Expression analysis of nuclear factor of activated T cells (NFAT) during myeloid differentiation of CD34+ cells: regulation of Fas ligand gene expression in megakaryocytes

The Orai1 channel is regulated by stromal interaction molecules STIM1 and STIM2 within endoplasmic reticulum (ER)-plasma membrane (PM) contact sites. Ca2+signals generated by Orai1 activate Ca2+-dependent gene expression. When compared with STIM1, STIM2 is a weak activator of Orai1, but it has been suggested to have a unique role in nuclear factor of activated T cells 1 (NFAT1) activation triggered by Orai1-mediated Ca2+entry. In this study, we examined the contribution of STIM2 in NFAT1 activation. We report that STIM2 recruitment of Orai1/STIM1 to ER-PM junctions in response to depletion of ER-Ca2+promotes assembly of the channel with AKAP79 to form a signaling complex that couples Orai1 channel function to the activation of NFAT1. Knockdown of STIM2 expression had relatively little effect on Orai1/STIM1 clustering or local and global [Ca2+]iincreases but significantly attenuated NFAT1 activation and assembly of Orai1 with AKAP79. STIM1ΔK, which lacks the PIP2-binding polybasic domain, was recruited to ER-PM junctions following ER-Ca2+depletion by binding to Orai1 and caused local and global [Ca2+]iincreases comparable to those induced by STIM1 activation of Orai1. However, in contrast to STIM1, STIM1ΔK induced less NFAT1 activation and attenuated the association of Orai1 with STIM2 and AKAP79. Orai1-AKAP79 interaction and NFAT1 activation were recovered by coexpressing STIM2 with STIM1ΔK. Replacing the PIP2-binding domain of STIM1 with that of STIM2 eliminated the requirement of STIM2 for NFAT1 activation. Together, these data demonstrate an important role for STIM2 in coupling Orai1-mediated Ca2+influx to NFAT1 activation.

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Reciprocal Regulation of DSCR1 (Down Syndrome Critical Region 1) Expression and NFAT (Nuclear Factor of Activated T Cells) Activation in Megakaryocytes.

Blood ◽

10.1182/blood.v110.11.2204.2204 ◽

2007 ◽

Vol 110 (11) ◽

pp. 2204-2204

Author(s):

Satu Kyttaelae ◽

Ivonne Habermann ◽

Martin Bornhaeuser ◽

Gerhard Ehninger ◽

Alexander Kiani

Keyword(s):

T Cells ◽

Down Syndrome ◽

Critical Region ◽

Fas Ligand ◽

Nuclear Translocation ◽

Specific Inhibitor ◽

Chromosome 21 ◽

Nuclear Factor ◽

Activated T Cells ◽

Retroviral Transduction

Abstract NFAT (Nuclear Factor of Activated T cells) is a family of calcium-induced, calcineurin-dependent transcription factors, well characterized as central regulators of inducible gene expression in T lymphocytes but now known to function also in several other cell types in various adaptation and differentiation processes. Activation of NFAT by the phosphatase calcineurin is counteracted by several inhibitory kinases and can be completely blocked by the immunosuppressant Cyclosporin A. The Down syndrome critical region 1 (DSCR1; also termed CSP1, MCIP1 or RCAN1) gene belongs to the calcipressin family of endogenous calcineurin inhibitors and is expressed in several isoforms, one of which (isoform C, coded by exons 4–7) has been described to be a transcriptional target for NFAT in striated muscle, endothelial, and neural cells. The DSCR1 gene is located within the Down syndrome critical region of human chromosome 21 and is, together with 200–300 other genes, overexpressed about 1.5-fold in patients with Down syndrome (DS). Previously, dysregulation of NFAT signaling by overexpression of DSCR1 has been implicated in causing various of the pathophysiological features observed in DS patients. Children with DS also suffer from an about 500-fold increased incidence of acute megakaryocytic leukemia; the respective roles of NFAT or DSCR1 in megakaryocytes of either normal individuals or those with DS, however, has not yet been established. Here we show that DSCR1 is upregulated during megakaryocytic differentiation in a lineage-specific manner, and in mature megakaryocytes is further strongly induced by calcineurin stimulation. DSCR1 expression in megakaryocytes is regulated by NFAT, since overexpression of NFATc2 enhances, while overexpression of the specific inhibitor of NFAT activation, VIVIT, suppresses expression of the gene. We further demonstrate that DSCR1 does not only represent an NFAT target in megakaryocytes, but itself acts an inhibitor of NFAT signaling in these cells. Overexpression of DSCR1 in CMK cells as well as in primary megakaryocytes by retroviral transduction profoundly suppressed ionomycin-induced dephosphorylation and nuclear translocation of NFATc2, as well as transactivation of an NFAT-dependent promoter construct. Finally, overexpression of DSCR1 in megakaryocytes markedly downregulated both the constitutive and induced expression of Fas Ligand, a pro-apoptotic gene recently established as a NFAT target in megakaryocytes. Together, these results suggest that DSCR1 acts as an NFAT-induced NFAT inhibitor in megakaryocytes and, when overexpressed, interferes with the expression of NFAT-dependent megakaryocytic genes.

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A potential role for nuclear factor of activated T-cells in receptor tyrosine kinase and G-protein-coupled receptor agonist-induced cell proliferation

Biochemical Journal ◽

10.1042/bj20020347 ◽

2002 ◽

Vol 368 (1) ◽

pp. 183-190 ◽

Cited By ~ 52

Author(s):

Chandrahasa R. YELLATURU ◽

Salil K. GHOSH ◽

R.K. RAO ◽

Lisa K. JENNINGS ◽

Aviv HASSID ◽

...

Keyword(s):

Gene Expression ◽

T Cells ◽

Dna Synthesis ◽

G Protein ◽

Receptor Tyrosine Kinase ◽

Binding Activity ◽

Nuclear Factor ◽

Activated T Cells ◽

Dna Binding Activity ◽

G Protein Coupled

We have studied the role of nuclear factor of activated T-cells (NFAT) transcription factors in the induction of vascular smooth muscle cell (VSMC) growth by platelet-derived growth factor-BB (PDGF-BB) and thrombin, the receptor tyrosine kinase (RTK) and G-protein-coupled receptor (GPCR) agonists, respectively. NFATc1 but not NFATc2 or NFATc3 was translocated from the cytoplasm to the nucleus upon treatment of VSMCs with PDGF-BB or thrombin. Translocation of NFATc1 was followed by an increase in NFAT—DNA binding activity and NFAT-dependent reporter gene expression. Cyclosporin A (CsA), a potent and specific inhibitor of calcineurin, a calcium/calmodulin-dependent serine phosphatase involved in the dephosphorylation and activation of NFATs, blocked NFAT—DNA binding activity and NFAT-dependent reporter gene expression induced by PDGF-BB and thrombin. CsA also completely inhibited PDGF-BB- and thrombin-induced VSMC growth, as measured by DNA synthesis and cell number. In addition, forced expression of the NFAT-competing peptide VIVIT for calcineurin binding significantly attenuated the DNA synthesis induced by PDGF-BB and thrombin in VSMCs. Together, these findings for the first time demonstrate a role for NFATs in RTK and GPCR agonist-induced growth in VSMCs.

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Foxp3 interacts with nuclear factor of activated T cells and NF- B to repress cytokine gene expression and effector functions of T helper cells

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.0501675102 ◽

2005 ◽

Vol 102 (14) ◽

pp. 5138-5143 ◽

Cited By ~ 378

Author(s):

E. Bettelli ◽

M. Dastrange ◽

M. Oukka

Keyword(s):

Gene Expression ◽

T Cells ◽

T Helper Cells ◽

Cytokine Gene Expression ◽

Nuclear Factor ◽

Cytokine Gene ◽

T Helper ◽

Activated T Cells ◽

Effector Functions ◽

Helper Cells

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Vascular Endothelial Growth Factor Activates Nuclear Factor of Activated T Cells in Human Endothelial Cells: a Role for Tissue Factor Gene Expression

Molecular and Cellular Biology ◽

10.1128/mcb.19.3.2032 ◽

1999 ◽

Vol 19 (3) ◽

pp. 2032-2043 ◽

Cited By ~ 138

Author(s):

Angel Luis Armesilla ◽

Elisa Lorenzo ◽

Pablo Gómez del Arco ◽

Sara Martínez-Martínez ◽

Arantzazu Alfranca ◽

...

Keyword(s):

Gene Expression ◽

Vascular Endothelial Growth Factor ◽

T Cells ◽

Endothelial Cells ◽

Growth Factor ◽

Vascular Endothelial ◽

Nuclear Factor ◽

Promoter Activation ◽

Activated T Cells ◽

Endothelial Growth Factor

ABSTRACT Vascular endothelial growth factor (VEGF) is a potent angiogenic inducer that stimulates the expression of tissue factor (TF), the major cellular initiator of blood coagulation. Here we show that signaling triggered by VEGF induced DNA-binding and transcriptional activities of nuclear factor of activated T cells (NFAT) and AP-1 in human umbilical vein endothelial cells (HUVECs). VEGF also induced TF mRNA expression and gene promoter activation by a cyclosporin A (CsA)-sensitive mechanism. As in lymphoid cells, NFAT was dephosphorylated and translocated to the nucleus upon activation of HUVECs, and these processes were blocked by CsA. NFAT was involved in the VEGF-mediated TF promoter activation as evidenced by cotransfection experiments with a dominant negative version of NFAT and site-directed mutagenesis of a newly identified NFAT site within the TF promoter that overlaps with a previously identified κB-like site. Strikingly, this site bound exclusively NFAT not only from nuclear extracts of HUVECs activated by VEGF, a stimulus that failed to induce NF-κB-binding activity, but also from extracts of cells activated with phorbol esters and calcium ionophore, a combination of stimuli that triggered the simultaneous activation of NFAT and NF-κB. These results implicate NFAT in the regulation of endothelial genes by physiological means and shed light on the mechanisms that switch on the gene expression program induced by VEGF and those regulating TF gene expression.

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Nuclear Factor of Activated T Cells and Cytokines Gene Expression of the T Cells in AIDS Patients with Immune Reconstitution Inflammatory Syndrome during Highly Active Antiretroviral Therapy

Mediators of Inflammation ◽

10.1155/2017/1754741 ◽

2017 ◽

Vol 2017 ◽

pp. 1-7 ◽

Cited By ~ 1

Author(s):

Jia Sun ◽

Heling Chen ◽

Yirui Xie ◽

Junwei Su ◽

Ying Huang ◽

...

Keyword(s):

Gene Expression ◽

T Cells ◽

Immune Reconstitution Inflammatory Syndrome ◽

Highly Active Antiretroviral Therapy ◽

Immune Reconstitution ◽

High Expression ◽

Nuclear Factor ◽

Aids Patients ◽

Activated T Cells ◽

Inflammatory Syndrome

Background. The etiology of immune reconstitution inflammatory syndrome (IRIS) in AIDS patients after the initiation of HAART remains unknown. Several researches indicated that the development of IRIS is associated with the production and variation of cytokines, whose gene expression are closely related to the Ca2+/CN-nuclear factor of activated T cells (NFAT) pathway.Methods. We studied the expression of NFAT isoforms and their major target cytokines genes in peripheral blood CD3+T cells of subjects through fluorescence quantitative PCR and explored the expression changes of these genes before and after HAART.Results. After the initiation of HARRT, NFAT1, IL-6, and IL-8 gene expression showed a reversal trend in the CD3+T cells of the IRIS group and changed from low expression before HARRT to high expression after HARRT. In particular, the relative gene expression of NFAT1 was markedly higher compared with the other three isoforms. The IRIS group also showed higher NFAT4, NFAT2, NFAT1, IL-1β, IL-10, IL-2, IL-18, and TNF-αgene expression than the non-IRIS group.Conclusion. This study suggested that high expression levels of IL-2, IL-6, IL-8, TNF-α, IL-1β, IL-10, IL-12, and IL-18 can predict the risk of IRIS. The increased expression of NFAT1 and NFAT4 may promote the expression of cytokines, such as IL-6, IL-8, and TNF-α, which may promote the occurrence of IRIS.

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Defective nuclear translocation of nuclear factor of activated T cells and extracellular signal-regulated kinase underlies deficient IL-2 gene expression in Wiskott-Aldrich syndrome

Journal of Allergy and Clinical Immunology ◽

10.1016/j.jaci.2005.09.006 ◽

2005 ◽

Vol 116 (6) ◽

pp. 1364-1371 ◽

Cited By ~ 44

Author(s):

A CIANFERONI ◽

M MASSAAD ◽

S FESKE ◽

M DELAFUENTE ◽

L GALLEGO ◽

...

Keyword(s):

Gene Expression ◽

T Cells ◽

Nuclear Translocation ◽

Nuclear Factor ◽

Activated T Cells ◽

Extracellular Signal Regulated Kinase ◽

Wiskott Aldrich Syndrome ◽

Extracellular Signal

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Hierarchy of signaling thresholds downstream of the T cell receptor and the Tec kinase ITK

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2025825118 ◽

2021 ◽

Vol 118 (35) ◽

pp. e2025825118

Author(s):

Michael P. Gallagher ◽

James M. Conley ◽

Pranitha Vangala ◽

Manuel Garber ◽

Andrea Reboldi ◽

...

Keyword(s):

Gene Expression ◽

T Cells ◽

T Cell ◽

T Cell Receptor ◽

Signal Strength ◽

Cell Receptor ◽

Early Gene ◽

Sequencing Analysis ◽

Nuclear Factor ◽

Activated T Cells

The strength of peptide:MHC interactions with the T cell receptor (TCR) is correlated with the time to first cell division, the relative scale of the effector cell response, and the graded expression of activation-associated proteins like IRF4. To regulate T cell activation programming, the TCR and the TCR proximal interleukin-2–inducible T cell kinase (ITK) simultaneously trigger many biochemically separate signaling cascades. T cells lacking ITK exhibit selective impairments in effector T cell responses after activation, but under the strongest signaling conditions, ITK activity is dispensable. To gain insight into whether TCR signal strength and ITK activity tune observed graded gene expression through the unequal activation of distinct signaling pathways, we examined Erk1/2 phosphorylation or nuclear factor of activated T cells (NFAT) and nuclear factor (NF)-κB translocation in naïve OT-I CD8+ cell nuclei. We observed the consistent digital activation of NFAT1 and Erk1/2, but NF-κB displayed dynamic, graded activation in response to variation in TCR signal strength, tunable by treatment with an ITK inhibitor. Inhibitor-treated cells showed the dampened induction of AP-1 factors Fos and Fosb, NF-κB response gene transcripts, and survival factor Il2 transcripts. ATAC sequencing analysis also revealed that genomic regions most sensitive to ITK inhibition were enriched for NF-κB and AP-1 motifs. Specific inhibition of NF-κB during peptide stimulation tuned the expression of early gene products like c-Fos. Together, these data indicate a key role for ITK in orchestrating the optimal activation of separate TCR downstream pathways, specifically aiding NF-κB activation. More broadly, we revealed a mechanism by which variations in TCR signal strength can produce patterns of graded gene expression in activated T cells.

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