Nuclear factor of activated T-cells (NFAT) is a family of transcription factors shown to be important in immune response. One or more members of the NFAT family is expressed in most cells of the immune system. NFAT is also involved in the development of cardiac, skeletal muscle, and nervous systems. NFAT was first discovered as an activator for the transcription of interleukin-2 in T cells, as a regulator for T cell immune response, but has since been found to play an important role in regulating many other body systems. NFAT transcription factors are involved in many normal body processes as well as in development of several diseases, such as inflammatory bowel diseases and several types of cancer. NFAT is also being investigated as a drug target for several different disorders.
The NFAT transcription factor family consists of five members NFATc1, NFATc2, NFATc3, NFATc4, and NFAT5. NFATc1 through NFATc4 are regulated by calcium signaling, and are known as the classical members of the NFAT family. NFAT5 is a more recently discovered member of the NFAT family that has special characteristics that differentiate it from other NFAT members. Calcium signaling is critical to NFAT activation because calmodulin (CaM), a well-known calcium sensor protein, activates the serine/threonine phosphatase calcineurin (CN). Activated CN rapidly dephosphorylates the serine-rich region (SRR) and SP-repeats in the amino termini of NFAT proteins, resulting in a conformational change that exposes a nuclear localization signal, resulting in NFAT nuclear import.
Signaling and Binding
Phosphorylation and dephosphorylation is key for controlling NFAT function by masking and unmasking nuclear localization signals, as shown by the high number of phosphorylation sites in the NFAT regulatory domain.
Nuclear import of NFAT proteins is opposed by maintenance kinases in the cytoplasm and export kinases in the nucleus. Export kinases, such as PKA and GSK-3β, must be inactivated for NFAT nuclear retention.
NFAT proteins have weak DNA-binding capacity. Therefore, to effectively bind DNA, NFAT proteins must cooperate with other nuclear resident transcription factors generically referred to as NFATn. This important feature of NFAT transcription factors enables integration and coincidence detection of calcium signals with other signaling pathways such as ras-MAPK or PKC. In addition, this signaling integration is involved in tissue-specific gene expression during development. A screen of ncRNA sequences identified in EST sequencing projects discovered a 'ncRNA repressor of the nuclear factor of activated T cells' called NRON.
The best known classes of binding sites for NFAT are the formation of a cooperative complex with AP-1 or other bZIP proteins to form a composite NFAT:AP-1 site that is involved in gene transcription in immune cells and the binding to sites for conventional Rel-family proteins.
NFAT-dependent promoters and enhancers tend to have 3-5 NFAT binding sites, which indicates that higher order, synergistic interactions between the relevant proteins in a cooperative complex is needed for effective transcription.
NFAT and AP-1
The best known classes of binding sites for NFAT are the formation of a cooperative complex with AP-1 or other bZIP proteins and the binding to sites for conventional Rel-family proteins. NFAT5 cannot form complexes with AP-1 proteins, however all NFAT proteins recognize similar DNA binding sites in gene regulatory regions.
Activation of NFAT and AP-1 is known to be required for productive immune responses. Cooperation of NFAT with AP-1 is required for many different genes to be transcribed including IL-2, GM-CSF, IL-3, and IFN-γ. In the thymus, FasL expression, which allows for potential cell death, also requires cooperation between NFAT and AP-1. This cooperation plays an important role in the cell survival or cell death checkpoint for developing T cells.
NFAT signaling in Neural Development
The Ca2+ dependent calcineurin/NFAT signaling pathway has been found to be important in neuronal growth and axon guidance during vertebrate development. Each different class of NFAT contributes to different steps in the neural development. NFAT works with neurotrophic signaling to regulate axon outgrowth in several neuronal populations. Additionally, NFAT transcription complexes integrate neuronal growth with guidance cues such as netrin to facilitate the formation of new synapses, helping to build neural circuits in the brain. NFAT is a known important player in both the developing and adult nervous system.
NFAT plays a role in the regulation of inflammation of inflammatory bowel disease (IBD). In the gene that encodes LRRK2 (leucine-rich repeat kinase 2), a susceptibility locus for IBD was found. The kinase LRRK2 is an inhibitor for the NFATc2 variety, so in mice lacking LRRK2, increased activation of NFATc2 was found in macrophages. This led to an increase in the NFAT-dependent cytokines that spark severe colitis attacks.
NFAT also plays a role in Rheumatoid Arthritis (RA), an autoimmune disease that has a strong pro-inflammatory component. TNF-α, a pro-inflammatory cytokine, activates the calcineurin-NFAT pathway in macrophages. Additionally, inhibiting the mTOR pathway decreases joint inflammation and erosion, so the known interaction between mTOR pathway and NFAT presents a key to the inflammatory process of RA.
NFAT is involved in a signaling pathway for cell transformation and tumorigenesis. The calcineurin/NFAT pathway signals for dephosphorylation of NFAT by activated calcineurin. This regulates the expression of several genes, including proteins helping with proliferation, growth, differentiation, migration, and survival, all processes commonly deregulated in cancer cells. Studies have shown that some forms of NFAT, such as the NFATc2 long isoform acts as a tumor suppressing gene whereas the NFATc1 short isoform functions as an oncogene. NFATc1 has been found in many aggressive B-cell lymphomas. Additionally, treating mice with calcineurin inhibitors that inactivate the calcineurin/NFAT pathway has been found to induce cell death in cancerous cells and inhibit the progression of the cell cycle. NFAT factors are promising therapeutic targets for these diseases.
In particular, NFAT transcription factors are implicated in breast cancer, more specifically in the process of cell motility at the basis of metastasis formation. Indeed NFAT1 (NFATC2) and NFAT5 are pro-invasive and pro-migratory in breast carcinoma and NFAT3 (NFATc4) is an inhibitor of cell motility. NFAT1 regulates the expression of the TWEAKR and its ligand TWEAK with the Lipocalin 2 to increase breast cancer cell invasion, and NFAT3 inhibits Lipocalin 2 expression to blunt the cell invasion.
As a drug target
Due to its essential role in the production of the T-cell proliferative cytokine interleukin-2, NFAT signaling is an important pharmacological target for the induction of immunosuppression. CN inhibitors, which prevent the activation of NFAT, including cyclosporine (CsA) and tacrolimus (FK506), are used in the treatment of rheumatoid arthritis, multiple sclerosis, Crohn's disease, and ulcerative colitis and to prevent the rejection of organ transplants. However, there is a toxicity associated with these drugs due to their ability to inhibit CN in non-immune cells, which limits their use in other situations that may call for immunosuppressing drug therapy, including allergy and inflammation. There are other compounds that target NFAT directly, as opposed to targeting the phosphatase activity of calcineurin, that may have broad immunosuppressive effects but lack the toxicity of CsA and FK506. Because individual NFAT proteins exist in specific cell types or affect specific genes, it may be possible to inhibit individual NFAT protein functions for an even more selective immune effect.
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