT2 in myotubes did not activate muscle-specific promoters, although it strongly activated an NFAT reporter. Thus overexpression of active calcineurin activates transcription of muscle-specific promoters in vitro but likely not via the NFAT2 transcription factor. Slow myosin light chain 2 (MLC2) and fast sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA1) reporter genes injected into rat soleus (slow) and extensor digitorum longus (EDL) (fast) muscles were not activated by coinjection of activated calcineurin or NFAT2 expression plasmids. However, an NFAT reporter was strongly activated by overexpression of NFAT2 in both muscle types. Calcineurin and NFAT protein expression and binding activity to NFAT oligonucleotides were different in slow vs. fast muscle. Taken together, these results indicate that neither calcineurin nor NFAT appear to have dominant roles in the induction and/or maintenance of slow or fast fiber type in adult skeletal muscle. Furthermore, different pathways may be involved in muscle-specific gene expression in vitro vs. in vivo.

NFAT1 Enhances HIV-1 Gene Expression in Primary Human CD4 T Cells

Cyclosporin A (CsA) is a potent inhibitor of the NFAT family of transcription factors that enhance T cell activation. The observation that human immunodeficiency virus type 1 (HIV-1)-positive transplant recipients have a reduced HIV-1 viral burden during treatment with CsA suggested that NFAT may play a direct role in enhancing transcription of the HIV-1 viral genome. Two sets of NFAT binding sites were identified in the HIV-1 long terminal repeat (LTR) promoter by in vitro footprinting with full-length recombinant NFAT protein, and gel shift analysis of nuclear protein from polyclonally activated primary CD4 T cells revealed specific binding of NFAT1 to the NFκB binding sites of the HIV-1 LTR. Activation of primary CD4 T cells transiently transfected with a HIV-1 LTR luciferase reporter plasmid, lacking the NFAT binding sites in the upstream putative negative regulatory element but maintaining the NFκB/NFAT sites, demonstrated increased HIV-1 gene expression when cotransfected with a NFAT1 expression vector. Moreover, CsA, FK506, and a dominant-negative NFAT1 protein independently inhibited HIV-1 LTR promoter activity in CD4 T cells stimulated with phorbol ester and calcium ionophore. In primary human CD4 T cells, CsA also inhibited promoter activity directed by multimers of binding sites for NFAT, while having no effect on NFκB multimer-driven promoter activity. Increasing NFAT1 levels in CD4 T cells transiently transfected with a HIV-1 provirus also increased p24 protein expression. Thus, NFAT may be a target for prevention of HIV-1 LTR-directed gene expression in human CD4 T cells.

FasL promoter activation by IL-2 through SP1 and NFAT but not Egr-2 and Egr-3.

Recently activated peripheral T cells treated with IL-2 for 4 days expressed Fas ligand (FasL)-mediated cytotoxicity. These IL-2-treated T cells had high nuclear expression of SP1 and NFAT, but lacked the Egr-2 and Egr-3 that could be induced by anti-CD3 stimulation and had been implicated in FasL gene activation. A minimal promoter region that responded to IL-2 was identified by transient transfection assays using deletion mutants. The data suggests that the GGGCGGAAA site present in the 5' end of the minimal FasL promoter is critical to IL-2-induced FasL gene activation. The GGGCGGAAA sequence contains an overlapping site used by two transcription factor families, one (GGGCGG) for the SP1 family and the other (GGAAA) for the NFAT family. FasL promoter activity was partially but statistically significantly reduced with constructs mutated at either site. More activity was lost with a construct mutated at both sites. In contrast, mutation at the Egr site had no effect on IL-2-induced FasL promoter activity. Our study identified a new FasL promoter site responding to IL-2-induced SP1 and NFAT factors. Furthermore, the nuclei of IL-2-treated cells express SP1 and NFAT, but not Egr-2 and Egr-3, for FasL gene activation.

FasL promoter activation by IL-2 through SP1 and NFAT but not Egr-2 and Egr-3

Recently activated peripheral T cells treated with IL-2 for 4 days expressed Fas ligand (FasL)-mediated cytotoxicity. These IL-2-treated T cells had high nuclear expression of SP1 and NFAT, but lacked the Egr-2 and Egr-3 that could be induced by anti-CD3 stimulation and had been implicated in FasL gene activation. A minimal promoter region that responded to IL-2 was identified by transient transfection assays using deletion mutants. The data suggests that the GGGCGGAAA site present in the 5′ end of the minimal FasL promoter is critical to IL-2-induced FasL gene activation. The GGGCGGAAA sequence contains an overlapping site used by two transcription factor families, one (GGGCGG) for the SP1 family and the other (GGAAA) for the NFAT family. FasL promoter activity was partially but statistically significantly reduced with constructs mutated at either site. More activity was lost with a construct mutated at both sites. In contrast, mutation at the Egr site had no effect on IL-2-induced FasL promoter activity. Our study identified a new FasL promoter site responding to IL-2-induced SP1 and NFAT factors. Furthermore, the nuclei of IL-2-treated cells express SP1 and NFAT, but not Egr-2 and Egr-3, for FasL gene activation.

Reduced expression of NF-AT and NF-κB transcription factors in tolerant recipients treated with tolerogenic allochimeric donor/recipient class I MHC protein

Reduced expression of NF-AT and NF-κB transcription factors in tolerant recipients treated with tolerogenic allochimeric donor/recipient class I MHC protein

T Cell Priming Enhances IL-4 Gene Expression by Increasing Nuclear Factor of Activated T Cells

The repetitive activation of T cells (priming) enhances the expression of many cytokines, such as IL-4, but not others, such as IL-2. Molecular mechanisms underlying selective expression of cytokines by T cells remain poorly understood. Here we show that priming of CD4 T cells selectively enhances IL-4 expression relative to IL-2 expression by a transcriptional mechanism involving nuclear factor of activated T cells (NFAT) proteins. As detected by in vivo footprinting, priming markedly increases the activation-dependent engagement of the P0 and P1 NFAT-binding elements of the IL-4 promoter. Moreover, each proximal P element is essential for optimal IL-4 promoter activity. Activated primed CD4 T cells contain more NFAT1 and support greater NFAT-directed transcription than unprimed CD4 T cells, while activator protein 1 binding and activator protein 1-mediated transcription by both cell types is similar. Increased expression of wild-type NFAT1 substantially increases IL-4 promoter activity in unprimed CD4 T cells, suggesting NFAT1 may be limiting for IL-4 gene expression in this cell type. Furthermore, a truncated form of NFAT1 acts as a dominant-negative, reducing IL-4 promoter activity in primed CD4 T cells and confirming the importance of endogenous NFAT to increased IL-4 gene expression by effector T cells. NFAT1 appears to be the major NFAT family member responsible for the initial increased expression of IL-4 by primed CD4 T cells.

The Bcl-2 gene is differentially regulated by IL-2 and IL-4: role of the transcription factor NF-AT.

The murine TS1alphabeta T cell line expresses the anti-apoptotic protein Bcl-2 upon IL-2 stimulation, whereas IL-4-mediated growth of this cell line proceeds in the absence of Bcl-2 expression. In addition, IL-4 stimulation inhibits Bcl-2 expression and modulates its mRNA level. IL-2-induced DNA binding activity for these transcription factors is sensitive to phosphatidylinositol 3 kinase inhibitor wortmannin and to Rho inhibitor Clostridium difficile toxin B, which inhibit IL-2-induced Bcl-2 expression. NF-AT transcription factor appears to be the most important in the control Bcl-2 expression, since inhibition of the calcium-calmodulin-dependent phosphatase calcineurin, which regulates NF-AT activity, downregulates Bcl-2 expression in IL-2-stimulated cells. Constitutive expression of this phosphatase also upregulates Bcl-2 expression in IL-4-stimulated cells. In addition, a dominant negative NF-AT expression vector downregulates Bcl-2 expression in IL-2-stimulated cells. These results suggest that IL-2 induction of Bcl-2 expression may be directly or indirectly mediated by NF-AT.

A regulatory element in the CD95 (APO-1/Fas) ligand promoter is essential for responsiveness to TCR-mediated activation.

Expression of the CD95 (APO-1/Fas) ligand (CD95L) in activated T cells is a major cause of T cell activation-induced apoptosis. To study the molecular mechanisms of transcriptional control of CD95L expression in T cells, we investigated the human CD95L promoter in Jurkat T cells. Deletion studies revealed that the CD95L proximal promoter sequence from -220 to the transcription start site is essential for T cell stimulation-induced expression of CD95L. In this study, we discovered a novel regulatory element located at -120 of the CD95L promoter which contains DNA binding sites for SP-1 and a yet unknown inducible factor. Mutation analysis demonstrated that binding of the inducible factor to the -120 region is crucial for the biological function of the CD95L promoter upon T cell stimulation. The DNA sequence at -120 also contains two DNA motifs homologous to the binding site for NF-AT. NF-AT does not directly bind to this element. However, cotransfection studies with an NF-AT expression vector showed that NF-AT may confer a strong inducible activity to the CD95L promoter at this regulatory region. Our data also show that the immunosuppressive agent cyclosporin A down-regulates CD95L transcription by inhibiting the function of this positive regulatory element.

Vav is a regulator of cytoskeletal reorganization mediated by the T-cell receptor

Background: Vav is a guanine-nucleotide exchange factor for the Rho-like small GTPases RhoA, Rac1 and Cdc42, which regulate cytoskeletal reorganization and activation of stress-activated protein kinases (SAPK/JNKs). Vav is expressed in hematopoietic cells and is phosphorylated in T and B cells following activation of various growth factor or antigen receptors. Vav interacts with several signaling molecules in T cells, but the functional relevance of these interactions is established only for Slp 76: they cooperate to induce activity of the transcription factor NF-AT and interleukin-2 expression. We have investigated the role of Vav in T cells by generating vav−/− mice.Results: Mice deficient for vav were viable and healthy, but had impaired T-cell development. In vav−/− T cells, in response to activation of the T-cell receptor (TCR), cell cycle progression, induction of NF-ATc1 activity, downregulation of the cell-cycle inhibitor p27Kip1, interleukin-2 production, actin polymerization and the clustering of TCRs into patches and caps – a cytoskeletal reorganization process – were defective. TCR-mediated activation of mitogen-activated protein kinase and SAPK/JNK was unaffected. Ca2+ mobilization was impaired in vav−/− thymocytes and T cells. In wild-type cells, Vav constitutively associated with the cytoskeletal membrane anchors talin and vinculin. In the absence of Vav, phosphorylation of Slp76, Slp76–talin interactions, and recruitment of the actin cytoskeleton to the CD3ζ chain of the TCR co-receptor were impaired.Conclusions: Vav is a crucial regulator of TCR-mediated Ca2+ flux, cytoskeletal reorganization and TCR clustering, and these are required for T-cell maturation, interleukin-2 production and cell cycle progression.

The Tpl-2 protooncoprotein activates the nuclear factor of activated T cells and induces interleukin 2 expression in T cell lines.

Tpl-2 expression is induced within 30-60 min after ConA stimulation of rat splenocytes, suggesting that it may contribute to the induction of IL-2 during T cell activation. Herein we show that wild-type and carboxyl-terminally truncated (activated) Tpl-2 activate the nuclear factor of activated T cells (NFAT) and induce interleukin 2 (IL-2) expression in EL4 cells. In Jurkat cells the truncated Tpl-2 activates NFAT and induces IL-2, whereas wild-type Tpl-2 activates NFAT only when cotransfected with NFAT expression constructs, suggesting that Tpl-2 may induce NFAT activation signals. Experiments in NIH 3T3 cells revealed that the NFATp isoform, but not the NFATc or NFATx isoform, undergoes nuclear translocation when coexpressed with wild-type Tpl-2 and confirmed this hypothesis. Activation of NFAT by anti-CD3 stimulation but not by phorbol 12-myristate 13-acetate and ionomycin in Jurkat cells was inhibited by the kinase-dead Tpl-2K167M, suggesting that Tpl-2 contributes to the transduction of NFAT activation signals originating in the T cell receptor. The Tpl-2-mediated induction of IL-2 was not observed in T cell lymphoma lines other than EL4 and Jurkat, as well as in normal T cells. NFAT activation by Tpl-2, however, was observed in several cell lines including some of nonhematopoietic origin. The activation of NFAT by Tpl-2 in different cell types defines a molecular mechanism that may contribute to its oncogenic potential.

S-acylation of LCK protein tyrosine kinase is essential for its signalling function in T lymphocytes.

LCK is a non-receptor protein tyrosine kinase required for signal transduction via the T-cell antigen receptor (TCR). LCK N-terminus is S-acylated on Cys3 and Cys5, in addition to its myristoylation on Gly2. Here the role of S-acylation in LCK function was examined. Transient transfection of COS-18 cells, which express a CD8-zeta chimera on their surface, revealed that LCK mutants that were singly S-acylated were able to target to the plasma membrane and to phosphorylate CD8-zeta. A non-S-acylated LCK mutant did not target to the plasma membrane and failed to phosphorylate CD8-zeta, although it was catalytically active. Fusion of non-S-acylated LCK to a transmembrane protein, CD16:7, allowed its plasma membrane targeting and also phosphorylation of CD8-zeta when expressed in COS-18 cells. Thus S-acylation targets LCK to the plasma membrane where it can interact with the TCR. When expressed in LCK-negative JCam-1.6 T cells, delocalized, non-S-acylated LCK was completely non-functional. Singly S-acylated LCK mutants, which were expressed in part at the plasma membrane, efficiently reconstituted the induced association of phospho-zeta with ZAP-70 and intracellular Ca2+ fluxes triggered by the TCR. Induction of the late signalling proteins, CD69 and NFAT, was also reconstituted, although at reduced levels. The transmembrane LCK chimera also supported the induction of tyrosine phosphorylation and Ca2+ flux by the TCR in JCam-1.6 cells. However, induction of ERK MAP kinase was reduced and the chimera was incapable of reconstituting induced CD69 or NFAT expression. These data indicate that LCK must be attached to the plasma membrane via dual acylation of its N-terminus to function properly in TCR signalling.

In vivo anergized CD4+ T cells express perturbed AP-1 and NF-kappa B transcription factors.

Anergy is a major mechanism to ensure antigen-specific tolerance in T lymphocytes in the adult. In vivo, anergy has mainly been studied at the cellular level. In this study, we used the T-cell-activating superantigen staphylococcal enterotoxin A (SEA) to investigate molecular mechanisms of T-lymphocyte anergy in vivo. Injection of SEA to adult mice activates CD4+ T cells expressing certain T-cell receptor (TCR) variable region beta-chain families and induces strong and rapid production of interleukin 2 (IL-2). In contrast, repeated injections of SEA cause CD4+ T-cell deletion and anergy in the remaining CD4+ T cells, characterized by reduced expression of IL-2 at mRNA and protein levels. We analyzed expression of AP-1, NF-kappa B, NF-AT, and octamer binding transcription factors, which are known to be involved in the regulation of IL-2 gene promoter activity. Large amounts of AP-1 and NF-kappa B and significant quantities of NF-AT were induced in SEA-activated CD4+ spleen T cells, whereas Oct-1 and Oct-2 DNA binding activity was similar in both resting and activated T cells. In contrast, anergic CD4+ T cells contained severely reduced levels of AP-1 and Fos/Jun-containing NF-AT complexes but expressed significant amounts of NF-kappa B and Oct binding proteins after SEA stimulation. Resolution of the NF-kappa B complex demonstrated predominant expression of p50-p65 heterodimers in activated CD4+ T cells, while anergic cells mainly expressed the transcriptionally inactive p50 homodimer. These alterations of transcription factors are likely to be responsible for repression of IL-2 in anergic T cells.

Signaling requirements for the expression of the transactivating factor NF‐AT in human T lymphocytes

A protein called nuclear factor of activated T cells (NF-AT) binds to DNA sequences within the enhancer region of the interleukin 2 (IL 2) gene and appears necessary for both the inducibility and T cell specificity of IL 2 expression. IL 2 production is regulated by multiple signals including those generated via activation of the T cell antigen receptor complex (TcR/CD3), CD2 antigen, protein kinase C (PKC) or elevation of intracellular free calcium concentration ([Ca2+]i). We have, therefore, explored the role of these different stimuli in regulating the nuclear expression of NF-AT in human peripheral blood-derived lymphocytes. Results presented herein indicate that maximal expression of NF-AT in T cells requires at least two signals: PKC activation and TcR/CD3 or CD2 triggering, [Ca2+]i increases and TcR/CD3 or CD2 triggering. Data are presented that indicate that either the [Ca2+]i or PKC signal generated via the TcR/CD3 complex would not alone induce NF-AT expression, and that the TcR/CD3 complex probably regulates NF-AT expression because of its ability to regulate multiple intracellular signals in T cells, and not via any single biochemical event. The combination of CD2 mAb GT2/OKT11 used in the present study to trigger the CD2 antigen is able to act in synergy with phorbol 12,13-dibutyrate or ionomycin to induce NF-AT expression. However, these CD2 mAb do not elevate [Ca2+]i or activate PKC, suggesting that signals other than [Ca2+]i or PKC can regulate NF-AT expression in peripheral blood-derived T cells.