NFATc1 autoregulation: a crucial stepfor cell-fate determinationEdgar Serfling1, Sergei Chuvpilo1, Jiming Liu1, Thomas Hofer2 andAlois Palmetshofer1

1 Department of Molecular Pathology, Institute of Pathology, University of Wuerzburg, D-97080 Wuerzburg, Germany2 Department of Theoretical Biophysics, Institute of Biology, Humboldt University Berlin, D-10115 Berlin, Germany

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Nuclear factor of activated T cell c (NFATc) transcriptionfactors appeared in evolution with the emergence oflymphocytes in jawed fish. They have decisive roles inthe development of the immune system and adaptiveimmune responses. Following immunoreceptorstimulation, NFAT factors control the expression of alarge set of genes and thereby the fate of peripherallymphocytes. NFATc1 and NFATc2 are the mostprominent NFAT factors in peripheral T cells; theyoverlap in their function but differ remarkably inthe mode of expression. NFATc2 is constitutivelysynthesized in T cells, whereas the expression ofNFATc1/aA, the most prominent of six NFATc1 isoformsin peripheral T cells, is strongly induced following T-cellreceptor and co-receptor stimulation and maintained bypositive autoregulation. Findings concerning NFATc1autoregulation in peripheral T lymphocytes and othercells suggest that positive autoregulation of NFATc1 is acrucial step in cell-fate determination.

IntroductionNuclear factor of activated T cell (NFAT) transcriptionfactors were originally described as binding to and control-ling the interleukin 2 (IL-2) promoter and other lympho-kine promoters/enhancers in T cells. However, despitetheir designation (Table 1), it is now recognized that theexpression of four Ca2+-dependent NFATc transcriptionfactors is not restricted to T cells. They are also expressedin numerous other cells and tissues and are cruciallyinvolved in the regulation of osteoclast formation, neuro-genesis and angiogenesis, and in the development of heartvalves and septa [1–3]. Similar to NF-kB factors [4],NFATs are essential in the regulation of lymphocyte func-tions. The central role of NFATc factors in adaptiveimmune responses is demonstrated by the suppressionof T-cell activation by cyclosporin A (CsA) and FK06,two immunosuppressants that are widely used in trans-plantation medicine to prevent organ rejection. Whencomplexed with immunophilin receptors, CsA andFK506 bind to calcineurin and inhibit its protein phospha-tase activity. Without active calcineurin, the dephosphor-ylation, nuclear transport and transcriptional activation ofNFATs is blocked and, ultimately, T-cell activation issuppressed.

Corresponding author: Serfling, E. (serfling.e@mail.uni-wuerzburg.de).Available online 22 August 2006.

www.sciencedirect.com 1471-4906/$ – see front matter � 2006 Elsevier Ltd. All rights reserve

Among the three NFATc proteins expressed in immunecells, the synthesis of NFATc1 is strongly induced by T-cellreceptor (TCR) and co-receptor signals in effector T cells.We and others have shown that NFAT factors contribute tothe induction of the nfatc1 gene [5,6]. Here, we discussdetails of this positive autoregulation in nfatc1 gene tran-scription and its consequence on the control of gene expres-sion in T lymphocytes and other cells.

The nfatc1 gene: control by two promoters, alternativesplicing and two poly A sitesThe human and murine nfatc1 genes span �150 kb ofchromosomal DNA (Figure 1). The immediate upstreamsequences of nfatc1 exons 1 and 2 are highly conservedbetween human and mouse and have characteristics oftypical eukaryotic promoters: they correspond to CpGislands that are hypermethylated in cells from variousnonlymphoid tissues and in several human lymphoma cellsbut are hypomethylated in T lymphocytes; they formDNase I hypersensitive chromatin sites; and they containnumerous transcription-factor-binding sites (Figure 1a). Inaddition, transfection assays have shown that bothupstream regions confer transcriptional activity whencloned in front of a reporter gene [5]. We have thereforedesignated these regions as the nfatc1 P1 and P2 promo-ters. Cloning and sequencing of NFATc1 cDNAs fromvarious human and murine hematopoietic cells showedthat both P1 and P2 direct the synthesis of three differentRNAs. P1 transcripts start at exon 1 and encode NFATc1proteins bearing the so-called N-terminal a peptide of 42amino acids (aa), whereas P2 transcripts start at exon 2,and code for proteinswith theN-terminal b peptide of 29 aa(Figure 2) [7,8]. The C termini of these proteins compriseeither a short stretch of 19 aa in the A isoform, or longerstretches of 128 aa in the B isoform or 246 aa in the Cisoform [9] (Figure 2). The B and C isoforms arise fromalternative splicing and poly A addition at the distal sitepA2, whereas the short isoform A results from polyadeny-lation at the proximal poly A site pA1 (Figure 1b,c).

Together with the inducible activity of the promoter P1,polyadenylation at the proximal poly A site pA1 results inthe strong induction of the short isoformNFATc1/aA [9]. Inresting T cells, the NFATc1/b RNAs are the most promi-nent nfatc1 transcripts and their synthesis is reduced uponT-cell activation (Figure 1d). However, following activationin primary effector T cells or in T-cell lines of human ormurine origin, a 15–20-fold induction of NFATc1/aA RNA

d. doi:10.1016/j.it.2006.08.005

Table 1. NFAT transcription factors: names, genes and proteins

Designationb Other names Chromosomal loci Proteins (aa)

Human Mouse Human Mouse

NFATc1 NFAT2, NF-ATc 18q23 18E3 aA 716 716

bA 703 703

aB 825 827

bB 812 814

aC 943 945

bC 930 932

NFATc2 NFAT1, NF-ATp 20q13 2H3 A 1064 927

B 921 673

C 925 452

NFATc3 NFAT4, NF-ATx 16q22 8D2 1075 1076

1068 1068

1065 N.d.a

NFATc4 NFAT3 14q11 14C2 902 901

NFAT5 TonEBP, OREBP 16q22 8D2 1531 1487

NFATz 1455 1225aAbbreviation: N.d., not determined.bThis designation was recommended by the HUGO nomenclature committee and can be found at: http://www.gene.ucl.ac.uk/nomenclature/genefamily/NFAT/NFAT.shtml.

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was detected, whereas only a 2–5-fold increase wasobserved for the NFATc1/aB or NFATc1/aC RNAs(Figure 1d).

Similar to the IL-2 promoter, the prototype promoter forwhich activity is specifically induced upon T-cell activation[10], the inducible P1 promoter needs more than one signalfor its optimal induction. One signal is provided by apersistent increase in free cytosolic Ca2+ induced by iono-mycin, which stimulates the nuclear translocation andtranscriptional activation of all NFATc factors [1–3];another signal is provided by phorbol esters, which acti-vate protein kinase C and other protein kinase pathways inT cells. This suggests that both TCR and co-receptorsignals contribute to give full P1 nfatc1 induction. How-ever, in transient transfection assays (Box 1) using EL-4 Tcells, the induction of the P1 promoter differed markedlyfrom that of the endogenous P1 promoter, suggesting thatadditional remote regulatory sequence elements contri-bute to nfatc1 transcription. Instead of ionomycin- andphorbol-ester-mediated signals (mimicking TCR and co-receptor signals), P1 requires ionomycin and cyclic AMP(cAMP) signals for its optimal induction in the transfectionmodel [5]. The cAMP dependence seems to be reflected intwo CRE sites (Figure 1a) within P1 to which CREB, Fosand ATF-2 factors were detected to bind in electrophoreticmobility shift assays (EMSAs) (Box 1). ATF-2 is a knowntarget of p38 protein kinase, which was shown to supportnfatc1 induction in T cells [11]. Other transcription-factor-binding sites include those for Sp1, NF-kB, GATA andNFATs (Figure 1a).

There are two NFAT-binding sites within P1. The distalsite around position �675 to which NFATs bind with highaffinity is composed of two repeats of the NFAT corebinding motif TGGAAA. However, it does not constituteany TPA-responsive (TRE)-like motif of palindromic struc-ture, TGAC/GTCA, for the binding of AP1 factors. Muta-tions that abolished NFAT binding to the repeat motifsresulted in a strong decrease in P1 induction [5], and inchromatin immunoprecipitation (ChIP) assays (Box 1),NFATc2 and NFATc1 were observed to sequentially bindto P1 in primary murine T cells. However, a strong P1

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induction was detected in effector T cells from NFATc2+c3double-deficient mice, indicating that these NFATs areof minor importance for P1 induction in these cells(S. Chuvpilo et al., unpublished). Despite the strong NFATbinding to the P1 NFAT repeat, no NFAT dimer formationcould be detected using this site as a probe in EMSAs(Box 1). It is conceivable that, supported by the closelylinked NF-kB-like site, NFATs could form largercomplexes at P1. The formation of NFAT dimers has beendescribed for the composite NF-kB/NFAT sites of HIV longterminal repeats (LTRs) [12] and for a pseudo-palindromicNF-kB-like site from the human il-8 promoter [13].

Additional regulatory sequences controlling nfatc1 P1induction have been mapped within intron 1 of the murinenfatc1 gene [5]. One of two DNase I hypersensitive regionsof intron 1 (HSS 3) (Figure 3) enhanced P1 activity up tofivefold in transfections but did not alter the inductionproperties [5]. Candidate regions for controlling T-cell-specific expression could correspond to insertion sites ofretroviral viruses that weremapped either at the promoteror close to polyadenylaton sites of the murine nfatc1 gene[5] (http://rtcgd.ncifcrf.gov/index.html). Similar retroviralinsertions were detected for the chromosomal nfatc3 geneand were found to decrease its expression [14].

The NFATc1 proteins: autoregulation results in thepredominant synthesis of NFATc1/aA‘Prototypical’ NFATc2 and NFATc1/bC proteins arepresented in Figure 2a. All NFAT proteins carry aDNA-binding domain of �300 aa that shares more than70% sequence homology among NFATc proteins. Althoughthis domain shows less than 20% sequence homology to theDNA-binding domain of Rel/NF-kB factors, it adopts a verysimilar conformation and therefore is often designated asthe Rel similarity (or homology) domain (RSD or RHD)[1–3]. The DNA binding domains of NFAT and NF-kBfactors comprise two independently folded immunoglobu-lin subdomains (RSD-N and RSD-C) that form ten anti-parallel b barrels and two loop structures at identical sites[15]. In addition to its interaction with DNA, the RSD alsointeracts with the AP1 proteins Fos and Jun, the most

Figure 1. The nfatc1 gene and its expression in murine T lymphocytes. (a) The sequence organization of the inducible nfatc1 P1 promoter. The two blocks of �300 bp from

the immediate upstream region of the murine and human genes, which are highly conserved between both species, are shown. There are two NFAT sites and two

composite CREB/Fos/ATF2 sites in both DNA blocks. Their factor binding has been determined in EMSAs using nuclear protein extracts from T cells [5]. (b) A schematic

representation of the chromosomal nfatc1 gene showing the two promoters P1 and P2 and the two poly A addition sites, pA1 and pA2. Red indicates sequences specific for

NFATc1/aA, green are sequences occurring in b isoforms, and blue are sequences that are common to all isoforms. (c,d) The expression of the murine nfatc1 gene as six

individual NFATc1 RNAs. (c) A model of nfatc1 gene expression. In resting (or naive) T cells, the activity of the promoter P2 results in the transcription of exon 2, splicing to

exon 3 and in the generation of three b isoforms. T-cell activation leads to a promoter switch, from P2 to P1, and the transcription of exon 1, splicing to exon 3 and the

generation of a isoforms. Optimal induction conditions lead to the predominant synthesis of NFATc1/aA (red) by the use of the proximal polyA site pA1. Unlike the two

promoters, this site is not conserved between mouse and human. Although in both species NFATc1 proteins of about the same sizes are synthesized, the human NFATc1/A

RNA spans �3 kb [9], whereas the murine RNA spans 4.5 kb. Moreover, the relatively strong pA1 site in mouse seems to generate more NFATc1/A transcripts in resting T

cells than the relatively weak human pA1 site [9]. NFATc1/aA lacks the C-terminal transactivation domain TAD-B. The induction of NFATc1/a RNAs is shown in (d) for murine

EL-4 thymoma cells. RNA from EL-4 cells stimulated by the phorbol ester TPA and ionomycin was isolated followed by cDNA synthesis and semi-quantitative long distance

PCR assays using the mMACS mRNA isolation and cDNA synthesis kits from Miltenyi.

Review TRENDS in Immunology Vol.27 No.10 463

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Figure 2. The structure of NFATc proteins. (a) The structures of NFAT proteins NFATc2 and NFATc1/bC, and sequences of their N-terminal b-peptides. The structure of

NFATc2 is adapted from Ref. [2]. The DNA-binding (Rel similarity) domain (RSD) is shown in yellow, and the N- and C-terminal transactivation domains (TAD-B and TAD-C)

are shown in green. The C-terminal TAD harbors two highly conserved sumoylation motifs (around aa positions 684 and 897 in NFATc2). In nonactivated T cells, the

regulatory region of NFATc2 contains 14 phosphorylated serine residues. Of these, 13 (red circles) are targets of protein kinases CK1, GSK3 and DYRK1A/2, and upon T cell

activation they are dephosphorylated by calcineurin [51,61,62] in a cooperative manner resulting in full NFAT activation [63]. Additional protein kinases were also described

to phosphorylate this region [63–66]. A and B indicate calcineurin-binding motifs [19,21]. NLS, a nuclear localization signal containing the core motif KRR; SP-1, SP-2 and

SP-3, SP motifs harboring multiple Ser/Pro sequences; SRR-1, SRR-2 and KTS, serine-rich motifs that are phosphorylated as indicated. For more details see Refs. [2,3]. (b)

The structure of NFATc1/aA and sequence of its N-terminal a-peptide. The sequence of the NFATc1/aA-specific a-peptide is shown in red; putative phosphorylation and

acetylation motifs are underlined; proline residues are shown in black.

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common nuclear partners of NFATs in DNA binding uponT-cell activation [16]. This is reflected in the compositeNFAT/AP1 binding motif 50-GGAAAaxxxxTGAxTCA-30,which occurs approximately ten times more frequentlyin promoters induced following T-cell activation than inpromoters active in other tissues [17]. The contactsbetween NFAT and Fos/Jun proteins support the bindingof each partner to DNA [16].

Another common feature of all NFATc proteins (but notof NFAT5 [18]) is their calcineurin-binding or regulatorydomain that harbors almost all of the NFATc phosphor-ylation sites. They are mostly organized within the serine-rich sequence motifs SRR and SP1–SP3 and seem to beconstitutively phosphorylated in resting T cells andsequentially dephosphorylated by calcineurin(Figure 2a). Two calcineurin-interaction motifs have beenidentified within the regulatory domain: the N-terminalSPRIEIT motif [19,20] and a second site in the C-terminal

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region [21]. In addition, nuclear-localization andnuclear-export signals have also been identified withinthe regulatory domain (Figure 2a).

The regulatory region of NFATc1 overlaps with thestrong N-terminal transactivation domain, TAD-A,which we mapped in Gal4-transactivation assays (Box1) using numerous N-terminal peptides of variouslengths from NFATc1/aA [22]. In these assays most ofthe transactivation potential was detected within a pep-tide spanning residues 113 to 205 and additional activitywas detected within the N-terminal peptide of residues1–108. Similar to the binding of the transcriptionalcofactor p300/CBP to NFATc2 [23], TAD-A interacts withp300/CBP, which enhances TAD-A activity [22]. A secondtransactivation domain, TAD-B, was mapped withinthe unique 240 aa C terminus of NFATc1/C. AlthoughTAD-B seemed to be markedly weaker in transactivationassays, its activity could be induced several fold by

Box 1. Basic techniques to study transcriptional activities

DNase I hypersensitivity mapping. The appearance of DNase I

hypersensitive chromatin sites indicate ‘open’ chromatin, a landmark

for active regulatory DNA elements. This technique should be used

first for transcriptional studies of an unknown gene in (primary)

lymphocytes. The nuclei are isolated and mildly digested by DNase I.

The DNA is isolated and digested to completion by a restriction

enzyme which creates DNA fragments of 4–20 kb around the gene (or

gene segment) of interest. The digest is fractionated on an agarose

gel, blotted and hybridized with a labelled probe that recognizes one

end of the gene fragment. The bands that appear in the autoradio-

graph in lanes of digested DNA indicate the position of hypersensitive

sites within the restriction enzyme fragment (HSS 1–4 in Figure 3). For

an excellent review on the method by Cockerill see [60].

DNase I footprinting protection and electrophoretic mobility shift

assay (EMSA). Studies on transcription factor (TF) binding should

begin by using DNase I footprint protection assays. DNase I

footprinting studies provide an overview on TF binding to DNA

fragments of 300–600 bp in length. For in vitro DNase footprinting, a

50- (or 30-) end-labelled DNA probe is incubated with an excess of

nuclear proteins or recombinant factor protein(s) followed by partial

DNase I digestion and DNA extraction. The DNA fragments are

separated on a 6% urea-polyacrylamide sequencing gel. Separation of

a chemical sequencing reaction of the probe in parallel indicates the

sites protected by the factors bound to the DNA probe. Footprinting

assays using cell-permeable, DNA-modifying agents, such as DMS,

have been developed for determining the occupancy of TF binding

sites in vivo. Details of TF binding in vitro can be studied in EMSAs.

Here, a labelled oligonucleotide probe (e.g. spanning the identified

footprint) is incubated with nuclear proteins (or recombinant factor

protein) and an excess of unlabelled polydI�dC. The reaction is

fractionated on a non-denaturing polyacrylamide gel. The generation

of retarded bands (in addition to the free probe) indicates the

formation of protein–DNA complexes, the specificity of which can

be determined by competition with an excess of a known factor

binding site, or by ‘supershifting’ with a TF-specific antibody.

Chromatin immunoprecipitation assay (ChIP). This technique allows

the identification of specific TF binding to regulatory DNA elements in

vivo. Cells are fixed with 1% formaldehyde to cross-link TFs to

chromatin. Nuclei are isolated and sonified to shear chromatin to

segments of �500 bp in length. The nuclear lysate is incubated with

an antibody directed against a TF (or its modification), and

chromatin–antibody complexes are precipitated by protein A sephar-

ose. After the reversal of cross-links, the DNA is isolated and used in

(semi-) quantitative PCR assays using primers that recognize DNA

fragments (promoters, enhancers) of interest. Controls (using no or

unspecific antibodies in precipitations, PCR of input DNAs and so on)

should be performed to show the specificity of PCR signals.

Transient transfections and luciferase reporter gene assays. For

functional testing transcriptional activities, DNA fragments are cloned

in front of a (firefly or renilla) luciferase reporter gene, for example, in

pGL3 or pGL4 vectors (Promega). Using an appropriate protocol, the

DNA of these constructs is transfected into cells where the gene of

interest will be expressed. Following 1–2 days incubation, the

transcriptional activity of the DNA segment of interest is determined

by measuring luciferase activities in lysates from transfected cells and

control cells transfected with the empty vector DNA. A transfection

control should be included using a known promoter/enhancer and a

second luciferase reporter gene. In the best case, the DNA (promoter)

fragment of interest behaves identically (with respect to cell-type

specificity, induction or repression) to the endogenous gene of

transfected cells. To demonstrate the contribution of individual TFs,

mutations in TF binding sites that abolish TF binding have to be

introduced and tested in transfections.

Gal4-transactivation assay. Gal4-transactivation assays have

been widely used for measuring the transactivation capacity of

(individual domains of) TFs (see [22,25,26,28] for studies on

NFATs). In such assays, the DNA-binding domain of yeast TF Gal4

(spanning aa 1–147 of its N-terminal DNA-binding domain) is fused

to the TF or a TF segment of interest. This expression vector is

co-transfected with a luciferase (or CAT) reporter gene under

the control of multiple Gal4-binding sites into tissue culture cells

(e.g. EL-4 T cells). The observed activity of the reporter gene will

reflect the transactivation capacity of the chimeric Gal4–TF protein

in a concentration dependent manner.

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phorbol esters and (other) stress signals, similar toTAD-A. Remarkably, neither stimulation of calcineurinactivity by ionomycin nor inhibition by CsA affectedinduction of TAD-A or TAD-B [22,24]. A transactivationdomain was also mapped within the C-terminal peptideof NFATc2 [25] that shares �30% homology with that ofNFATc1/C. Near their C termini, the TAD-B domains inNFATc1/C and NFATc2 harbor a conserved hydrophobicdecapeptide that also occurs in other NFAT membersand exerts, in a somewhat extended 15 aa version, strongtransactivation activity when tested in Gal4 assays [26](Box 1). In addition, the TAD-B domains in NFATc1/Cand NFATc2 contain two potential CKxE sumoylationmotifs (where C corresponds to a hydrophobic aa, mainlyisoleucine or valine) within highly conserved aa stretchesaround aa positions 684 and 897 (in NFATc2). ForNFATc2, it has been shown that both motifs are sumoy-lated in vivo, and mutating the proximal 684 site to anon-sumoylated motif resulted in a strong decrease inNFATc2 activity and localization in nuclear (SUMO-1/PML?) bodies [27]. Following ectopic expression,NFATc2 and NFATc1/aC facilitated activation inducedcell death (AICD) of primary murine T cells followinganti-CD3 antibody stimulation, whereas NFATc1/aA hadno effect. This supports the view that AICD of T cells iscontrolled by mechanisms involving the C-terminal TAD-B of NFATc proteins [5].

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Contrary to NFATc1/bB and NFATc1/bC, the inducibleshort NFATc1/aA isoform does not harbor either the con-served hydrophobic decapeptide or sumoylation sites and,therefore, its activity is not controlled by sumoylation.Instead of containing the acidic N-terminal b-peptide of29 aa, it bears the N-terminal a-peptide of 42 aa thatharbors several motifs for potential protein modifications(Figure 2b). These motifs are seven Ser/Thr residues(whereas only one threonine is present in the b-peptide)and additional sites that, with high probability (accordingto computer-based predictions; http://www.expasy.org),include acetylation and glycosylation motifs. In mast cells,the a-peptide remained inactive, whereas the b-peptideexerted some transacting potency on its own inGal4 assays[28] (Box 1).

Autoregulation of NFATc1/aA synthesis: a crucial stepin tissue differentiationOsteoclasts: RANKL induces NFATc1/aA synthesis in

osteoclastogenesis

Homeostasis of the skeletal system is maintained by abone-remodelling process that is dependent on a fine-tunedbalance between bone formation by osteoblasts and boneresorption by osteoclasts. The receptor activator of nuclearfactor kB ligand (RANKL), a member of the TNF super-family, is a crucial inducer of osteoclast differentiationfrom bone marrow precursor cells [29]. One prominent

Figure 3. A model of activation of the nfatc1 gene locus during T-cell development. Portions of the nfatc1 promoter and polyA site regions are shown. (a) Opening of the

locus. NFATc1 expression can be detected in early DN thymocytes, before the expression of pre-TCR in late DN3 thymocytes. We assume that (lymphokine?) signals will

lead to the generation of DNase I hypersensitive sites, DNA hypomethylation and histone acetylation at the nfatc1 promoter region, resulting in the activation of the P2

promoter. This will lead to the generation of NFATc1/b transcripts. It is currently unknown whether at any stage in thymocyte development NFATc1 expression is controlled

by positive autoregulation. (b) The activation of the locus in naive CD4+ T cells. TCR (and co-receptor?) signals stimulate transcription from P2 into NFATc/b transcripts. This

is supported by Sp1/Sp3, Octamer (Oct) and other transcription factors that bind to P2 (and, probably, other regulatory DNA elements) and trigger the synthesis of low

NFATc1/b RNA levels. The pA2 site is predominantly used. (c) Transcription of the locus in effector T cells by positive NFATc1 autoregulation. At high concentrations of

NFATs and other transcription factors, splicing and polyA addition factors, upon TCR and co-receptor stimulation, transcription intiation switches from the use of promoter

P2 to P1, and polyadenylation from the distal pA2 site to the proximal pA1 site. Prominent transcription factors binding to P1 include NFATs (NFATc2, NFATc1), NF-kB,

CREB, ATF2 and Fos members. This results in the predominant synthesis of high levels of NFATc1/aA, the short NFATc1 isoform containing an N-terminal a peptide

(encoded in exon 1) and lacking the C-terminal TAD-B (encoded in exons 10 and 11). Thus, for optimal NFATc1/aA (auto-) induction, the coordinated activities of Ca2+/

calcineurin, NF-kB- and MAP/p38-kinase signaling pathways are necessary to induce P1 activity, transcription and polyadenylation at pA1. Because NFATc1/aA induction is

unaffected in NFATc2+c3 double-deficient T cells, NFATc1 autoregulates its own synthesis by controlling P1 activity and NFATc1/aA induction.

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signaling pathway induced by RANKL is theTAK1–MKK6–p38 kinase cascade, which leads to the acti-vation of NF-kB (by phosphorylating p65/RelA at Ser536)and, in combination with Ca2+ signals, to NFATc1 induc-tion in bone-marrow-derived macrophages [30–32].Although NFATc2 also facilitates the differentiation ofosteoclasts [33] and regulates chondrocyte differentiation[34], it does not seem to be required for osteoclastogenesisbecause NFATc2-deficient mice have a normal number ofosteoclasts [35]. In contrast, the inducible synthesis ofNFATc1/aA is essential for the generation of osteoclasts.This has convincingly been shown by the adoptive transferof fetal liver cells from NFATc1-deficient mice into c-Fos-deficient mice, which cannot form osteoclasts because of acell autonomous defect [36]. Although the transfer ofnfatc1+/� cells resulted in the amelioration of bone defects,nfatc1�/� cells were unable to cure these defects upontransfer into Fos-deficient mice. However, ectopic expres-sion of either NFATc1 or NFATc2 led to the rescue ofosteoclasts, suggesting that not the functional propertiesof the two NFAT factors but their expression levelsdetermine the development of bone marrow precursor cellsto mature osteoclasts [35]. This conclusion is supported bythe observations that: RANKL predominantly induces the

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synthesis of NFATc1/aA but not of NFATc2 or of any otherNFATc1 protein; this synthesis can be blocked by theimmunosuppressant FK506, an efficient inhibitor of osteo-clast formation, illustrating the NFAT-mediated autore-gulation of NFATc1/aA induction in osteoclasts [35].

Endocardial cells: an NFAT-driven intronic enhancer

contributes to pro-valve formation

Several members of the NFATc family have beenimplicated in cardiovascular development and cardiachypertrophy (e.g. [37,38]). Lethal defects have beenobserved in the formation of embryonic heart valves andsepta in mice deficient for NFATc1 [39,40]. In the devel-oping heart, NFATc1/aA seems to act as an endocardialtranscription factor that is essential for cardiac valvedevelopment [41]. When RNA from cultured primaryE11.5 endocardial cells was investigated using PCRassays, NFATc1/a RNA was found to be the predominantNFATc1 RNA. By investigating various DNA segmentsfrom the nfatc1 promoter region in transgenic mice, atissue-specific enhancer was identified located near the30 end of the first intron of the nfatc1 gene. By activatingthe P1 promoter, this enhancer activates and directs geneexpression in a subpopulation of pro-valve endocardial

Review TRENDS in Immunology Vol.27 No.10 467

cells. A 250 bp DNA fragment within the enhancer wasdetected to be crucial for active expression in developingendocardial cells. Within this region, binding sites wereidentified for GATA, Smad, Hox and NFAT factors.Whereas Hox proteins provide endocardial specificity sup-pressing NFATc1 expression outside pro-valve endocardialcells, cell-autonomous autoregulation of NFATc1 isrequired for maintaining high NFATc1 expression inpro-valve endocardial cells during valve development [41].

T lymphocytes: NFATc1 autoregulation is a property of

effector T helper cells

In murine T-cell development, NFATc1 expression can beobserved as early as in double-negative DN1 thymocytes(A. Patra et al. unpublished), that is, before the expressionof pre-TCR at the late DN3 stage [42]. This shows that(pre-)TCR signals do not control the ‘opening’ of the nfatc1locus in early thymocytes or their progenitors (Figure 3).However, TCR (and co-receptor) signals induce the tran-scription from the seemingly ‘repressed’ nfatc1 locus innaive CD4+ T cells, and persistent Ca2+ signals are neces-sary for maintaining full NFATc1 transcription. The initialphase of nfatc1 transcription in naive CD4+ T cells iscontrolled by the promoter P2which is constitutively activein resting T cells. The activation of resting T cells results ina decrease of P2 and the induction of P1 activity (Figure 1d)and, under optimal conditions, in the predominant synth-esis of NFATc1/aA in effector T cells (Figure 3). In additionto the high concentrations of poly A factors required foroptimal pA1 function [9], the levels of transcription factors,in particular NFATs, must also increase for P1 induction.Using single-cell assays, it was shown, by the Herzenberglaboratory more than 15 years ago [43], that NFATs mustexceed certain threshold levels for transcriptional activa-tion. Although the inactivation of one NFAT factor, eitherNFATc1 or NFATc2, in mice did not affect IL-2 production[44–48], the inactivation of both factors completely abol-ished IL-2 production [49]. This illustrates that a thresholdlevel provided by one or both NFAT factors was sufficientfor IL-2 promoter/enhancer induction, whereas below acertain threshold (provided byNFATc3, which is expressedat low levels in peripheral T cells) the IL-2 promoter/enhancer remained inactive. The P1 promoter might alsobe controlled by this binary (‘all or nothing’) mode ofinduction.

In addition to TCR signals leading to NFAT activation,CD28 (and further co-receptor) mediated signals exert a(co-)stimulatory effect on P1 induction, in particular in Th1cells. In Foxp3-expressing murine CD4+CD25+ T regula-tory cells (Tregs), the NFATc1 RNA levels were decreased�20-fold comparedwith levels in conventional CD4+ T cells[50], which might be owing to a low P1 activity in these(‘semi-anergic’) cells. Moreover, human T cells from SCIDpatients that are mutated in Orai, a CRAC channel com-ponent [51], and deficient in Ca2+ influx were unable toexpress NFATc1/aA [52].

Conclusions and perspectivesThe induction of CD4+ effector T cells by TCR and co-receptor signals leads to the synthesis of NFATc1/aA, ashort NFATc1 isoform with an altered transactivation

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potential. In T cells, NFATc1/aA is the only NFAT proteinfor which synthesis is controlled by positive autoregula-tion, resulting in high NFATc1 threshold levels. For thegeneration of osteoclasts it has been demonstrated thatthese high NFATc1 levels generated by autoregulation arenecessary for cell lineage commitment, and it is likely thatNFATc1/aA autoregulation has a similar crucial role in thedifferentiation of naive T cells to the various T effector cellpopulations. In addition, the altered transactivation poten-tial of NFATc1/aA suggests a specific role for this NFATc1protein in gene control, such as in Th1 effector cells whereNFATc1/aA is synthesized at high concentrations.

On the basis of our experimental data on NFATc1regulation in T cells, we have developed a mathematicalmodel (see the online supplementary material). Thetheoretical analysis demonstrates that NFATc1/aAexpression has a switch-like dependence on calcineurinactivity that is owing to the autoactivation feedback loop[53]. By contrast, the feedforward activators NF-kB,CREB, ATF-2 and Fos exert a graded effect on NFATc1/a levels. Provided that the calcineurin activity hasexceeded a threshold value, these activators will determinethe actual level of NFATc1/a expression. Thus, the expres-sion of the inducible NFATc1/aA isoform can be regulatedboth in a switch-like and in a gradual manner by differentstimuli.

Positive autoregulation has been described for severaltranscription factors, such as for Myo-D, Pit-1 and GATA-1in the commitment of nonlymphoid cells and for GATA-3 inthe commitment of naive CD4+ T cells to Th2 effector cells[53–58]. Recently, a positive autoregulatory loop has alsobeen postulated for NFATc4 in non-lymphoid cells inDown’s syndrome [59], further illustrating how importantmedical consequences might arise from subtle changes inautoregulatory networks. Positive autoregulation ensuresthe formation and maintenance of high concentrations ofimportant ‘master regulators’ of cell differentiation formaintaining a committed differentiation state. Althoughthe conditions for the induction of NFATc1/aA and othercommitment factors differ in detail, their common strategyto enhance the factor levels by autoregulation suggeststhat NFATc1/aA determines the fate of peripheralCD4+ T-cell subsets in a similar decisive way.

AcknowledgementsWe thank Anneliese Schimpl, Kirsty McPherson and Andris Avots forcritically reading the manuscript and for helpful suggestions. Theexperimental work of the Department of Molecular Pathology has beenthe supported by the DFG, the Wilhelm-Sander-Foundation, the Mildred-Scheel-Foundation for Cancer Research and the Volkswagen Foundation.

Supplementary dataSupplementary data associated with this article canbe found, in the online version, at doi:10.1016/j.it.2006.08.005.

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