Nuclear Factor Of Activated T-cells Proteins Research Articles

Near-UV Light Induced ROS Production Initiates Spatial Ca2+ Spiking to Fire NFATc3 Translocation.

Ca2+-dependent gene regulation controls several functions to determine the fate of the cells. Proteins of the nuclear factor of activated T-cells (NFAT) family are Ca2+ sensitive transcription factors that control the cell growth, proliferation and insulin secretion in β-cells. Translocation of NFAT proteins to the nucleus occurs in a sequence of events that starts with activating calmodulin-dependent phosphatase calcineurin in a Ca2+-dependent manner, which dephosphorylates the NFAT proteins and leads to their translocation to the nucleus. Here, we examined the role of IP3-generating agonists and near-UV light in the induction of NFATc3 migration to the nucleus in the pancreatic β-cell line INS-1. Our results show that IP3 generation yields cytosolic Ca2+ rise and NFATc3 translocation. Moreover, near-UV light exposure generates reactive oxygen species (ROS), resulting in cytosolic Ca2+ spiking via the L-type Ca2+ channel and triggers NFATc3 translocation to the nucleus. Using the mitochondria as a Ca2+ buffering tool, we showed that ROS-induced cytosolic Ca2+ spiking, not the ROS themselves, was the triggering mechanism of nuclear import of NFATc3. Collectively, this study reveals the mechanism of near-UV light induced NFATc3 migration.

Sumoylation regulates the transcriptional activity of different human NFAT isoforms in neurons.

In the nervous system, four calcium/calcineurin-regulated members of the nuclear factor of activated T-cells (NFAT) family of transcription factors, NFATc1-c4, are involved in many developmental and functional processes, such as corticogenesis, synaptogenesis, synaptic plasticity and neurotransmission, that all need precise gene regulation. Therefore it is important to understand molecular events that contribute to the regulation of the transcriptional activity of specific NFAT isoforms. Previously, we have shown that there are a number of alternative splice variants of NFAT genes expressed in the brain and that neuronal activity leads to isoform-specific transactivation capacities of different human NFAT proteins. Here we looked at the effect of sumoylation as a possible regulator of the transcriptional activity of different human NFAT isoforms in rat primary cortical and hippocampal neurons in response to membrane depolarization and compared the results to those obtained from non-neuronal HEK293-FT and BHK-21 cells in response to calcium signaling. Our results show that in primary hippocampal neurons, sumoylation represses the transcriptional activity of NFATc1, NFATc2, and NFATc3 isoforms, whereas in cortical neurons, transactivation capacity of only NFATc1 and NFATc2 is repressed by sumoylation. In non-neuronal cells, however, transcriptional activity of all four NFAT isoforms is repressed by sumoylation in HEK293-FT cells, while only NFATc1 and NFATc2 isoforms are affected by sumoylation in BHK-21 cells. Altogether, our results show that sumoylation represses the transcription activation capacities of NFAT isoforms and that the effect is cell type-specific.

Regulation of different human NFAT isoforms by neuronal activity.

Nuclear factor of activated T-cells (NFAT) is a family of transcription factors comprising four calcium-regulated members: NFATc1, NFATc2, NFATc3, and NFATc4. Upon activation by the calcium-dependent phosphatase calcineurin (CaN), NFATs translocate from cytosol to the nucleus and regulate their target genes, which in the nervous system are involved in axon growth, synaptic plasticity, and neuronal survival. We have shown previously that there are a number of different splice variants of NFAT genes expressed in the brain. Here, we studied the subcellular localizations and transactivation capacities of alternative human NFAT isoforms in rat primary cortical or hippocampal neurons in response to membrane depolarization and compared the induced transactivation levels in neurons to those obtained from HEK293 cells in response to calcium signaling. We confirm that in neurons the translocation to the nucleus of all NFAT isoforms is reliant on the activity of CaN. However, our results suggest that both the regulation of subcellular localization and transcriptional activity of NFAT proteins in neurons is isoform specific. We show that in primary hippocampal neurons NFATc2 isoforms have very fast translocation kinetics, whereas NFATc4 isoforms translocate relatively slowly to the nucleus. Moreover, we demonstrate that the strongest transcriptional activators in HEK293 cells are NFATc1 and NFATc3, but in neurons NFATc3 and NFATc4 lead to the highest induction, and NFATc2 and NFATc1 display isoform-specific transcription activation capacities. Altogether, our results indicate that the effects of calcium signaling on the action of NFAT proteins are isoform-specific and can differ between cell types. We show that the effects of calcium signaling on the action of NFAT proteins are isoform-specific and differ between cell types. Although nuclear localization of all NFAT isoforms in neurons requires calcineurin, the subcellular distributions, neuronal activity-induced nuclear translocation extent and kinetics, and transcription activation capacities of alternative NFAT proteins vary.

A novel role for an RCAN3-derived peptide as a tumor suppressor in breast cancer.

The members of the human regulators of calcineurin (RCAN) protein family are endogenous regulators of the calcineurin (CN)-cytosolic nuclear factor of activated T-cells (NFATc) pathway activation. This function is explained by the presence of a highly conserved calcipressin inhibitor of calcineurin (CIC) motif in RCAN proteins, which has been shown to compete with NFATc for the binding to CN and therefore are able to inhibit NFATc dephosphorylation and activation by CN. Very recently, emerging roles for NFATc proteins in transformation, tumor angiogenesis and metastasis have been described in different cancer cell types. In this work, we report that the overexpression of RCAN3 dramatically inhibits tumor growth and tumor angiogenesis in an orthotopic human breast cancer model. We suggest that RCAN3 exerts these effects in a CN-dependent manner, as mutation of the CIC motif in RCAN3 abolishes the tumor suppressor effect. Moreover, the expression of the EGFP-R3(178-210) peptide, spanning the CIC motif of RCAN3, is able to reproduce all the antitumor effects of RCAN3 full-length protein. Finally, we show that RCAN3 and the EGFP-R3(178-210) peptide inhibit the CN-NFATc signaling pathway and the induction of the NFATc-dependent gene cyclooxygenase-2. Our work suggests that the EGFP-R3(178-210) peptide possess potent tumor suppressor properties and therefore constitutes a novel lead for the development of potent and specific antitumoral agents. Moreover, we propose the targeting of the CN-NFATc pathway in the tumor cells constitutes an effective way to hamper tumor progression by impairing the paracrine network among tumor, endothelial and polymorphonucleated cells.

Ets-1 facilitates nuclear entry of NFAT proteins and their recruitment to the IL-2 promoter

E26 transformation-specific sequence 1 (Ets-1), the prototype of the ETS family of transcription factors, is critical for the expression of IL-2 by murine Th cells; however, its mechanism of action is still unclear. Here we show that Ets-1 is also essential for optimal production of IL-2 by primary human Th cells. Although Ets-1 negatively regulates the expression of Blimp1, a known suppressor of IL-2 expression, ablation of B lymphocyte-induced maturation protein 1 (Blimp1) does not rescue the expression of IL-2 by Ets-1-deficient Th cells. Instead, Ets-1 physically and functionally interacts with the nuclear factor of activated T-cells (NFAT) and is required for the recruitment of NFAT to the IL-2 promoter. In addition, Ets-1 is located in both the nucleus and cytoplasm of resting Th cells. Nuclear Ets-1 quickly exits the nucleus in response to calcium-dependent signals and competes with NFAT proteins for binding to protein components of noncoding RNA repressor of NFAT complex (NRON), which serves as a cytoplasmic trap for phosphorylated NFAT proteins. This nuclear exit of Ets-1 precedes rapid nuclear entry of NFAT and Ets-1 deficiency results in impaired nuclear entry, but not dephosphorylation, of NFAT proteins. Thus, Ets-1 promotes the expression of IL-2 by modulating the activity of NFAT.

Identification of Two Nuclear Factor of Activated T-cells (NFAT)-response Elements in the 5′-Upstream Regulatory Region of the ET-1 Promoter

Collecting duct-derived ET-1 regulates salt excretion and blood pressure. We have reported the presence of an inner medullary collecting duct (IMCD)-specific enhancer region in the 5'-upstream ET-1 promoter (Strait, K. A., Stricklett, P. K., Kohan, J. L., Miller, M. B., and Kohan, D. E. (2007) Am. J. Physiol. Renal Physiol. 293, F601-F606). The current studies provide further characterization of the ET-1 5'-upstream distal promoter to identify the IMCD-specific enhancer elements. Deletion studies identified two regions of the 5'-upstream ET-1 promoter, -1725 to -1319 bp and -1319 to -1026 bp, which were required for maximal promoter activity in transfected rat IMCD cells. Transcription factor binding site analysis of these regions identified two consensus nuclear factor of activated T-cells (NFAT) binding sites at -1263 and -1563. EMSA analysis using nuclear extracts from IMCD cells showed that both the -1263 and the -1563 NFAT sites in the ET-1 distal promoter competed for NFAT binding to previously identified NFAT sites in the IL-2 and TNF genes. Gel supershift analysis showed that each of the NFAT binding sites in the ET-1 promoter bound NFAT proteins derived from IMCD nuclear extracts, but they selectively bound different NFAT isoforms; ET-1263 bound NFATc1, whereas ET-1563 bound NFATc3. Site-directed mutagenesis of either the ET-1263 or the ET-1563 sites prevented NFAT binding and reduced ET-1 promoter activity. Thus, NFAT appears to be an important regulator of ET-1 transcription in IMCD cells, and thus, it may play a role in controlling blood pressure through ET-1 regulation of renal salt excretion.

Nuclear factor of activated T-cells transcription factors in the vasculature: the good guys or the bad guys?

The nuclear factor of activated T-cells (NFAT) proteins are a family of Ca/calcineurin-dependent transcription factors that were first characterized in T-lymphocytes as inducers of cytokine gene expression. Since then, NFAT proteins have been shown to play varied roles outside of the immune system, including in the cardiovascular system. Cells in the vessel wall display a diverse array of Ca signaling modalities, which are subject to change during disease. The fact that NFAT proteins are able to decode and translate these signals into changes in gene expression makes them potential regulators of vascular pathogenesis. It is now clear that NFAT signaling is required for normal vascular patterning during embryogenesis and for vascular endothelial growth factor-induced angiogenesis. The overall role of NFAT signaling in the vasculature, however, is less clear during adult life. This review aims to give an update on mechanisms that regulate NFAT activation in vascular cells, with an emphasis on the role of mitochondria and of upstream activators such as lipids and glucose. It also addresses recent work implicating NFAT proteins as mediators of vascular disease. A better understanding of the NFAT-signaling pathway in the vasculature may open up an unexplored area for the development of new therapeutic approaches for treating vascular disease.

Dimer Composition and Promoter Context Contribute to Functional Cooperation between AP-1 and NFAT

The transcription factors activator protein 1 (AP-1) and nuclear factor of activated T-cells (NFAT) cooperate to induce the expression of cytokines during the immune response. While much is known about the signaling pathways and physical interactions between NFAT and AP-1 dimers following lymphocyte activation, few studies have addressed the role of AP-1 composition in modulating NFAT:AP-1-dependent transcription. We examined the function of specific AP-1 complexes using “tethered” AP-1 dimers with defined composition. We found that NFAT can functionally cooperate with all AP-1 dimers tested. Noteworthy, Jun∼Jun-containing dimers, which are relatively inactive when tested on an AP-1-dependent promoter, are effective co-activators of an NFAT:AP-1-dependent promoter. Interestingly, specific AP-1 dimer combinations behave differently when tested on interleukin 2 ( IL2) and interleukin 4 ( IL4) gene regulatory regions. Moreover, the requirement for NFAT to activate each of the promoters is different. Our results suggest that higher NFAT levels are necessary to activate the IL4 promoter. Hence changes in AP-1 composition and the level of participating NFAT proteins can differentially influence cytokine gene expression, resulting in biological consequences for the modulation and dynamics of the immune response.

The Essential Role of MEKK3 Signaling in Angiotensin II-induced Calcineurin/Nuclear Factor of Activated T-cells Activation

Calcineurin is a serine/threonine protein phosphatase that plays a critical role in many physiologic processes, such as T-cell activation, apoptosis, skeletal myocyte differentiation, and cardiac hypertrophy. We determined that active MEKK3 was capable of activating calcineurin/nuclear factor of activated T-cells (NFAT) signaling in cardiac myocytes and reprogramming cardiac gene expression. In contrast, small interference RNA directed against MEKK3 and a dominant negative form of MEKK3 caused the reduction of NFAT activation in response to angiotensin II in cardiac myocytes. Genetic studies showed that MEKK3-deficient mouse embryo fibroblasts failed to activate calcineurin/NFAT in response to angiotensin II, a potent NFAT activator. Conversely, restoring MEKK3 to the MEKK3-deficient cells restored angiotensin II-mediated calcineurin/NFAT activation. We determined that angiotensin II induced MEKK3 phosphorylation. Thus, MEKK3 functions downstream of the AT1 receptor and is essential for calcineurin/NFAT activation. Finally, we determined that MEKK3-mediated activation of calcineurin/NFAT signaling was associated with the phosphorylation of modulatory calcineurin-interacting protein 1 at Ser(108) and Ser(112). Taken together, our studies reveal a previously unrecognized novel essential regulatory role of MEKK3 signaling in calcineurin/NFAT activation.

Identification and Characterization of a Novel Nuclear Factor of Activated T-cells-1 Isoform Expressed in Mouse Brain

The nuclear factor of activated T-cells (NFAT) family transcription factors play a key role in the control of cytokine gene expression in T-cells. Although initially identified in T-cells, recent data have unveiled unanticipated roles for NFATs in the development, proliferation, and differentiation of other tissues. Here we report the identification, cDNA cloning, and functional characterization of a new isoform of NFAT1 highly expressed in mouse brain. This isoform, which we named NFAT1-D, is identical to NFAT1 throughout the N-terminal regulatory domain and the portion of the Rel domain which includes the minimal region required for specific binding to DNA and interaction with AP-1. The homology stops sharply upstream of the 3'-boundary of the Rel homology domain and is followed by a short unique C-terminal region. NFAT1-D was expressed at high levels in all brain districts and was found as a constitutively active transcription complex. Transfection of a NFAT/luciferase reporter in the neuronal cell line PC12, which also expresses NFAT1-D, showed that these cells expressed a constitutive NFAT activity that was enhanced after nerve growth factor-induced differentiation but was resistant to the immunosuppressant cyclosporin A. NFAT1-D was, however, inducibly activated in a cyclosporin A-sensitive manner when expressed in T-cells, suggesting that the activity of NFAT proteins might be controlled by their specific cellular context.