Activation Of NFATc4 Research Articles

Ethanol inhibition of undifferentiated rat neural progenitor cell replication can be prevented by chlorogenic acid via the NFATc4/CSE signaling pathway.

Prenatal ethanol exposure hinders oxidative stress-mediated neuroblast/neural progenitor cell proliferation by inhibiting G1-S transition, a process vital to neocortical development. We previously showed that ethanol elicits this redox imbalance by repressing cystathionine γ-lyase (CSE), the rate-limiting enzyme in the transsulfuration pathway in fetal brain and cultured cerebral cortical neurons. However, the mechanism by which ethanol impacts the CSE pathway in proliferating neuroblasts is not known. We conducted experiments to define the effects of ethanol on CSE regulation and the molecular signaling events that control this vital pathway. This enabled us to develop an intervention to prevent the ethanol-associated cytostasis. Spontaneously immortalized undifferentiated E18 rat neuroblasts from brain cerebral cortex were exposed to ethanol to mimic an acute consumption pattern in humans. We performed loss- and gain-of-function studies to evaluate whether NFATc4 is a transcriptional regulator of CSE. The neuroprotective effects of chlorogenic acid (CGA) against the effects of ethanol were assessed using ROS and GSH/GSSG assays as measures of oxidative stress, transcriptional activation of NFATc4, and expression of NFATc4 and CSE by qRT-PCR and immunoblotting. Ethanol treatment of E18-neuroblast cells elicited oxidative stress and significantly reduced CSE expression with a concomitant decrease in NFATc4 transcriptional activation and expression. In parallel, inhibition of the calcineurin/NFAT pathway by FK506 exaggerated ethanol-induced CSE loss. In contrast, NFATc4 overexpression prevented loss of ethanol-induced CSE. CGA increased and activated NFATc4, amplified CSE expression, rescued ethanol-induced oxidative stress, and averted the cytostasis of neuroblasts by rescuing cyclin D1 expression. These findings demonstrate that ethanol can perturb CSE-dependent redox homeostasis by impairing the NFATc4 signaling pathway in neuroblasts. Notably, ethanol-associated impairments were rescued by genetic or pharmacological activation of NFATc4. Furthermore, we found a potential role for CGA in mitigating the ethanol-related neuroblast toxicity with a compelling connection to the NFATc4/CSE pathway.

Stretching Promotes Wound Contraction Through Enhanced Expression of Endothelin Receptor B and TRPC3 in Fibroblasts.

The conditioned medium from stretched keratinocyte was added to the fibroblast populated collagen lattice. Then, we analyzed the levels of endothelin receptor in the human hypertrophic scar tissue and stretched fibroblasts. To address the function of TRPC3, we have used an overexpression system with the collagen lattice. Finally, the TRPC3 overexpressing fibroblasts were transplanted to mouse dorsal skin, and the rate of skin wound contraction was assessed. Conditioned medium from stretched keratinocytes increased the rate of contraction of fibroblast populated collagen lattice. In human hypertrophic scar and stretched fibroblasts, endothelin receptor type B was increased. Cyclic stretching of TRPC3 overexpressing fibroblasts activated NFATc4, and stretched human fibroblasts showed more activation of NFATc4 in response to ET-1. The wound treated with TRPC3 overexpressing fibroblasts showed more contraction than control wound. These findings suggest that cyclical stretching of wounds have an effect on both keratinocytes and fibroblasts, where keratinocytes secret more ET-1, and fibroblasts develop more sensitivity to ET-1 by expressing more endothelin receptors and TRPC3.

Trypanosoma cruzi down-regulates adiponectin expression in mouse adipocytes via the NFAT signaling pathway

Upon infection by Trypanosoma cruzi, adipocytes adopt a clearly defined inflammatory phenotype with concomitant down-regulation of adiponectin expression, which influences the pathogenesis of Chagas heart disease. Herein, we examined how T. cruzi interferes with transcriptional regulation of adiponectin production in mouse adipocytes. The invading pathogen activates the Ca2+/calcineurin/NFATc4 signaling pathway in 3T3-L1 cells. Parasite-induced early activation of NFATc4 is involved in repressing adiponectin expression through recognition of the specific response element located at (−363 to −344) of the gene promoter. Nuclear import of dephosphorylated NFATc4 and decreased adiponectin levels were further demonstrated in white adipose tissue from acutely infected mice. Our current findings point to better clarify the complex role of adipose tissue in the modulation of inflammatory mechanisms operative during T. cruzi infection.

Targeting NFATc4 attenuates non-alcoholic steatohepatitis in mice

The nuclear factor of activated T-cells (NFAT) family was first recognised to play an important role in the differentiation of T cells, but has since been shown to regulate multiple pathophysiological processes. However, whether it is involved in the pathogenesis of non-alcoholic steatohepatitis (NASH) remains unknown. Hepatic NFATc expression and localisation were analysed in C57BL/6 mice on a methionine-choline-deficient diet, as well as in samples from non-alcoholic fatty liver disease patients. Gain- or loss-of-function approaches were used to investigate the role of NFATc4 in NASH. NFATc4 translocates from the cytoplasm to the nucleus in hepatocytes of both humans and rodents with NASH. NFATc4 knockdown resulted in decreased hepatic steatosis, inflammation, and fibrosis during NASH progression. Mechanistically, we found that activated NFATc4 directly bound to peroxisome proliferator-activated receptor α (PPARα) in the nucleus and negatively regulated its transcriptional activity, thereby impairing the hepatic fatty acid oxidation pathway and increasing lipid deposition in the liver. Moreover, NFATc4 activation increased the production and secretion of osteopontin (OPN) from hepatocytes, which subsequently enhanced the macrophage-mediated inflammatory response and hepatic stellate cell-mediated fibrosis progression via paracrine signalling. Hepatic NFATc4 activation accelerates the progression of NASH by suppressing PPARα signalling and increasing OPN expression. Genetic or pharmacological inhibition of NFATc4 may have potential for future therapy of NASH. NFATc4 is activated in the non-alcoholic steatohepatitis of mice and patients. Inhibition of NFATc4 activation alleviates lipid deposition, inflammatory response, and fibrosis progression in the liver.

Nfatc4 Deficiency Attenuates Ototoxicity by Suppressing Tnf-Mediated Hair Cell Apoptosis in the Mouse Cochlea.

The loss of sensory hair cells in the cochlea is the major cause of sensorineural hearing loss, and inflammatory processes and immune factors in response to cochlear damage have been shown to induce hair cell apoptosis. The expression and function of Nfatc4 in the cochlea remains unclear. In this study, we investigated the expression of Nfatc4 in the mouse cochlea and explored its function using Nfatc4−/− mice. We first showed that Nfatc4 was expressed in the cochlear hair cells. Cochlear hair cell development and hearing function were normal in Nfatc4−/− mice, suggesting that Nfatc4 is not critical for cochlear development. We then showed that when the hair cells were challenged by ototoxic drugs Nfatc4 was activated and translocated from the cytoplasm to the nucleus, and this was accompanied by increased expression of Tnf and its downstream targets and subsequent hair cell apoptosis. Finally, we demonstrated that Nfatc4-deficient hair cells showed lower sensitivity to damage induced by ototoxic drugs and noise exposure compared to wild type controls. The Tnf-mediated apoptosis pathway was attenuated in Nfatc4-deficient cochlear epithelium, and this might be the reason for the reduced sensitivity of Nfatc4-deficient hair cells to injury. These findings suggest that the amelioration of inflammation-mediated hair cell apoptosis by inhibition of Nfatc4 activation might have significant therapeutic value in preventing ototoxic drug or noise exposure-induced sensorineural hearing loss.

SIRT6 Suppresses NFATc4 Expression and Activation in Cardiomyocyte Hypertrophy.

NFATc4, a member from the Nuclear Factor of Activated T cells (NFATs) transcription factor family, plays a pivotal role in the development of cardiac hypertrophy. NFATc4 is dephosphorylated by calcineurin and translocated from the cytoplasm to the nucleus to regulate the expression of hypertrophic genes, like brain natriuretic polypeptide (BNP). The present study identified SIRT6, an important subtype of NAD+ dependent class III histone deacetylase, to be a negative regulator of NFATc4 in cardiomyocyte hypertrophy. In phenylephrine (PE)-induced hypertrophic cardiomyocyte model, overexpression of SIRT6 by adenovirus infection or by plasmid transfection repressed the protein and mRNA expressions of NFATc4, elevated its phosphorylation level, prevented its nuclear accumulation, subsequently suppressed its transcriptional activity and downregulated its target gene BNP. By contrast, mutant of SIRT6 without deacetylase activity (H133Y) did not demonstrate these effects, suggesting that the inhibitory effect of SIRT6 on NFATc4 was dependent on its deacetylase activity. Moreover, the effect of SIRT6 overexpression on repressing BNP expression was reversed by NFATc4 replenishment, whereas the effect of SIRT6 deficiency on upregulating BNP was recovered by NFATc4 silencing. Mechanistically, interactions between SIRT6 and NFATc4 might possibly facilitate the deacetylation of NFATc4 by SIRT6, thereby preventing the activation of NFATc4. In conclusion, the present study reveals that SIRT6 suppresses the expression and activation of NFATc4. These findings provide more evidences of the anti-hypertrophic effect of SIRT6 and suggest SIRT6 as a potential therapeutic target for cardiac hypertrophy.

Abstract 2003: P38α suppresses tumor initiating cell phenotypes through inhibition of NFATc4 in non-small cell lung cancer

Abstract Lung cancer development is often mediated through oxidative and inflammatory stresses caused by tobacco and environmental carcinogens, suggesting dysregulation of stress mediators might play a role during carcinogenesis. The p38 MAPK pathway is activated by multiple stress stimuli and inflammatory cytokines, and mediates a wide range of cellular processes in a context-specific manner. P38α, a member of the p38 family, is known to regulate the self-renewal and differentiation of adult lung stem cells. While p38α has been reported to mediate tumor suppressive or supportive roles in different cancers, inactivation of p38α in the lung epithelium is involved in KRASG12V induced tumorigenesis. However, whether p38α could be involved in regulating tumor initiating cells (TIC) of lung cancer remains unclear. To investigate this possibility, immunohistochemistry was first used to analyse the nuclear expression of phosphorylation-activated p38α (p-p38α) in 90 human primary lung adenocarcinomas. Results showed higher expression of p-p38α was significantly correlated with longer progression free and overall survivals, as well as a more differentiated tumor histological phenotype, indicating p38α might play a tumor suppressive role and drive tumor differentiation in lung cancer. To further address its functional role on lung TIC, p38α was suppressed by siRNA or pharmacologically inhibited by SB203580. This led to increase of the TIC subset that coexpresses ALDH and CD44 (ALDH+/CD44+-TIC), as well as up-regulated expression of the pluripotency genes SOX2, OCT4, and NANOG in lung cancer cell lines. Functionally, p38α inhibition by SB203580 promoted tumor spheres formation for two consecutive generations. NFAT is a family of transcription factors reported to regulate TIC in various cancers which are activated via de-phosphorylation leading to nuclear translocation. We hypothesize the TIC supportive function of p38α inhibition might be mediated through loss of phosphorylation and subsequent activation of NFATc4. In line with this hypothesis, we showed, using Western blot, p38α inhibition increased the expression of nuclear NFATc4. Moreover, NFATc4 knockdown led to suppressed sphere formation and pluripotency genes expression in lung cancer cell lines. Together, our results indicated p38α mediates a tumor suppressor pathway through downstream NFATc4 inactivation and TIC inhibition. The p38α -NFATc4 stress pathway might serve as a potential target for long term lung cancer treatment through TIC regulation. Citation Format: Zhi-Jie Xiao, Jing Liu, Si-Qi Wang, Xu-Yuan Gao, Vicky Pui-Chi Tin, Maria Pik Wong. P38α suppresses tumor initiating cell phenotypes through inhibition of NFATc4 in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2003.

A GTPase-activating protein–binding protein (G3BP1)/antiviral protein relay conveys arteriosclerotic Wnt signals in aortic smooth muscle cells

In aortic vascular smooth muscle (VSM), the canonical Wnt receptor LRP6 inhibits protein arginine (Arg) methylation, a new component of noncanonical Wnt signaling that stimulates nuclear factor of activated T cells (viz NFATc4). To better understand how methylation mediates these actions, MS was performed on VSM cell extracts from control and LRP6-deficient mice. LRP6-dependent Arg methylation was regulated on >500 proteins; only 21 exhibited increased monomethylation (MMA) with concomitant reductions in dimethylation. G3BP1, a known regulator of arteriosclerosis, exhibited a >30-fold increase in MMA in its C-terminal domain. Co-transfection studies confirm that G3BP1 (G3BP is Ras-GAP SH3 domain-binding protein) methylation is inhibited by LRP6 and that G3BP1 stimulates NFATc4 transcription. NFATc4 association with VSM osteopontin (OPN) and alkaline phosphatase (TNAP) chromatin was increased with LRP6 deficiency and reduced with G3BP1 deficiency. G3BP1 activation of NFATc4 mapped to G3BP1 domains supporting interactions with RIG-I (retinoic acid inducible gene I), a stimulus for mitochondrial antiviral signaling (MAVS) that drives cardiovascular calcification in humans when mutated in Singleton-Merten syndrome (SGMRT2). Gain-of-function SGMRT2/RIG-I mutants increased G3BP1 methylation and synergized with osteogenic transcription factors (Runx2 and NFATc4). A chemical antagonist of G3BP, C108 (C108 is 2-hydroxybenzoic acid, 2-[1-(2-hydroxyphenyl)ethylidene]hydrazide CAS 15533-09-2), down-regulated RIG-I-stimulated G3BP1 methylation, Wnt/NFAT signaling, VSM TNAP activity, and calcification. G3BP1 deficiency reduced RIG-I protein levels and VSM osteogenic programs. Like G3BP1 and RIG-I deficiency, MAVS deficiency reduced VSM osteogenic signals, including TNAP activity and Wnt5-dependent nuclear NFATc4 levels. Aortic calcium accumulation is decreased in MAVS-deficient LDLR-/- mice fed arteriosclerotic diets. The G3BP1/RIG-I/MAVS relay is a component of Wnt signaling. Targeting this relay may help mitigate arteriosclerosis.

Astaxanthin Protects Primary Hippocampal Neurons against Noxious Effects of Aβ-Oligomers.

Increased reactive oxygen species (ROS) generation and the ensuing oxidative stress contribute to Alzheimer's disease pathology. We reported previously that amyloid-β peptide oligomers (AβOs) produce aberrant Ca2+ signals at sublethal concentrations and decrease the expression of type-2 ryanodine receptors (RyR2), which are crucial for hippocampal synaptic plasticity and memory. Here, we investigated whether the antioxidant agent astaxanthin (ATX) protects neurons from AβOs-induced excessive mitochondrial ROS generation, NFATc4 activation, and RyR2 mRNA downregulation. To determine mitochondrial H2O2 production or NFATc4 nuclear translocation, neurons were transfected with plasmids coding for HyperMito or NFATc4-eGFP, respectively. Primary hippocampal cultures were incubated with 0.1 μM ATX for 1.5 h prior to AβOs addition (500 nM). We found that incubation with ATX (≤10 μM) for ≤24 h was nontoxic to neurons, evaluated by the live/dead assay. Preincubation with 0.1 μM ATX also prevented the neuronal mitochondrial H2O2 generation induced within minutes of AβOs addition. Longer exposures to AβOs (6 h) promoted NFATc4-eGFP nuclear translocation and decreased RyR2 mRNA levels, evaluated by detection of the eGFP-tagged fluorescent plasmid and qPCR, respectively. Preincubation with 0.1 μM ATX prevented both effects. These results indicate that ATX protects neurons from the noxious effects of AβOs on mitochondrial ROS production, NFATc4 activation, and RyR2 gene expression downregulation.

Regulation of type 1 IP3 receptor expression by dopamine D2-like receptors via AP-1 and NFATc4 activation

Type 1 inositol 1,4,5-trisphosphate receptors (IP3Rs-1), together with ryanodine receptors, are major calcium channels to regulate intracellular Ca2+ concentration. Although our recent report demonstrates the essential involvement of IP3R-1 up-regulation induced by dopamine D1-like and D2-like receptor (D1 and D2R) stimulation in psychological dependence, exact regulatory mechanisms of IP3R-1 expression by D2Rs have not yet been clarified. Mouse cerebral cortical neurons were treated with inhibitor of Ca2+-related signal transduction pathways coupling to D2Rs and used to analyze the mechanisms of IP3R-1 expression regulated by transcriptional factor. A selective D2R agonist, quinpirole, up-regulated IP3R-1 protein following its mRNA increase, which was significantly inhibited by gallein (a Gβγ modulator), U73122 (a phospholipase C inhibitor), BAPTA-AM (an intracellular calcium chelating reagent), W7 (a calmodulin inhibitor), KN-93 (a calmodulin-dependent protein kinases inhibitor), and FK506 (a calcineurin inhibitor). Immunocytochemical assessment showed that quinpirole increased expression of both cFos and phosphorylated-cJun in nucleus and enhanced translocation of NFATc4 complex to nucleus from cytoplasm. In addition, quinpirole directly recruited bindings between AP-1 and IP3R-1 promoter region and between NFATc4 and IP3R-1 promoter region. These results indicate that D2Rs enhance IP3R-1 gene transcription via increased bindings of AP-1 and NFATc4 to IP3R-1 promoter region after Gβγ activation.

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.

Genome-wide association study of inbred murine strains discovered nuclear factor of activated T-cells Nfatc4 as a major gene controlling severity of inflammatory arthritis. (47.14)

Abstract Nuclear factor of activated T cells (NFAT) is a family of transcription factors critical in regulating gene expression in development and disease. NFAT is an important target for cyclosporine immunosuppression in transplant rejection. The NFAT pathway is also important in inflammatory and autoimmune diseases such as rheumatoid arthritis. Using genome-wide association study (GWAS) of antibody-mediated arthritis in inbred murine strains, we found NFATc4 within the major arthritis severity peak at 56.35-56.46 Mb of chromosome 14 with genetic association of log(1/p) > 5.5. To confirm the genetic linkage and functional significance of the NFATc4 gene, we performed collagen antibody-induced arthritis (CAIA) and found that NFATc4-deficient mice exhibited significantly reduced inflammation as compared to wild-type counterparts (55% downregulation, p < 0.01). Immunoblotting analysis demonstrated the presence of NFATc4 in articular cartilage and chondrocytes derived from femoral heads of arthritic and naïve mice. Notably, NFATc4 in chondrocytes isolated from arthritic mice was hypo-phosphorylated as compared to naïve cells, indicating activation of NFATc4. NFATc4, as expected, was not found in splenocytes. We also performed genome-wide Affymetrix oligonucleotide array studies in CAIA experimental arthritis model using wild type and NFATc4-deficient mice. Taking together, we uncovered a novel role of NFATc4 in inflammatory arthritis in chondrocytes.

Enhanced hypertrophy in ob/ob mice due to an impairment in expression of atrial natriuretic peptide

Rationale We investigated the molecular mechanism(s) that play a role in leptin signaling during the development of left ventricular hypertrophy (LVH) due to pressure overload. To this end, ob/ob leptin deficient and C57BL/6J control mice were subjected transverse aortic constriction (TAC). Methods Control sham C57BL/6J and ob/ob mice, along with C57BL/6J and ob/ob leptin deficient mice were subjected transverse aortic constriction (TAC) for 15 days and then evaluated for morphological, physiological, and molecular changes associated with pressure overload hypertrophy. Results Evaluation by echocardiography revealed a significant increase in left ventricular mass (LVmass) and wall thickness in ob/ob mice subjected to transverse aortic constriction (TAC) as compared to C57BL/6J. Analysis of the expression of molecular markers of LVH, such as atrial natriuretic peptide (ANP), revealed a blunted increase in the level of ANP in ob/ob mice as compared to C57BL/6J mice. We observed that leptin plays a role in modulating the transcriptional activity of the promoter of the ANP gene. Leptin acts by regulating NFATc4, a member of the nuclear factor activated T cell (NFAT) family of transcription factors in cardiomyocytes. Our in vivo studies revealed that ob/ob mice subjected to TAC failed to activate the NFATc4 in the heart, however, intraperitoneal injection of leptin in ob/ob mice restored the NFATc4 DNA-binding activity and induced expression of the ANP gene. Conclusion This study establishes the role of leptin as an anti-hypertrophic agent during pressure overload hypertrophy, and suggests that a key molecular event is the leptin mediated activation of NFATc4 that regulates the transcriptional activation of the ANP gene promoter.

Interleukin-18 Suppresses Adiponectin Expression in 3T3-L1 Adipocytes via a Novel Signal Transduction Pathway Involving ERK1/2-dependent NFATc4 Phosphorylation

An inverse correlation between the pro-inflammatory cytokine interleukin-18 and the anti-atherogenic adipokine adiponectin has been reported in the chronic pathological conditions obesity, insulin resistance, coronary artery disease, and metabolic syndrome. We investigated whether this relationship is coincidental or has a causal basis. Here we show that interleukin-18 (IL-18) suppresses adiponectin transcription, mRNA expression, and secretion by 3T3-L1 adipocytes. IL-18 suppresses adiponectin promoter-reporter activity, an effect reversed by deletion or mutation of the NFATc4 core DNA-binding site. IL-18 induces NFATc4 phosphorylation (Ser(676)), nuclear translocation, and in vivo DNA binding. IL-18 induces ERK1/2 phosphorylation and enzyme activity, and pretreatment with the MEK inhibitor U0126, ERK1/2 inhibitor PD98059, or small interference RNA targeted to ERK1/2 attenuates ERK1/2 activation and NFATc4 phosphorylation. Finally, inhibition of ERK1/2 or NFATc4 knockdown reverses IL-18-mediated adiponectin suppression. In contrast to its inhibitory effects on adiponectin expression, IL-18 potently stimulates PAI-1 secretion. These data demonstrate for the first time that IL-18 selectively suppresses adiponectin expression via ERK1/2-dependent NFATc4 activation and suggest that the inverse relationship observed between IL-18 and adiponectin in various chronic pathological conditions is causally related. Thus, targeting IL-18 expression may enhance adiponectin expression and mitigate disease progression.

Neurotrophin activation of NFAT‐dependent transcription contributes to the regulation of pro‐nociceptive genes

Nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) play key roles in the development of inflammation-induced hyperalgesia by triggering the expression of pro-nociceptive genes within primary afferent and spinal neurons. However, the mechanisms by which neurotrophins elicit gene expression remain largely unknown. Recently, neurotrophins have been shown to activate members of the calcineurin (CaN)-regulated, nuclear factor of activated T-cells (NFATc) family of transcription factors within brain. Thus, we hypothesized that NFATc transcription factors couple neurotrophin signaling to gene expression within primary afferent and spinal neurons. In situ hybridization revealed NFATc4 mRNA within the dorsal root ganglion and spinal cord. In cultured dorsal root ganglion cells, NGF triggered NFAT-dependent transcription in a CaN-sensitive manner. Further, increased BDNF expression following NGF treatment relied on CaN, thereby suggesting that NGF regulates BDNF transcription via activation of NFATc4. Within cultured spinal cells, BDNF also activated CaN-dependent, NFAT-regulated gene expression. Interestingly, BDNF stimulation increased the expression of the pro-nociceptive genes cyclooxygenase-2, neurokinin-1 receptor, inositol trisphosphates receptor type 1, and BDNF itself, through both NFAT-dependent and NFAT-independent transcriptional mechanisms. Our results suggest that regulation of pro-nociceptive genes through activation of NFAT-dependent transcription is one mechanism by which NGF and BDNF signaling contributes to the development of persistent pain states.