Nuclear Factor Of Activated T-cells Isoforms Research Articles

Targeting CaN/NFAT in Alzheimer's brain degeneration.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by a progressive loss of cognitive functions. While the exact causes of this debilitating disorder remain elusive, numerous investigations have characterized its two core pathologies: the presence of β-amyloid plaques and tau tangles. Additionally, multiple studies of postmortem brain tissue, as well as results from AD preclinical models, have consistently demonstrated the presence of a sustained inflammatory response. As the persistent immune response is associated with neurodegeneration, it became clear that it may also exacerbate other AD pathologies, providing a link between the initial deposition of β-amyloid plaques and the later development of neurofibrillary tangles. Initially discovered in T cells, the nuclear factor of activated T-cells (NFAT) is one of the main transcription factors driving the expression of inflammatory genes and thus regulating immune responses. NFAT-dependent production of inflammatory mediators is controlled by Ca2+-dependent protein phosphatase calcineurin (CaN), which dephosphorylates NFAT and promotes its transcriptional activity. A substantial body of evidence has demonstrated that aberrant CaN/NFAT signaling is linked to several pathologies observed in AD, including neuronal apoptosis, synaptic deficits, and glia activation. In view of this, the role of NFAT isoforms in AD has been linked to disease progression at different stages, some of which are paralleled to diminished cognitive status. The use of classical inhibitors of CaN/NFAT signaling, such as tacrolimus or cyclosporine, or adeno-associated viruses to specifically inhibit astrocytic NFAT activation, has alleviated some symptoms of AD by diminishing β-amyloid neurotoxicity and neuroinflammation. In this article, we discuss the recent findings related to the contribution of CaN/NFAT signaling to the progression of AD and highlight the possible benefits of targeting this pathway in AD treatment.

Hepatic NFAT signaling regulates the expression of inflammatory cytokines in cholestasis

The nuclear factor of activated T-cells (NFAT) plays an important role in immune responses by regulating the expression of inflammatory genes. However, it is not known whether NFAT plays any role in the bile acid (BA)-induced hepatic inflammatory response. Thus, we aimed to examine the functional role of NFATc3 in cholestatic liver injury in mice and humans. Gene and protein expression and cellular localization were assessed in primary hepatocyte cultures (mouse and human) and cholestatic liver tissues (murine models and patients with primary biliary cholangitis [PBC] or primary sclerosing cholangitis [PSC]) by quantitative PCR, western blot and immunohistochemistry. Specific NFAT inhibitors were used invivo and invitro. Gene reporter assays and ChIP-PCR were used to determine promoter activity. NFAT isoforms c1 and c3 were expressed in human and mouse hepatocytes. When treated with cholestatic levels of BAs, nuclear translocation of NFATc3 was increased in both human and mouse hepatocytes and was associated with elevated mRNA levels of IL-8, CXCL2, and CXCL10 in these cells. Blocking NFAT activation with pathway-specific inhibitors or knocking down Nfatc3 expression significantly decreased BA-driven induction of these cytokines in mouse hepatocytes. Nuclear expression of NFATc3/Nfatc3 protein was increased in cholestatic livers, both in mouse models (bile duct ligation or Abcb4-/- mice) and in patients with PBC and PSC in association with elevated tissue levels of Cxcl2 (mice) or IL-8 (humans). Gene reporter assays and ChIP-PCR demonstrated that the NFAT response element in the IL-8 promoter played a key role in BA-induced human IL-8 expression. Finally, blocking NFAT activation invivo in Abcb4-/- mice reduced cholestatic liver injury. NFAT plays an important role in BA-stimulated hepatic cytokine expression in cholestasis. Blocking hepatic NFAT activation may reduce cholestatic liver injury in humans. Bile acid induces liver injury by stimulating the expression of inflammatory genes in hepatocytes through activation of the transcription factor NFAT. Blocking this activation invitro (in hepatocyte cultures) and invivo (in cholestatic mice) decreased the expression of inflammatory genes and reduced liver injury.

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.

NFATc3 and VIP in Idiopathic Pulmonary Fibrosis and Chronic Obstructive Pulmonary Disease.

Idiopathic pulmonary fibrosis (IPF) and chronic obstructive pulmonary disease (COPD) are both debilitating lung diseases which can lead to hypoxemia and pulmonary hypertension (PH). Nuclear Factor of Activated T-cells (NFAT) is a transcription factor implicated in the etiology of vascular remodeling in hypoxic PH. We have previously shown that mice lacking the ability to generate Vasoactive Intestinal Peptide (VIP) develop spontaneous PH, pulmonary arterial remodeling and lung inflammation. Inhibition of NFAT attenuated PH in these mice suggesting a connection between NFAT and VIP. To test the hypotheses that: 1) VIP inhibits NFAT isoform c3 (NFATc3) activity in pulmonary vascular smooth muscle cells; 2) lung NFATc3 activation is associated with disease severity in IPF and COPD patients, and 3) VIP and NFATc3 expression correlate in lung tissue from IPF and COPD patients. NFAT activity was determined in isolated pulmonary arteries from NFAT-luciferase reporter mice. The % of nuclei with NFAT nuclear accumulation was determined in primary human pulmonary artery smooth muscle cell (PASMC) cultures; in lung airway epithelia and smooth muscle and pulmonary endothelia and smooth muscle from IPF and COPD patients; and in PASMC from mouse lung sections by fluorescence microscopy. Both NFAT and VIP mRNA levels were measured in lungs from IPF and COPD patients. Empirical strategies applied to test hypotheses regarding VIP, NFATc3 expression and activity, and disease type and severity. This study shows a significant negative correlation between NFAT isoform c3 protein expression levels in PASMC, activity of NFATc3 in pulmonary endothelial cells, expression and activity of NFATc3 in bronchial epithelial cells and lung function in IPF patients, supporting the concept that NFATc3 is activated in the early stages of IPF. We further show that there is a significant positive correlation between NFATc3 mRNA expression and VIP RNA expression only in lungs from IPF patients. In addition, we found that VIP inhibits NFAT nuclear translocation in primary human pulmonary artery smooth muscle cells (PASMC). Early activation of NFATc3 in IPF patients may contribute to disease progression and the increase in VIP expression could be a protective compensatory mechanism.

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.

Transcriptional regulation of BACE1 by NFAT3 leads to enhanced amyloidogenic processing.

Deposition of amyloid β (Aβ) to form neuritic plaques in the brain is the pathological hallmark of Alzheimer's disease (AD). Aβ is generated from sequential cleavages of the β-amyloid precursor protein (APP) by the β- and γ-secretases, and β-site APP-cleaving enzyme 1 (BACE1) is the essential β-secretase for Aβ generation. Vulnerable regions in AD brains show increased BACE1 protein levels. However, the underlying mechanism how BACE1 is regulated remains to be further illustrated. Nuclear Factor of Activated T-cells (NFAT) has been implicated in AD pathogenesis. Despite the increasing appreciation for the importance of NFAT-dependent transcription in the nervous system, the regulation and function of specific NFAT isoforms in neurons is poorly understood. In this report we found that both BACE1 and NFAT3 levels were significantly increased in the brains of APP/PS1 transgenic mice. We found that overexpression of NFAT3 resulted in increase of BACE1 promoter activity and BACE1 transcription, while disruption of NFAT3 expression decreased BACE1 gene transcription and protein expression in SAS1 cells. In a addition, overexpression of NFAT3 leads to increase levels of Aβ production. Chromatin immunoprecipitation analysis revealed direct binding of NFAT3 to specific DNA sequences within BACE1 promoter region. Taken together, our results indicate that NFAT is a BACE1 transcription factor. Our study suggests that inhibition of NFAT-mediated BACE1 expression may be a valuable drug target for AD therapy.

The role of NFATc1 in prostate cancer progression: cyclosporine A and tacrolimus inhibit cell proliferation, migration, and invasion.

The functional role of nuclear factor of activated T-cells (NFAT), a well-characterized regulator of the immune response, in prostate cancer progression remains largely unknown. We aim to investigate biological significance of NFATc1, a NFAT isoform shown to function as an oncogene in a sarcoma model, in human prostate cancer. We first determined the expression levels of NFAT in prostate cell lines and tissue specimens. We then assessed the effects of NFAT inhibition via NFATc1-small interfering RNA (siRNA) as well as immunosuppressants including cyclosporine A (CsA) and tacrolimus (FK506) on prostate cancer cell proliferation, apoptosis, migration, and invasion in vitro and in vivo. Immunohistochemistry revealed that the expression levels of NFATc1 were significantly elevated in prostatic carcinomas, compared with non-neoplastic prostate or high-grade prostatic intraepithelial neoplasia tissues, and in high-grade (Gleason scores ≥7) tumors. NFATc1 positivity in carcinomas, as an independent prognosticator, also correlated with the risk of biochemical recurrence after radical prostatectomy. In prostate cancer cell lines, CsA and FK506 inhibited NFATc1 expression and its nuclear translocation, NFAT transcriptional activity, and the expression of c-myc, a downstream target of NFAT. NFAT silencing or treatment with these NFAT inhibitors resulted in decreases in cell viability/colony formation and cell migration/invasion, as well as increases in apoptosis, in androgen receptor (AR)-negative, AR-positive/androgen-sensitive, and AR-positive/castration-resistant lines. No significant additional inhibition in the growth of NFAT-siRNA cells by CsA and FK506 was seen, whereas these agents, especially FK506, further inhibited their invasion. In xenograft-bearing mice, CsA and FK506 significantly retarded tumor growth. Our results suggest that NFATc1 plays an important role in prostate cancer outgrowth. Thus, NFATc1 inactivation, especially using CsA and FK506, has the potential of being a therapeutic approach for not only hormone-naïve but also castration-resistant prostate cancers.

Cyclosporine A and tacrolimus inhibit urothelial tumorigenesis.

The functional role of nuclear factor of activated T-cells (NFAT), while it has been extensively investigated in the immune system, remains uncertain in bladder cancer development. We here aim to assess the effects of cyclosporine A (CsA) and tacrolimus (FK506), immunosuppressants known to specifically inactivate the NFAT pathway in immune cells, on neoplastic transformation of urothelial cells. Immunohistochemistry revealed that the expression levels of NFATc1, a NFAT isoform shown to function as an oncogene in a sarcoma model, were elevated in urothelial neoplasms, compared with non-neoplastic urothelial tissues, and in low-grade and high-grade papillary urothelial carcinomas, compared with papillary urothelial neoplasms of low malignant potential. In an immortalized normal urothelial cell line SVHUC, CsA and FK506 reduced NFATc1 expression, NFAT transcriptional activity, and the expression of c-myc, a downstream target of NFATc1 signals. Treatment with CsA or FK506 in the SVHUC cells undergoing neoplastic transformation induced by exposure to a chemical carcinogen 3-methylcholanthrene resulted in strong inhibition in colony formation in vitro as well as tumor formation in NOD-SCID mice. CsA and FK506 were additionally found to up-regulate the expression of several molecules that play a protective role in bladder tumorigenesis, including p53, p21, and p27, and down-regulate that of oncogenic genes, such as cyclin D1, cyclin D3, and cyclin E, in SVHUC cells with the carcinogen challenge. Thus, CsA and FK506 likely inhibit urothelial tumorigenesis. These findings offer a potential chemopreventive approach for urothelial tumors using NFAT inhibitors.

NFAT transcription factor regulation by urocortin II in cardiac myocytes and heart failure

Urocortin II (UcnII), a cardioactive peptide with beneficial effects in normal and failing hearts, is also arrhythmogenic and prohypertrophic. We demonstrated that cardiac effects are mediated by a phosphatidylinositol-3 kinase (PI3K)/Akt kinase (Akt)/endothelial nitric oxide synthase (eNOS)/nitric oxide (NO) signaling pathways. Nuclear factor of activated T-cells (NFAT) transcription factors play a key role in the regulation of gene expression in cardiac development, maintenance of an adult differentiated cardiac phenotype, and remodeling processes in cardiac hypertrophy and heart failure (HF). We tested the hypothesis that UcnII differentially regulates NFAT activity in cardiac myocytes from both normal and failing hearts through the PI3K/Akt/eNOS/NO pathway. Isoforms NFATc1 and NFATc3 revealed different basal subcellular distribution in normal and HF rabbit ventricular myocytes with a nuclear NFATc1 and a cytosolic localization of NFATc3. However, in HF, the nuclear localization of NFATc1 was less pronounced, whereas the nuclear occupancy of NFATc3 was increased. In normal myocytes, UcnII induced nuclear export of NFATc1 and attenuated NFAT-dependent transcriptional activity but did not affect the distribution of NFATc3. In HF UcnII facilitated nuclear export of both isoforms and reduced transcriptional activity. NFAT regulation was mediated by a PI3K/Akt/eNOS/NO signaling cascade that converged on the activation of several kinases, including glycogen synthase kinase-3β (GSK3β), c-Jun NH2-terminal kinase (JNK), p38 mitogen-activated kinase (p38), and PKG, resulting in phosphorylation, deactivation, and nuclear export of NFAT. In conclusion, while NFATc1 and NFATc3 reveal distinct subcellular distribution patterns, both are regulated by the UcnII-PI3K/Akt/eNOS/NO pathway that converges on the activation of NFAT kinases and NFAT inactivation. The data reconcile cardioprotective and prohypertrophic UcnII effects mediated by different NFAT isoforms.

Digitoxin induces apoptosis in cancer cells by inhibiting nuclear factor of activated T-cells-driven c-MYC expression

Background:Cardiac glycosides such as digitoxin have been shown to directly cause apoptotic death of cancer cells both in vitro, and in vivo. However, the mechanism connecting cardiac glycoside action to apoptosis is not known. It has been reported that compounds resembling digitoxin are able to reduce c-MYC expression. Furthermore, it has been previously shown that the transcription of c-MYC depends on nuclear factor of activated T-cells (NFAT) binding sites in the c-MYC promoter. We have therefore hypothesized that NFAT might mediate digitoxin effects on c-MYC mRNA message.Materials and Methods:We have chosen to study this process in HeLa cells where structurally intact c-MYC genes in 8q24 co-localize with human papilloma virus 18 at all integration sites.Results:Here we show that within the 1st h following treatment with digitoxin, a significant reduction in c-MYC mRNA occurs. This is followed by a precipitous loss of c-MYC protein, activation of caspase 3, and subsequent apoptotic cell death. To test the NFAT-dependence mechanism, we analyzed the effects of digitoxin on NFAT isoform-dependent auto-activation of a NFAT-luciferase expression system. Drug dependent effects on expression varied according to each of the four canonical NFAT isoforms (1, 2, 3 or 4). The most digitoxin-sensitive NFAT isoform was NFAT1. Using c-MYC chromatin immune precipitation, we find that digitoxin inhibits interaction of NFAT1 with the proximal c-MYC promoter.Conclusions:These results suggest that the carcinotoxic activity of digitoxin includes suppression of NFAT-driven c-MYC expression.

Distinct Activation Properties of the Nuclear Factor of Activated T-cells (NFAT) Isoforms NFATc3 and NFATc4 in Neurons

The Ca(2+)/calcineurin-dependent transcription factor NFAT (nuclear factor of activated T-cells) is implicated in regulating dendritic and axonal development, synaptogenesis, and neuronal survival. Despite the increasing appreciation for the importance of NFAT-dependent transcription in the nervous system, the regulation and function of specific NFAT isoforms in neurons are poorly understood. Here, we compare the activation of NFATc3 and NFATc4 in hippocampal and dorsal root ganglion neurons following electrically evoked elevations of intracellular Ca(2+) concentration ([Ca(2+)](i)). We find that NFATc3 undergoes rapid dephosphorylation and nuclear translocation that are essentially complete within 20 min, although NFATc4 remains phosphorylated and localized to the cytosol, only exhibiting nuclear localization following prolonged (1-3 h) depolarization. Knocking down NFATc3, but not NFATc4, strongly diminished NFAT-mediated transcription induced by mild depolarization in neurons. By analyzing NFATc3/NFATc4 chimeras, we find that the region containing the serine-rich region-1 (SRR1) mildly affects initial NFAT translocation, although the region containing the serine-proline repeats is critical for determining the magnitude of NFAT activation and nuclear localization upon depolarization. Knockdown of glycogen synthase kinase 3β (GSK3β) significantly increased the depolarization-induced nuclear localization of NFATc4. In contrast, inhibition of p38 or mammalian target of rapamycin (mTOR) kinases had no significant effect on nuclear import of NFATc4. Thus, electrically evoked [Ca(2+)](i) elevation in neurons rapidly and strongly activates NFATc3, whereas activation of NFATc4 requires a coincident increase in [Ca(2+)](i) and suppression of GSK3β, with differences in the serine-proline-containing region giving rise to these distinct activation properties of NFATc3 and NFATc4.

The NFATc2 transcription factor is required for normal heart function and signaling in adult mice

The Ca2+- dependent phosphatase, calcineurin (Cn) and its downstream transcriptional effectors, nuclear factor of activated T-cells (NFAT), are major intracellular modulators of cardiac hypertrophy. In young mice, transcription factor NFATc2 has been identified as the major NFAT isoform responsible for Cn-mediated cardiac hypertrophy. We observed that 7–9 month old NFATc2−/− mice have a higher susceptibility to cardiac dilatation and sudden death, prompting us to monitor changes in heart morphology and growth pathways in this model. Using histology, we show that hearts of adult NFATc2−/− mice display a more dilated left ventricle and thinner interventricular wall. In addition, NFATc2 null hearts have increased GATA-4 expression, higher nuclear transit of GATA-4 and NFATc1, and decreased GSK3-β expression, thus supporting the finding that adult NFATc2−/− mice are more prone to heart failure. Furthermore, angiotensin II-induced cardiac growth reveals that hearts of NFATc2−/− mice hypertrophy to the same extent as wild-type mice, display a thinner myocardial wall and have decreased active AKT and eIF2α expression, suggesting altered translational regulation of myocardial growth. Our collective results propose an uncharacterized role of NFATc2 for normal heart function and growth signaling in adult mice, providing evidence of Cn-signaling being crucial at later stages of life. Supported by CIHR, NSERC and CRC to RNM.

Response of transcription factor NFATc3 to excitotoxic and traumatic brain insults: Identification of a subpopulation of reactive astrocytes

Astrocytes react to brain injury triggering neuroinflammatory processes that determine the degree of neuronal damage. However, the signaling events associated to astrocyte activation remain largely undefined. The nuclear factor of activated T-cells (NFAT) is a transcription factor family implicated in activation of immune cells. We previously characterized the expression of NFAT isoforms in cultured astrocytes, and NFAT activation in response to mechanical lesion. Here we analyze NFATc3 in two mouse models of inflammatory brain damage: hippocampal excitotoxicity induced by intracerebral kainic acid (KA) injection and cortical mechanical lesion. Immunofluorescence results demonstrated that NFATc3 is specifically induced in a subset of reactive astrocytes, and not in microglia or neurons. In KA-treated brains, NFATc3 expression is transient and NFATc3-positive astrocytes concentrate around damaged neurons in areas CA3 and CA1. Complementary Western blot and RT-PCR analysis revealed an NFAT-dependent induction of RCAN1-4 and COX-2 in hippocampus as soon as 6 h after KA exposure, indicating that NFAT activation precedes NFATc3 over-expression. Moreover, activation of NFAT by ATP increased NFATc3 mRNA levels in astrocyte cultures, suggesting that NFATc3 expression is controlled through an auto-regulatory loop. Meanwhile, stab wound enhanced NFATc3 expression specifically in a subclass of reactive astrocytes confined within the proximal layer of the glial scar, and GFAP immunoreactivity was attenuated in NFATc3-expressing astrocytes. In conclusion, our work establishes NFATc3 as a marker of activation for a specific population of astrocytes in response to brain damage, which may have consequences for neuronal survival.

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.

TECHNIQUES FOR PHYSIOLOGY: A fluorescence-based assay to monitor transcriptional activity of NFAT in living cells

Ca(2+)-sensitive NFAT (nuclear factor of activated T-cells) transcription factors are implicated in many pathophysiological processes in different cell types. The precise control of activation varies with NFAT isoform and cell type. Here we present feasibility of an in vivo assay (NFAT-RFP) that reports transcriptional activity of NFAT via expression of red fluorescent protein (RFP) in individual cells. This new tool allows continuous monitoring of transcriptional activity of NFAT in a physiological context in living cells. Furthermore, NFAT-RFP can be used simultaneously with NFAT-GFP fusion proteins to monitor transcriptional activity and subcellular localization of NFAT in the same cell.

Requirement of Nuclear Factor of Activated T-cells in Calcineurin-mediated Cardiomyocyte Hypertrophy

The calcium-activated phosphatase calcineurin has been implicated as a critical intracellular signal transducer of cardiomyocyte hypertrophy. Although previous data suggested the nuclear factor of activated T-cells (NFAT) as its sole transcriptional effector, the absolute requirement of NFAT as a mediator of calcineurin signaling has not been examined in the heart. We therefore investigated the expression and activation profile of NFAT genes in the heart. Four members (NFATc1-c4) are expressed in cardiomyocytes, elicit nuclear translocation upon calcineurin activation, and are able to drive transactivation of cardiac promoter luciferase constructs. To define the necessary function of NFAT factors as hypertrophic transducers, a dominant negative NFAT construct was created, encompassing part of the N-terminal region of NFATc4 containing a conserved calcineurin-binding motif. Cotransfection of this construct dose-dependently abrogated promoter activation, irrespective of the NFAT isoform used, whereas a control construct with the calcineurin-binding motif mutated displayed no such effects. Adenoviral gene transfer of dominant negative NFAT rendered cardiomyocytes resistant toward all aspects of calcineurin or agonist-induced cardiomyocyte hypertrophy, whereas adenoviral gene transfer of the control construct had no discernable effect on these parameters. These results indicate that multiple NFAT isoforms are expressed in cardiomyocytes where they function as necessary transducers of calcineurin in facilitating cardiomyocyte hypertrophy.

Opposing Actions of Inositol 1,4,5-Trisphosphate and Ryanodine Receptors on Nuclear Factor of Activated T-cells Regulation in Smooth Muscle

The nuclear factor of activated T-cells (NFAT), originally identified in T-cells, has since been shown to play a role in mediating Ca(2+)-dependent gene transcription in diverse cell types outside of the immune system. We have previously shown that nuclear accumulation of NFATc3 is induced in ileal smooth muscle by platelet-derived growth factor in a manner that depends on Ca(2+) influx through L-type, voltage-dependent Ca(2+) channels. Here we show that NFATc3 is also the predominant NFAT isoform expressed in cerebral artery smooth muscle and is induced to accumulate in the nucleus by UTP and other G(q/11)-coupled receptor agonists. This induction is mediated by calcineurin and is dependent on sarcoplasmic reticulum Ca(2+) release through inositol 1,4,5-trisphosphate receptors and extracellular Ca(2+) influx through L-type, voltage-dependent Ca(2+) channels. Consistent with results obtained in ileal smooth muscle, depolarization-induced Ca(2+) influx fails to induce NFAT nuclear accumulation in cerebral arteries. We also provide evidence that Ca(2+) release by ryanodine receptors in the form of Ca(2+) sparks may exert an inhibitory influence on UTP-induced NFATc3 nuclear accumulation and further suggest that UTP may act, in part, by inhibiting Ca(2+) sparks. These results are consistent with a multifactorial regulation of NFAT nuclear accumulation in smooth muscle that is likely to involve several intracellular signaling pathways, including local effects of sarcoplasmic reticulum Ca(2+) release and effects attributable to global elevations in intracellular Ca(2+).