NFATc1 (Nuclear factor of activated T-cells cytoplasmic calcineurin-dependent 1; NFAT2; NFATc, NFAT cytoplasmic); NFATc2 (Nuclear factor of activated T-cells cytoplasmic calcineurin-dependent 2; NFAT1; NFATp, NFAT preexisting); NFATc3 (Nuclear factor of activated T-cells cytoplasmic calcineurin-dependent 3; NFAT4; NFATx); NFATc4 (Nuclear factor of activated T-cells cytoplasmic calcineurin dependent 4, NFAT3); NFAT5 (Nuclear factor of activated T-cells 5; TonEBP, Tonicity-responsive enhancer binding protein; OREBP, Osmotic response element-binding protein)
The gene encoding for the first nuclear factor of activated T cells (NFAT) protein was cloned more than 20 years ago and termed NFATp, as it was shown to be “preexistent” in the cytosol of resting T cells. In activated T cells, NFATp interacted with the transcription factors Fos and Jun in the nucleus to induce the expression of interleukin (IL-) 2 (Jain et al. 1993). Soon after, new members of the NFAT family were isolated, and the characterization of the calcineurin-mediated dephosphorylation and activation of NFAT proteins identified them as the targets of the immunosuppressive effects of cyclosporine A. Although initial efforts were focused on studying the role of NFAT proteins in the regulation of T cell activation, it soon became clear that members of this ubiquitously expressed family of transcription factors were involved in the regulation of a multitude of programs of development and differentiation in many cell types and tissues. This chapter reviews the current understanding of the mechanisms that regulate the activity of NFAT proteins and the functions that these proteins have in different cells and tissues.
Family and Structure
NFAT activation is mainly regulated by its subcellular localization. The net result of the rate of nuclear import and export of NFAT proteins, which is controlled by their phosphorylation status, determines the overall level of activation of NFAT. Other mechanisms have also been described to contribute to the fine regulation of the transcriptional activity of NFAT.
NFAT-kinases: Phosphorylation of NFAT proteins is required to promote nuclear export and to maintain cytosolic localization in resting conditions. Several kinases have been reported to be responsible for the phosphorylation of different serine-containing motifs in NFAT. Casein kinase1 (CK1) binds the N-terminal region of NFAT and regulates its nuclear export and cytosolic retention through phosphorylation of a serine-rich motif. Glycogen synthase kinase 3 (GSK3) phosphorylates serine-proline motives in NFATc2 and NFATc1, promoting NFAT nuclear export. These phosphorylation sites appear to be created by previous priming by cyclic-AMP dependent protein kinase A (PKA)-mediated phosphorylation of NFAT. Activation of the AKT kinase negatively regulates GSK3 and prolongs NFAT residence time in the nucleus. The dual-specificity tyrosine-(Y)-phosphorylation-regulated kinases DYRK1A and DYRK2 also regulate NFAT nuclear export through phosphorylation of an SP motif that primes for subsequent phosphorylation by CK1 or GSK3 (Muller and Rao 2010). LRRK2 may also phosphorylate NFAT proteins and contribute to the regulation of NFAT activation (Liu et al. 2011). Interestingly, it has been shown that many of these kinases, including CK1 and GSK3, exist in a complex with a noncoding RNA repressor of NFAT (NRON) that inhibits nuclear import of NFAT by facilitating interactions of those kinases with NFAT (Sharma et al. 2011). Recently, in response to IL-7, JAK3 has been reported to induce phosphorylation of NFATc1 in CD4−CD8− thymocytes; however, in this case, phosphorylation of this NFAT protein induces its translocation into the nucleus in a calcineurin-independent manner (Patra et al. 2013).
Transcriptional regulation of NFAT: NFATc1A is an isoform of NFATc1 that is regulated at a transcriptional level by an autoregulatory loop under the control of an inducible NFAT-dependent promoter. In T cells, it has been shown that the activation of constitutively expressed isoforms of NFATc1 and NFATc2 induces the expression and accumulation of this inducible NFATc1A isoform (Serfling et al. 2006).
Posttranslational regulation: Several posttranslational mechanisms that contribute to the regulation of NFAT activity have been described. Sumoylation of NFAT is an NFAT nuclear retention mechanism, which might also regulate NFAT transcriptional activity. Evidence has also been presented supporting that poly(ADP-ribose) polymerase-1 (PARP-1) interacts with NFATc1 and NFATc2, regulating NFAT nuclear export and transcriptional activity (Muller and Rao 2010). Furthermore, NFATc2 has also been shown to be ubiquitinated by the E3 ubiquitin ligase MDM2 in breast cancer cells, which leads to proteasome-mediated degradation (Mancini and Toker 2009).
NFAT proteins form transcriptional complexes, in which they cooperate with other transcription factors to activate or repress the expression of specific genes. This allows cells to integrate calcium signaling with other signaling pathways to regulate the expression of specific programs of gene expression. Initially identified as the nuclear component of the NFAT activity that was responsible for the expression of IL-2 in activated T cells, activator protein 1 (AP-1) complexes are the best characterized NFAT partners and are one of the main transcription factors that interacts with NFAT during T cell activation. In response to the engagement of the TCR and costimulatory receptors, calcium signaling and the Ras-MAPK pathway converge in the activation of NFAT and Fos and Jun proteins, which form the AP-1 complex. The DNA-binding domain of NFAT interacts with AP-1, forming a quaternary complex on DNA that activates the expression of activation-induced genes, including numerous cytokines (Macian et al. 2001). The number of transcription factors that have been identified to cooperate with NFAT has grown in the last few years. In many cases, these interactions occur in specific cells of tissues and are responsible for the regulation of different programs of activation, differentiation, or development. NFAT proteins are also able to form homodimers. These complexes bind to κB-like sites that contain two tandem NFAT-binding sites separated by one or two bases. Dimers formed by NFATc2 have been implicated in regulating the expression of genes that induce a hyporesponsive state in T helper cells. In addition, NFAT5 needs to form homodimers to bind to DNA and exert its transcriptional activity (Lopez-Rodriguez et al. 2001; Baine et al. 2009).
The innate immune system is our first line of defense against invading pathogens and, therefore, it must react quickly and efficiently. Cell types involved in this process include eosinophils, neutrophils, mast cells, and macrophages. NFAT proteins have been shown to be expressed in all these cell types and serve a pivotal role in transforming molecular signals to expression of genes. Ligation of pattern recognition receptors in myeloid cells by microbes can lead to NFAT activation in different cells of the innate immune system, including neutrophils, dendritic cells, and macrophages. Indeed, fungal infections that are commonly seen in cyclosporine A-treated individuals appear to be linked to inhibition of NFAT-mediated gene expression in neutrophils. Interestingly, not only calcineurin regulated members of the NFAT family of transcription factors but also NFAT5 participate in the regulation of gene expression in response to pattern recognition receptors in cells that participate in the innate immune response. NFAT expression also plays an integral part of the mast cell response and survival, as ligation of the Fcε receptor results in an influx of intracellular calcium, causing mast cells to release histamine-containing granules and produce cytokines (Muller and Rao 2010; Fric et al. 2012).
The adaptive immune response is highly specific and efficient at targeting pathogens and infected cells for elimination. The activation of effector cells is governed by the recognition of an antigen by the B cell receptor (BCR) or its presentation in the context of an MHC molecule to cognate TCRs on T cells. NFAT has been most extensively studied in T cells, although the participation of NFAT in the regulation of B cells has also been characterized. NFAT proteins are activated in B cells in response to BCR engagement and participate in the induction of the programs of gene expression that regulate B cell activation and differentiation. In T cells, coordinated engagement of the TCR and the costimulatory receptor CD28 allows NFAT to cooperate with other transcription factors, such as Fos and Jun, to induce the expression of many genes that are required to ensure effective T cell activation (Macian 2005; Muller and Rao 2010). However, in the absence of costimulation, TCR triggering causes NFAT nuclear localization, which, without the opportunity to cooperate with Fos and Jun, forms homodimers that regulate the expression of genes that maintain T cells in a tolerant hyporesponsive state termed anergy (Baine et al. 2009). The involvement of NFAT proteins in the regulation of immune tolerance is also mediated through their participation in the development of regulatory T cells (Treg), a distinct population of T cells that express the transcription factor FoxP3 and have the capacity to suppress the activation of other immune cell populations. NFAT proteins not only regulate the expression of FoxP3 but also cooperate with this transcription factor to activate (CTLA-4, CD25, and GITR) or inhibit (IL-2) the expression of genes in Treg and, therefore, regulate the differentiation and function of these suppressor cells (Wu et al. 2006).
Development of thymocytes is also dependent on NFAT activity. In the thymus, immature thymocytes cells rearrange both the α and β chains of the TCR and mature from a double negative (CD4-CD8-) thymocytes into a double positive thymocyte that expresses both CD4 and CD8 coreceptors. As mentioned above, NFATc1 is crucial in the regulation of this process (Patra et al. 2013). Double positive thymocytes then undergo a rigorous process that involves being positively selected for the presence of a TCR that can interact with MHC molecules but negatively selected for self-reactive TCR that can recognize self-antigens. The lack of calcineurin activity in immature thymocytes leads to a block in positive selection, suggesting that NFAT proteins are also involved in this step of thymocyte development. Supporting this fact, NFATc2 and NFATc3 appear to be responsible for regulating the thresholds of signal transduction that govern positive selection in the thymus (Gallo et al. 2007).
CD4+ T cells can differentiate into many different effector subsets, such as Th1, Th2, or Th17, a process that is regulated by the cytokine profile of the extracellular environment. In all these differentiation events, NFAT proteins regulate the expression of specific genes that help define the particular T cell subtype. The ability of NFAT to direct opposite programs of differentiation depends on the activation of transcriptional copartners. For instance, T-bet is an integral cotranscriptional partner for NFAT in the development of Th1 cells and the secretion of their signature cytokine IFN-γ. Likewise, GATA3–NFAT interactions are necessary for IL-4 expression and maturation into Th2 cells. Similarly, the presence of IL-6 in the extracellular milieu induces the expression of RORγt, which synergizes with NFAT in the expression of IL-17, IL-21, and IL-22 to allow Th17 differentiation (Muller and Rao 2010). As mentioned above, NFAT proteins participate in the regulation of Treg generation and have also been implicated in the differentiation of follicular helper T cells (Wu et al. 2006; Martinez et al. 2016).
Outside of the immune system, NFAT expression has been reported in almost all tissues, although the expression of any individual NFAT protein is often limited to specific tissues. For instance, whereas NFATc1, NFATc2, and NFATc3 are expressed in cells of the immune system and in many nonlymphoid cells and tissues, NFATc4 expression has not been reported in immune cells. An increasing number of reports have characterized the role that different NFAT proteins have as regulators of development, differentiation, and function in many cells and tissues, including skeletal muscle differentiation, myocardial hypertrophy, control of heart valve formation, vascular development, cartilage formation, neuronal development, and the regulation of stem cell quiescence (Wu et al. 2007). The expression of different NFAT proteins is developmentally regulated in skeletal muscle and these proteins control progression from immature precursors to mature myocytes and contribute to the specification of muscle fiber type. NFAT proteins also regulate cartilage growth and bone remodeling. The role that NFAT proteins play in the regulation of osteoclast differentiation has been amply documented. In these cells, RANKL-mediated activation of NFATc1 directs the expression of a set of genes required for osteoclast differentiation. Initial characterization of a mouse model that lacked expression of NFATc1 unequivocally showed that NFATc1 played a key role in the formation of the heart’s valves. In the adult heart, NFAT proteins partner with members of the GATA and MEF2 families of transcription factors to regulate myocardial hypertrophy. NFATc3 and NFATc4 are expressed in perivascular mesenchymal cells, which regulate the assembly of blood vessels during embryogenesis, and mice that lack those NFAT proteins present an abnormal vascular development. Vascular endothelial growth factor is a major activator of NFAT proteins in endothelial cells and engagement of its receptor leads to the activation of NFAT-dependent genes such as COX2. NFAT proteins also regulate neuronal axon growth and are essential for neuronal development and the differentiation of Schwann cells. NFAT has also been shown to control beta cell growth in the endocrine pancreas and regulate insulin-signaling pathways and adipogenesis. NFATc1 has also been implicated in the maintenance of stem cell quiescence in the skin follicle by repressing the expression of the cell cycle kinase CDK4 (Wu et al. 2007; Sitara and Aliprantis 2010).
NFAT proteins are key regulators in the control of cell development and differentiation in part by modulating proliferation and cell death. As such, altered NFAT signaling has been described in cancer cells. As expected due to their pivotal role in lymphocyte development, dysregulated NFAT activity have been associated with several forms of B and T cell lymphoma and leukemia. Furthermore, NFAT proteins have been shown to be involved in the regulation of specific properties of different cancers. For instance, NFATc2 and NFAT5 appear to positively regulate migration and invasion of breast cancer cells. NFAT proteins have also been proposed to regulate tumor-associated angiogenesis and may directly activate the expression of oncogenes, such as Myc (Mancini and Toker 2009; Muller and Rao 2010).
Pharmacological Modulation of NFAT Activity
Pharmacological agents used to modulate NFAT activity have been tremendously beneficial in the clinic. These agents, which inhibit the activation of T cells and act as immunosuppressants, are widely used in therapies ranging from treatment of autoimmune disease to prevention of organ transplant rejection. However, their widespread use is limited by their costly side effects, such as nephrotoxicity. The activation of NFAT is dependent on its dephosphorylation, and, therefore, the phosphatase calcineurin has been an important area of focus for inhibitor development. Nevertheless, the specificity of NFAT inhibition is limited by the fact that NFAT is not the only target of calcineurin. Cyclosporine A and FK506 are the most widely used and studied suppressors of NFAT activity. Both are calcineurin inhibitors whose mechanism of action is quite similar. They bind separate intracellular peptidyl prolyl isomerases (cyclosporine A-cyclophilin; FK506-FKBP12). These complexes then bind to distinct regions of calcineurin and inhibit its phosphatase activity. The fact that these compounds not only affect NFAT but also the activity of other endogenous targets also limits their use as specific NFAT inhibitors (Li et al. 2011). The search for more specific inhibitors has centered on targeting specifically the interaction of NFAT and calcineurin. Calcineurin-binding sites on NFAT have been mapped to the N-terminal regulatory domain and include the amino acid sequence SPRIET. A closer analysis of this sequence among NFAT family members revealed the consensus-binding sequence PxIxIT, which laid the groundwork for the discovery using combinatorial libraries of the highly potent VIVIT peptide (Hogan et al. 2003). This peptide has been successfully used in mouse models of graft rejection and tumor progression. The major benefit of the VIVIT peptide is in its higher specificity for the inhibition of NFAT function; however, this sequence is still conserved in other proteins, such as AKAP79, Cabin1, or MCIP1, that have calcineurin-binding activity. Due to the severe side effects caused by long-term administration of cyclosporine A or FK506, finding inhibitors with more specificity for NFAT has been also pursued. Disrupting the ability of NFAT to bind DNA or enhancing the nuclear export of NFAT has been explored. As binding partners and gene targets of NFAT are being discovered, further inhibitors with higher specificity could be developed, which should lead to a higher degree of precision to specifically block NFAT activity.
Members of the NFAT family of transcription factors have been established as crucial regulators of numerous programs of development, differentiation, and activation in many cell types and tissues. NFAT activation is induced by the engagement of calcium-coupled receptor. This eventually leads to the translocation of NFAT into the nucleus, where it cooperates with several transcriptional partners so that signals that emanate from different inputs can be integrated to ensure specific regulation of distinct programs of gene expression. Circuits of regulation that fine-tune NFAT activation have also been characterized and are likely to play important roles in the regulation of NFAT activity. Given the wide range of tissue expression of NFAT proteins, it is clear that novel functions and targets of these transcription factors still remain to be discovered. The identification of specific functions for individual NFAT family members and the characterization of the differential spatial and temporal expression of specific NFAT proteins during unique programs of development should also enhance our understanding on how NFAT-regulated programs of development are orchestrated. The development of new, highly specific inhibitors of NFAT activity to resolve the inherent toxic effects associated with the use of calcineurin inhibitors is an ongoing challenge that could have great clinical impact. Given the increasing amount of evidence that implicates NFAT signaling in oncogenesis and cancer progression, these new therapeutic approaches could prove of great value not only to suppress NFAT-regulated immune responses but also to design new interventions for the treatment of certain types of cancer and other pathologies controlled by this family of transcription factors.
- Aramburu J, Drews-Elger K, Estrada-Gelonch A, Minguillon J, Morancho B, Santiago V, et al. Regulation of the hypertonic stress response and other cellular functions by the Rel-like transcription factor NFAT5. Biochem Pharmacol. 2006;72:1597–604. doi: 10.1016/j.bcp.2006.07.002.PubMedCrossRefGoogle Scholar
- Martinez GJ, Hu JK, Pereira RM, Crampton JS, Togher S, Bild N, et al. Cutting edge: NFAT transcription factors promote the generation of follicular helper T cells in response to acute viral infection. J Immunol. 2016;196:2015–9. doi: 10.4049/jimmunol.1501841.PubMedPubMedCentralCrossRefGoogle Scholar
- Sharma S, Findlay GM, Bandukwala HS, Oberdoerffer S, Baust B, Li Z, et al. Dephosphorylation of the nuclear factor of activated T cells (NFAT) transcription factor is regulated by an RNA-protein scaffold complex. Proc Natl Acad Sci USA. 2011;108:11381–6. doi: 10.1073/pnas.1019711108.PubMedPubMedCentralCrossRefGoogle Scholar