General Overview:
The ubiquitous transcription factor NFAT (nuclear factor of activated T-cells) associates with other proteins to bind DNA and induce genes responsible for cell-cell interactions (Crabtree et al., 2002). NFAT is expressed in a variety of lymphocytes: T cells, B cells, natural killer (NK) cells, monocytes and non-immune related cells: muscle, cardiac, and neuronal (Scott et al., 2001, Janeway et al., 2001). In the literature there are 4 reported NFAT isoforms, which are designated as NFAT1, NFAT2, NFAT3, NFAT4 (Masuda et al., 1998). The presence and redundancy of these homologs suggests the critical nature of NFAT expression in immune and non-immune related cells. NFAT transcriptional activity is modulated by cytoplasmic Ca2+ concentration through various Ca2+ associated signaling pathways (Masuda et al., 1998, Crabtree et al., 2002). Increases in cytoplasmic Ca2+ concentration induce NFAT dephosphorylation and NFAT translocation to the nucleus where it binds to cis regulatory elements of target genes as a monomer (Masuda E.S. et al., 1998). Figure 1 outlines a general signaling mechanism for NFAT activation.

Figure 1: General signaling pathway for NFAT. ATP hydrolysis is not included in the cartoon (Crabtree et al., 2002) Permission Pending for image use.

NFAT Structure:
NFAT secondary structure consists of b sheets and random coils forming three functional domains: a Rel-similarity domain (RSD) for DNA binding and association with AP-1 transcription factors, a NFAT homolog region (NHR) domain, containing intracellular localization signaling sequences, and transcriptional activation domains (TAD), which recruit coactivators of NFAT transcription CBP, p300 and JAB1 after NFAT:AP-1 protein complex formation (Crabtree et al., 2002). Phosphorylating and dephosphorylating the NHR domain determines intracellular localization. NHR phosphorylation results in nuclear export and cytoplasmic localization and NHR dephosphorylation results in nuclear import and nuclear localization. NFAT also contains two sequence motifs: a nuclear location signal sequence (NLS) and a nuclear export signal sequence (NES) that permit the import and export of NFAT from the nucleus, respectively (Janeway C. A. et al., 2001). NFAT contains two NLS motifs, one in the NHR domain and one in the RSD domain, the latter, however, is less efficient at translocating NFAT to the nucleus. Also mutations in the basic NLS domain reduce NFAT translocation. (Masuda et al., 1998)
The tertiary structure of NFAT has two domains that resemble immunoglobulin (Ig) folds. One domain, in the RSD, specifically binds to DNA and is a beta barrel comprised of three beta sheets. Within the beta barrel there are two prominent loop structures, the first loop (designated as the DNA binding loop) is more constrained and binds to the DNA major grove at the 5’ recognition sequence GGAAAA. The second more mobile loop (designated as the Rel-insert region) binds to the minor grove at the 3’ recognition sequence GGAAAA. (Masuda et al., 1998) The anatomical structure, topology and protein interactions of NFAT are depicted in figures 2 and 3.

Figure 2: A 2.7 Angstrom resolved crystal structure of NFAT, Ap-1 heterodimer (Fos:Jun), and a distal portion of the IL-2 gene promoter (Chen et al., 1998). Image from PDB.

Figure 3: Cartoon of NFAT crystallized with AP-1 proteins and DNA. The DNA promoter bound to the NFAT, AP-1 protein complex is ARRE2, part of the IL-2 promoter (Chen et al., 1998). The protein-protein interactions for this complex are largely polar and hydrogen bonding and salt bridges cooperatively link the protein-protein-DNA complex. (Szilak et al., 1999) These extensive interactions permit the bending of Fos and DNA to form an extended grove which interacts with 15 DNA base pairs (Chen et al., 1998). Note the Rel-homology region (RHR) N domain of NFAT extensively interacts with the Fos-Jun heterodimer. As Jun moves away from NFAT, Fos’s luecine zipper moiety interacts with the NFAT RHR-N domain (Szilak et al., 1999). Premission pending for image use.

A mechanism for NFAT regulation: Ca2+ signalling via the TCR leads to NFAT activation:
Two enzymes, calcineurin, a cytoplasmic serine/threonine phosphotase and nuclear glycogen synthase kinase 3 (GSK3) are responsible for the regulation of NFAT transcriptional activity. Calineurin upregulates NFAT by dephosphorylating serines in the SP-repeats and in the serine rich N terminus region of NFAT (Crabtree et al., 2002). This induces a conformational change exposing the NLS (Scott et al., 2001), allowing NFAT to translocate to the nucleus. Calcineurin activity is regulated by cytoplasmic Ca2+ concentration. In T cells, TCR stimulation spurs a biphasic increase in intracellular Ca2+ concentration. The initial wave of Ca2+ concentration from Ca2+ sequestered ions in the ER, the second results from an influx of extra-cellular calcium ions from Ca2+ release activated Ca2+ channels (CRAC) (Crabtree G. R. et al., 2002). CRAC activation is required for upregulation of NFAT controlled genes since transient increases in Ca2+ concentration from the ER is not sufficient to promote NFAT activation (Crabtree et al., 2002). This indirectly suggests that translocated NFAT must overwhelm GSK3 in the nucleus in order for NFAT to bind to target genes.
GSK3, on the other hand, downregulates NFAT by phosphorylating NFAT and inducing a conformation change exposing the NES (Masuda et al., 1998).

NFAT regulates….
NFAT activation regulates a variety of immune processes: apoptosis, anergy, T-cell development, and ageing of immune system. In particular, NFAT isoforms are responsible for regulating interluekin (IL) 2, IL-3, IL-4, IL-5, granulocyte macrophage colony-stimulation factor (GM-CSF), interferon (IFN)-y, tumor necrosis factor (TNF) alpha and the cell surface receptors CD40L, CTLA-4 and FasL expression (Masuda et al., 1998). Macian et al. further characterized NFAT regulatory roles, hypothesizing that transcriptional activity mediates two distinct gene expression patterns which are independent and dependent upon NFAT:AP1 interactions. NFAT1 mutagenesis studies demonstrated that NFAT:Ap-1 complexes are required for inducing IL-2, GM-CSF, IL-3, IL-4, MIP1 alpha, and FasL. Conversely, the expression of TNF alpha and IL13 promoter activity does not require NFAT:Ap-1 interactions (Macian et al. 2002). The NFAT:Ap-1 complex is also required to induce NFAT activated cell death. (Macian et al. 2000) Additional regulatory roles have been observed for NFAT. For example, Gomez et al. reported that NFAT translocation regulates expression of the anti-apoptotic protein Bcl-2. Inhibition of calcineurin downregulates Bcl-2 expression in IL-2 cells. Conversely, constitutive calcineurin expression upregulates Bcl-2 expression, indirectly implicating the function of NFAT in Bcl-2 expression (Gomez J. et al., 1998). Sheng Xiao et al. observed that FasL transcription could be induced by two different promoters which are mediated by NFAT binding. Only one promoter, however, required Sp1 and NFAT binding. Thus, the aforementioned studies suggest that NFAT has a variety of regulatory roles in cells that are dependent upon promoter and NFAT:protein complexes.

Receptors inducing NFAT activity:
The most prominent and studied NFAT activation pathway is through the TCR. TCR stimulation by MHC molecules, in conjunction with CD28:B7 costimulatory signal induces a biphasic intracellular Ca2+ influx that induces NFAT dephosphorylation and translocation to the nucleus (Janeway et al., 2001). In B cells, BCR stimulation or CD40 and IL-4R stimulation induces NFAT activation. Stimulation of FceR1 in mast and basophil, and FcgRIII in NK cells also induces NFAT dephosphorylation and translocation, respectively. (Masuda et al., 1998)

Inhibitors of NFAT:
NFAT inhibitors can be divided into two class, protein inhibitors and small molecule inhibitors. Most of these inhibitors bind calcineurin and suppress dephosphorylating activity. To date, there are four protein inhibitors which prevent NFAT nuclear translocation: AKAP79, a scaffold protein that prevents calcineurin substrate interactions, CABIN protein, which blocks calcineurin activity, a calcineurin B homolog, CHP, and MCIP1,2,3 proteins which have the ability to prevent NFAT2 phosphorylation and nuclear import (Crabtree et al., 2002).
The two most prominent NFAT small molecule inhibitors are cyclosporin A and FK506. Mechanistically, cyclosporin A and FK506 indirectly repress NFAT by inhibiting calcineurin activity. These drugs target NFAT specific pathways and act as immunosuppressants by inhibiting alloreactive T-cells. Clinically, these drugs are administered to patients to prevent graft reject (Janeway C. A. et al., 2001) and have reduced joint erosion and disease progression, yet there are also side effects: nephrotoxicity, neurotoxicity, diabetogenicity, and gastrointestinal toxicity (Trevillyan et al., 2001). Several new drug candidates, 3,5-bistriflouromethyl pyrazole (BTP) derivatives inhibit Th1 and Th2 cytokine gene expression thereby indirectly inhibiting the nuclear localization of NFAT. These BTP derivates, however, do not dephosphorylate calcineurin and a mechanistic model for inhibition of NFAT pathways has not been elucidated (Trevillyan J. M. et al. 2001).

NFAT knockout mice:
Several studies have been conducted using knockout NFAT mice to determine NFAT’s functional role in the immune and non-immune related cells. Masuda et al. reports that NFAT1 (-/-) mice have deregulated transcription of certain genes accompanied by the hyperproliferation of splenic B and T cells. This hyperproliferation is due to a lack of FasL expression which would normally induce cell death in surviving cells (Crabtree et al., 2002) In T-cells stimulated with anti-CD3 there was noticeably reduced IL-4 transcription. Hence, Masuda et al, posits that NFAT1 is a positive and negative regulator of cytokine expression. NFAT4 is also highly expressed in CD4+CD8+ thymocytes and NFAT4(-/-) mice have reduced CD4+CD8+ cell counts (Amaskaki et al., 2002). Crabtree et al. suggests that the loss of double positive thymocytes results from Bcl-2 suppression during thymic development. Mice with mutant NFAT1/4 undergo spontaneous differentiation in to T helper 2 cells with excess IgE production resulting in strong allergic responses. These mice also have spontaneous T-cell hyperproliferation, which does not depend on CD28 stimulation for activation (Crabtree et al. 2002).
Subsequent studies with knockout mice have revealed the additional importance of NFAT activity in cell differentiation and development. NFAT1 (-/-) mice also have small-multinucleated muscle cells, while older mice have excess cartilage resulting in decreased joint mobility. Horsley et al. observed that NFAT2 (-/-) mice have “defective heart valve development and abnormalities in the cardiac septum”. In NFAT3/4 double knockout mice, embryo fatality result from disrupted blood vessel organization and instability in the vessel walls. NFAT4 knockout mice have defective embryonic myofibers (Horsley V. et al, 2002)

NFAT and T-cell’s:
NFAT expression and trancscriptional activity are critical for the proliferation and differentiation of armed effector T-cells (Janaway et al. 2001). NFAT has also been implicated not only in inducing genes for an immune response but also a set of anergy associated genes. Weak TCR and BCR stimulation can induce NFAT activation by low sustained intracellular Ca2+ concentrations. Macian et al observed that null T cells for NFAT1 do not undergo anergy and show lower expression levels of anergy-genes. For T cells with “a constitutively active NFAT1” where AP-1 is not induced, or NFAT:AP-1 interactions do not occur had increased expression of anergy related genes indicative of an anergy associated phenotype with lower TCR responsiveness (Macian et al. 2002).

NFAT and Disease:
The ubiquitous nature of NFAT expression and its regulatory role in cell differentiation and development makes it a critical enzyme in adaptive immune responses. In patients with defective T-cell proliferation and IL-2 synthesis, researchers noticed detectable Ca2+ release from the ER where as Ca2+ release from the plasma membrane was not detected. This data supports the hypothesis that a sustained intracellular calcium concentration is required for NFAT translocation via the calcineurin GSK3 signaling pathway (Masuda et al, 1998).
Various viral infections also positively and negatively interfere with NFAT signaling pathways. Scott et al. observed that GFP-NFAT nuclear translocation was blocked in the early stages of herpes simplex virus (HSV) infection. HIV hepatitis C and African swine fever are also believed to interfere and repress NFAT transcriptional activity. In particular, the protein A238L of African swine fever virus binds to calcineurin, thereby blocking NFAT translocation (Crabtree et al., 2002). Kaposi sarcoma associated herpes virus and rhesus monkey rhadinoviruss, which, infect B cells, however, stimulate NFAT transcriptional pathways and may be responsible for lymphoproliferative disorders (Scott et al. 2001).

Janeway C. A., Travers P., Walport M., Shlomchik M. J. 2001. Immunobiology. New York, NY: Garland Publishing. p. 204-205

Masuda E. S., Imamura R., Amasaki Y, Arai K, Arai N. 1998. Signalling into the T-Cell Nucleus: NFAT regulation. Cell Signal. 10 (9): 599-611.

Xiao S, Matsui K., Fine A., Zhu B., Marshak-Rothstein A., Widom R. L., Ju S-T. 1999. FasL promoter activation by IL-2 through SP1 and NFAT but not Egr-2 and Egr-3. Eur. J. Immunol. 29:3456-3465.

Szilak L., Moll J. R., Vinson C. 1999. Structure of the DNA-Binding Domains from NFAT, FOS and JUN Bound Specifically to DNA. Chemtracts. 12: 768-773

Trevillyan J. M., Chiou X. G., Chen Y. W., Ballarom S. J., Sheets M. P., Smith M. L., Wiedeman P. E., Warrior U., Wilkins J., Gubbins E. J., Gagne G. D., Fagerland J., Carter G. W., Jay R. L., Mollison K. W., Djuric S. W. 2001 December 21. Potent Inhibition of NFAT activation and T Cell Cytokine Production by Novel Low Molecular Weight Pyrazole Compounds. Journal of Biological Chemistry 276(51): 48118-48126.

Scott E. S., Malcomber S., O’Hare P. 2001 October. Nuclear Translocation and Activation of the Transcription factor NFAT Is Blocked by Herpres Simplex Virus Infection. Journal of Virology 75(20) 9955-9965.

Amasaki Y., Adachi S., Ishida Y., Iwata M., Arai N., Arai K., Miyatake S. 2002 July 12. A Constitutively Nuclear Form of NFATx Shows Efficient Transactivation Activity and Induces Differentiation of CD4+CD8+ T cells. The Journal of Biological Chemistry 227:28 25640-25648.

Horsley V., Pavlath G. K. 2002 March 5. NFAT: ubiquitous regulator of cell differentiation and adaptation. The Journal of Cell Biology: 156(5) 771-774.

Chen L, Mark Glover J. N., Hogan P. G., Rao A., Harrison S. C. 1998 March 5. Structure of the DNA-binding domains from NFAT, Fos and Jun bound specifically to DNA. Nature 392: 42-48

Macian F., Carmen G-R., Rao A. 2000. Gene expression elicited by NFAT in the presence or absence f cooperative recruitment of Fos and Jun. The EMBO Journal 19(17): 4783-4795.

Gomez J., Martinez-A C., Gonzalez A., Garcia A., Rebollo A. 1998. The Bcl-2 gene is idfferentiall regulated by IL-2 and IL-4: role of the transcription factor NFAT. Oncogene 17:1235-1243.

Macian F., Garcia-Cozar F., Im S-H., Horton H. F., Byrne M. C., Rao A. 2002 June 14. Transcriptional Mechanisms Underlying Lymphocyte Tolerance. Cell 109: 719-731.

Crabtree G. R., Olson E. N. 2002. NFAT Signaling: Choreographing the Social Lives of Cells. Cell 109: S67-S79.

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