Báo cáo Y học: Repression of FasL expression by retinoic acid involves a novel mechanism of inhibition of transactivation function of the nuclear factors of activated T-cells pptx

Repression of FasL expression by retinoic acid involves a novel
mechanism of inhibition of transactivation function of the nuclear
factors of activated T-cells
Mi-Ock Lee
1,
*, Hyo-Jin Kang
1,
*, Young Mi Kim
1
, Ji-Hyun Oum
2
and Jungchan Park
2
1
Department of Bioscience and Biotechnology, Institute of Bioscience, Sejong University, Seoul, Korea;
2
Department of Bioscience
and Biotechnology, Hankuk University of Foreign Studies, Kyounggi-do, Korea
Retinoids are potent immune modulators that inhibit Fas
ligand (FasL) e xpression and t hereby repress t he activation -
induced apoptosis of immature thymocytes and T-cell
hybridomas. In this study, we demonstrate that all-trans-
retinoic acid ( all-trans-RA) directly represses t he transcrip-
tional activity of the nuclear factors of activated T-cells
(NFAT), which is an important transactivator of the FasL
promoter. The analysis of reporter constructs containing the
FasL promoter and wild-type or mutant NFAT binding-
sites indicated that all-trans-RA repression was mediated via
an NFAT binding element located in the promoter. A
reporter construct comprising the NFAT binding sequence
linked to a heterologous SV-40 promoter showed that

NFAT transc riptional activity was significantly inhibited by
all-trans-RA. Furthermore, all- trans-RA inhibited a ctiva-
tion of the distal NFAT binding motif present in the inter-
leukin (IL)-2 p romoter, s uggesting that t he inhibition of
NFAT function by all-trans-RA was not specific to the FasL
promoter. Gel shift assays corroborated the results of the
gene reporter studies by showing that all-trans-RA decreased
the NFAT binding to DNA. All-trans-RA blocked trans-
location of NFATp from the cytosol into the nucleus, which
was induced by PMA/ionomycin treatment in HeLa cells
transfected with a Flag-tagged NFATp. Taken together, our
results indicate that FasL inhibition by all-trans-RA involves
a novel mechanism whereby the transcriptional function of
NFAT is blocked.
Keywords: r etinoic acid; NFAT; FasL.
The CD95 (Fas) ligand (FasL) is a type-II transmembrane
protein expressed on highly activated T-lymphocytes [1,2].
Activated T-lymphocytes undergo apoptosis following
homotypic interaction of F asL and its receptor, Fas [3–5].
Thus, the elimination of highly activated T-cells by the Fas/
FasL system is critical for the downregulation of immune
responses, the homeostasis of lymphocytes, and the main-
tenance of peripheral tolerance. Retinoids, vitamin A and its
natural and synthetic derivatives, regulate a wide array of
biological processes, includin g cellular proliferation, differ-
entiation, and immune modulation. All-trans-retinoic acid
(RA) and 9 -cis-RA inhibit FasL e xpression, and thereby
suppress the activation-induced apoptosis of immature
thymocytes and T -cell hybridomas [ 6–9]. The inhibitory
effects o f RA are mediated through two classes of nuclear

receptors, retinoic a cid receptors (RARs) and retinoid X
receptors (RXRs), both of which are ligand-dependent
transcriptional factors of the steroid/thyroid hormone
receptor superfamily [9–11]. However, the molecular details
of RA-mediated repression of FasL gene expression have
not been elucidated.
Nuclear factors of activated T-cells (NFAT) is a family of
related transcription factors that play a c entral role in
regulating the immune response by modulating the expres-
sion of important cytokines such as interleukin (IL)-2 in the
activated T-cells [12]. Five members of the NFAT family are
currently known, NFATp ( NFAT1, NFATc2), NFATc
(NFAT2, NFATc1), NFAT3 (NFATc4), NFAT4
(NFATc3, NFATx), and NFAT5, which share homology
within a region of t he DNA binding domain that is distantly
related to the Rel domain [13–17]. Moreover, various lines
of biochemical evidence, including knock-out studies and
tissue distribution patterns of t he proteins, indicate that
three of the NFAT family members, NFATp, NFATc, and
NFAT4, play important roles in the modulation and
development of the immune system [12,18]. Although
NFAT5 appears to be constitutively localized in the nucleus
and under t he regulation of osmotic shock, the other NFAT
family members are primarily controlled by their subcellular
localization depending on their phosphorylation status. In
resting T-cells, NFAT proteins are present in the cytoplasm
in a phosphorylated state. Activation via the T-cell receptor
(TCR) o r other stimulus results in an influx of calcium and
induces the dephosphorylation of NFAT, and r apid trans-
location of the protein into the nucleus [19,20]. D ephos-

phorylated NFAT binds to specific response elements and
thereby a ctivates a number o f genes, including those
Correspondence to M O. Lee, Department of Bioscience and
Biotechnology, Sejong University, 98 Kunja-dong, Kwangjin-gu,
Seoul 143-747, Korea. Fax: + 82 2 3408 3768,
Tel.: + 82 2 3408 3768, E-mail:
Abbreviations: FasL, Fas ligand; RA, retinoic acid; RARs, retinoic
acid receptors; RXRs, retinoid X receptors; NFAT, nuclear factors
of activated T-cells; TCR, T-cell receptor; CsA, cyclosporin A;
PBMCs, peripheral blood mononuclear cells; PMA, 4b-phorbol
12-myristate 13-acetate; b-gal, b-galactosidase; IL, interleukin; VDR,
vitamin D receptor.
*Note: both authors co ntributed equally to this work.
(Received 30 July 2001, revised 18 December 2001, accepted 19
December 2001)
Eur. J. Biochem. 269, 1162–1170 (2002) Ó FEBS 2002
encoding cytokines, cell surface receptors, signaling mole-
cules, and other, as y et unidentified, targets. As NFAT
dephosphorylation is mediated by the Ca
2+
/calmodulin-
dependent phosphatase, calcineurin, NFAT-regulated genes
are sensitive to inhibition by immunosuppressive agents that
inhibit calcineurin, such as cyclosporin A (CsA) and FK506
[21].
Recently, several studies have demonstrated the in volve-
ment of NF AT in the t ranscriptional activation of F asL
[22–25]. Therefore, w e speculated that N FAT inhibition
might be an important mechanism through which RA
inhibited the expression of FasL. In this study, we show that

all-trans-RA inhibits FasL expression by blocking tran-
scriptional activation by NFAT. Our r esults suggest t he
therapeutic potential of targeting NFAT function with RA
to achieve immunosuppression.
EXPERIMENTAL PROCEDURES
Cells and reagents
The Jurkat human T-cell leukemia (ATCC, CRL1990), and
HeLa human cervical carcinoma (ATCC, CCL-2) cell lines
were obtained from the American Type Culture Collection.
Cells were maintained in RPMI 1640 medium containing
10% fetal bovine serum. Human peripheral blood mono-
nuclear cells (PBMCs) were isolated from healthy donors by
density gradient centrifugation of heparinized blood on a
layer of Ficoll/Hypaque (Sigma, St. Louis, MO, USA). All-
trans-RA, 9-cis-RA, 4b-phorbol 12-myristate 13-acetate
(PMA) and CsA were purchased from Sigma. Ionomycin
was obtained from Calbiochem (La Jolla, CA, USA). All
other chemicals used were of the purest grade available from
Sigma.
RT-PCR for FasL
Jurkat cells (2 · 10
6
cells) were treated with a mixture of
PMA (10 ngÆmL
)1
) and ionomycin (0.5 l
M
) for 6 h with or
without a 24-h pretreatment with various concentrations of
all-trans-RA. Total R NA w as prepared using Q iagen

RNeasy kit (Qiagen Inc., Chatsworth, CA, USA) following
the m anufacturer’s instructions. RT-PCR was performed
essentially as described previously [26]. cDNA was synthe-
sized from 4 lg total RNA using 100 ng random hexamer
(Pharmacia, Uppsala, Sweden). The PCR primer sequences
used were as follows. FasL (forward: 5¢-ATGTTTCAGC
TCTTCCACCTACAGAAGGA-3¢,reverse:5¢-CAGAGA
GAGCTCAGATACGTTGAC-3¢); and b-actin (forward:
5¢-CGTGGGCCGCCCTAGGCACCA-3¢,reverse: 5¢-TTG
GCCTTAGGGTTCAGGGGGG-3¢. PCR cycling condi-
tions were: de-naturation at 94 °C for 30 s, annealing at
52 °C for 30 s and extension at 72 °C for 30 s. Twenty-eight
cycles were carried out for amplification of FasL and 22
cycles for b-actin.
Plasmids and reporter gene assay
The luciferase reporter constructs containing a 2 .3-kb
fragment (from n ucleotide s )2365 to )2) and a 320-bp
fragment (nucleotides )318 to )2) of genome region located
5¢ upstream of the FasL translation initiation s ite, and the
luciferase reporters containing mutatio ns in the NFAT
(DNFAT) or SP1 (DSP-1) sites, were previously described
[22]. The luciferase reporter constructs containing deleted
promoter fragments (nucleotides )1783 to )2) and (nucleo-
tides )1703 to )2), were constructed b y r estricting the
2.3-kb full promoter using XhoIandNcoI/XhoI, respectively.
The NFAT-Luc reporter was constructed by inserting an
oligonucleotide en coding the NFAT binding site of the
FasL promoter (5¢-ATTGTGGGCGGAAACTTCCAG-3¢)
with additional GATC motifs at the 5¢ endintotheBglII site
of the pGL2-promoter (Promega, Madison, WI) that

carries an SV40 promoter. The eukaryotic expression
vectors carrying Flag-NFATp, RARa,RARb, RARc,
and RXRa have been reported previously [27,28]. Jurkat
cells (1–2 · 10
7
cells) were transfected with reporter plas-
mids (7.5 lg) or with a b-galactosidase (b-gal) expression
vector (2.5 lg) by electroporation. CV-1 cells were seeded in
a 24-well culture p late at 5 · 10
4
cells per well, and
transfected with DNA mixtures (1 lg per well) containing
reporter plasmids (0.1 lg), the eukaryotic expression vector
encoding Flag-NFATp (25 ng), the retinoid receptor
expression plasmid (25 ng), or the b-gal expression vector
(0.15 lg) with carrier DNA (pBluescript). The cell cultures
were incubated for 6 h with PMA (10 ngÆmL
)1
)and
ionomycin (0.5 l
M
), in the presence or a bsence of all-
trans-RA. At the end of the incubation period, luciferase
activity was determined using a luminometer according to
the manufacturer’s instructions. The luciferase activity was
normalized for transfection efficiency using the correspond-
ing b-gal activity.
To examine the effects of all-trans-RA on IL-2 NFAT
site-dependent transcription, we employed a Jurkat cell line
that was s tably transfected with the NFATZH reporter

construct (Oum, J H. & Park, J., unpublished r esults). The
reporter construct contained three copies of the distal
NFAT binding site in the human I L-2 promoter and a
minimal IL-2 promoter, upstream of the b- gal gene [29]. The
Jurkat-NFAT cells (1 · 10
5
cells per well) were cultured in a
24-well plate and stimulated for 6 h with PMA
(10 ngÆmL
)1
) and ionomycin (0.5 l
M
), in the presence or
absence of a ll-trans-RA (2.0 l
M
). The b-gal activity was
determined using t he fluorogenic substrate 4-methyl-lum-
bellifery-b-galactoside, and was normalize d for protein
content [30]. A one-way analysis of variance was performed
using GraphPad
INSTAT
Ò (GraphPad Software, San Diego,
CA, USA). A value of P < 0.05 was considered statistically
significant.
Electrophoretic mobility shift assay (EMSA)
PBMCs (7 · 10
6
cells) obtained from healthy dono rs were
stimulated in a 100-cm
2

plates precoated with anti-CD3 Ig
(100 lgÆmL
)1
) for 4 h with or without various concentra-
tions of all-trans-RA p retreatment. A mouse antibody
against human C D3 was prepared from the supernatants of
OKT3 hybridoma cell cultures [28]. Nuclear extracts were
prepared from the PBMCs and gel-shift assays were carried
out using p reviously desc ribed meth ods [28]. Nuclear
extracts (5 lg) were incubated for 20 min at 25 °Cwith
32
P-labeled oligonucleotides encoding either t he NFAT or
SP-1 binding sequences in a 20-lL reaction mixture
containing 10 m
M
Tris buffer ( pH 7.5), 1 00 m
M
KCl,
1m
M
dithiothreitol, 1 m
M
EDTA, 0.2 m
M
phenyl-
methanesulfonyl fluoride, 1 mgÆmL
)1
BSA, and 5%
Ó FEBS 2002 Repression of NFAT by retinoic acid (Eur. J. Biochem. 269) 1163
glycerol. The sequences of oligonucleotides used as probe in

the experiments were: NFAT, 5¢-GATCATTGTGGGCG
GAAACTTCC AG-3¢; and SP-1, 5¢-GATCGATCGGGG
CGGGGCGAG-3¢.
Immunofluorescence studies
For the subcellular localization studies, HeLa cells (1 · 10
6
per well) were transie ntly transfected with 4 lg Flag-
NFATp using LipofectaminePlus
TM
(Gibco BRL, Grand
Island, NY, USA) according to t he manufacturer’s instruc-
tions. The transfected HeLa cells were cultured for 24 h on
poly
L
-lysine-coated 11-mm coverslips. Th e cells were
stimulated with PMA (10 ngÆmL
)1
) and ionomycin
(0.5 l
M
), in the presence or absence of all-trans-RA
(1.0 l
M
). Following treatment, the cells were fixed overnight
at )20 °C in a methanol/acetone (1 : 1) solution. The cells
were then stained with an anti-(Flag M 2) Ig (Upstate
Biotech., Lake Placid, NY, USA) a t a co ncentration of
1 lgÆmL
)1
in NaCl/P

i
and 1% bovine serum albumin,
followed by a biotin-labeled, anti-(mouse Ig) Ig (1 : 1000,
Vector Laboratories, I nc., Burlingame, CA, USA), and
streptavidin–fluorescein isothiocyanate (1 : 200, Vector
Laboratories). Fluorescent cells were washed with NaCl/P
i
and visualized by confocal microscopy (Nikon, Japan).
RESULTS
All-
trans
-RA represses FasL expression
As RA has been shown to inhibit the expression of FasL in
the immature thymocytes and T-cell hybridomas [6–8], we
confirmed these data using a human leukemia cell line,
Jurkat. The addition of PMA and ionomycin into culture
media remarkably induced the expression of FasL in Jurkat
cells and the induction was r epressed by all-trans-RA
treatment in a dose-dependent manner (Fig. 1A). FasL
transcription was decreased at all-trans-RA concentrations
as low as 0.01 l
M
, and was almost completely abolished at
1.0 l
M
. To further establish the inhibitory effect of all-trans-
RA on FasL gene expression, we employed a luciferase
reporter system containing the 2 .3-kb genomic DNA
fragment that is sufficient for transcriptional activation of
the FasL gene [22]. Transient transfection of the reporter

into Jurkat cells produced a 3.25-fold increase in reporter
gene activity in response to PMA and iono mycin treatmen t,
a finding that was consistent with previously rep orted results
[22]. Approximately 80% of the reporter gene activity was
repressed i n the presence of all- trans-RA (Fig. 1B). In
summary, the results f rom R T-PCR and r eporter g ene
analyses clearly showed that RA decreased the transcrip-
tional expression of FasL in Jurkat cells.
The NFAT binding motif in the FasL promoter
confers responsiveness to all-
trans
-RA
We studied the RA-responsive, cis-regulatory elements in
the FasL promoter, in order to elucidate t he molecular
mechanism through which RA represses FasL expression.
First, we tested the responsiveness to all-trans-RAoffour
reporter constructs c ontaining serially deleted FasL pro-
moters (Fig. 2A). As shown in Fig. 2B, all-trans-RA
significantly repressed the transcriptional induction of the
four reporter genes that were induced by PMA a nd
ionomycin treatment. These results suggested that the
putative R A-responsive elements were located w ithin the
nucleotides )318 to )2 region of the FasL pro moter.
The FasL promoter (nucleotides )318 to )2) contains
several potential cis-acting r egulatory ele me nts, including
binding sites for NFAT and SP-1 [22–25]. However, there
are no consensus retinoid-responsive elements prese nt in
this region, suggesting that retinoid receptors may not bind
directly to this portion of the FasL promoter. Therefore, it is
possible that the activities of RA are m ediated through

transcriptional modulation by other nuclear transcriptional
factors, such as NFAT and SP-1. To test this hypothesis, we
employed reporters encoding mutated DNA-bind ing
sequences fo r NFAT o r SP-1 (Fig. 3A). When the wild-
type or SP-1-mutated reporter was transfected into Jurkat
cells, PMA and ionomycin treatment induced an approxi-
mately 3.5-fold increase in reporter gene activation
(Fig. 1 B). Co-treatment with all-trans-RA of cells carrying
either of these reporter constructs repressed the PMA and
ionomycin-induced reporter gene a ctivity by approximately
80% (Fig. 3B). In contrast, neither PMA and ionomycin
nor all-trans-RA treatment meaningfully modulated the
transcriptional activity of a reporter g ene c ontaining the
Fig. 1 . All-trans-RA represses the induction of FasL expression. (A)
The effects of all-trans-RA on FasL transcription were examined using
RT-PCR. Jurkat cells were incubated with the indicated concentra-
tions o f all- trans -RA f or 24 h a nd then treated with PMA
(10 ngÆmL
)1
)andionomycin(0.5l
M
) for 6 h. T h e expression of
b-actin was monitored as a co ntrol. (B) The FasL (nucleotides )2306
to )2)-Luc reporter, together with the b-gal expression vector, was
transiently transfected into Jurkat cells as described in the Experi-
mental p rocedu res. Transfected cells were trea ted with PMA
(10 ngÆmL
)1
) and ionomycin (0.5 l
M

) in t he presence or absence of
1.0 l
M
RA for 6 h. The luciferase activity was measured and nor-
malized by b-gal activity. Data are shown as the mean ± S E of three
independent measurements.
1164 M O. Lee et al. (Eur. J. Biochem. 269) Ó FEBS 2002
mutated NFAT sequence. These results indicated that all-
trans-RA repressed the FasL promoter, mainly through the
inhibition of NFAT a ctivity. T o further c onfirm t he
involvement of NFAT, we generated a reporter construct,
NFAT(FasL)-Luc, in w hich an NFAT binding site from the
FasL promoter was subcloned upstream of a heterologous
SV40 promoter and luciferase. When t his construct was
transfected into Jurkat cells, the reporter gene activity was
increased about threefold by P MA and ionomycin treat-
ment; approximately 60% a nd 70% of the P MA and
ionomycin-induced reporter gene activity was repressed by
the addition of all-trans-RA and 9-cis-RA, respectively
(Fig. 3C). We then tested whether all-trans-RA inhibited the
transcriptional activation driven b y NFAT binding
sequences present in other NFAT target genes. F or this
purpose, we employed a Jurkat cell line in which b-galacto-
sidase expression was under the control of three copies of
the distal IL-2 NFAT site upstream of the minimal IL-2
promoter. As shown in Fig. 3D, approximately 65% and
85% of the reporter gene transcriptional activity induced by
PMA and ionomycin was repressed by treatment with all-
trans-RA and 9-cis-RA, respectively (Fig. 3 D), indicating
that all-trans-RA-induced repression of NFAT binding

motifs was not specific for the FasL promoter, and further
supporting our contention that RA modulates the transac-
tivation function of NFAT.
We also cotransfected the NFAT(FasL)-Luc reporter,
along with the retinoid receptor expression plasmid, into
CV-1 cells, in order to investigate whether the modulatory
activities of all-trans-RA were mediated by retinoid recep-
tors. As shown in Fig. 4, NFAT-Luc was strongly induced
by PMA and ionomycin in the p resence of NFATp.
Although all-trans-RA d id not induce a significant repres-
sion of the reporter g ene a ctivity in t he absence o f
cotransfection with the retinoid receptor plasmid, repression
was greater when plasmids containing RARa,RARb,
Fig. 2 . Delineation of all-trans-RA-responsive cis-actingelementsinthe
FasL promoter. (A) Schematic representation of the deletions in the
5¢ term inus of the FasL promoter that were cloned upstream of a
luciferase report er gene. The 3¢ end of the FasL promoter contains
nucleotide )2, counted from the translation initiation site, and tran-
scription s tarts from nucleotide )181 [22]. (B) Each reporter construct
was transiently transfected into Jurkat cells. Transfected cells were
stimulated with PMA (25 ngÆmL
)1
)andionomycin(0.5l
M
)inthe
absence (empty bar) or presence (filled bar) of all-trans-RA ( 2.0 l
M
)
for 6 h. Luciferase activity was measured and normalized by b-gal
activity. To establish the reporter construct basal expression, pTK-luc,

which contains a minimal promoter of thymidine kinase, was also used
in the transfection assay.
Fig. 3 . The effect of all-trans-RA is mediated by an NFAT binding motif
present in the FasL promoter region. (A ) S chema tic representation of
the FasL promoter (nucleotides )318 to )2) reporter construct, along
with NFAT and SP-1 binding sites. The nucleotide sequences of the
NFAT- and SP-1- bindin g sites and of mutations in these sites are
shown. (B) The indicated rep orter constructs together with a b-gal
expression vector were transiently t ransfected into Jurkat cell s as
described in the E xperimental procedures. Transfected c ells were
treated with PMA (10 ng ÆmL
)1
) and iono mycin (0.5 l
M
)inthepres-
ence or absence of R A (1.0 l
M
) for 6 h. Luciferase activity was mea-
sured and normalized by b-gal activity. (C) The NFAT(FasL)-Luc
construct was transfected into Jurkat cells and incubated for 6 h with
PMA (10 ngÆmL
)1
)andionomycin(0.5l
M
) in the absence or presence
of all-trans-RA (1.0 l
M
). Luciferase activity was measured and nor-
malized by b-gal act ivity. (D) Jurkat-NFAT cells were treated with
PMA (25Æng mL

)1
)/ionomycin (0.5 l
M
), CsA (1 lgÆmL
)1
), and RA
(2.0 l
M
)for6h,asindicated.b-Gal activity was measured and nor-
malized with the protein concentrations of cell extracts. All data from
the reporter gene assays are shown as the mean ± SE of more than
three indepen dent me asurem ents.
Ó FEBS 2002 Repression of NFAT by retinoic acid (Eur. J. Biochem. 269) 1165
and/or RXRa were cotransfected (Fig. 4 ). Interstingly,
ligand-dependent repression was observed when RXRa or
RARa/RXRa was c otransfected, i mplicating that RXR
plays an important role in repressing NFAT activity.
However, RARc expression did not induce a significan t
change in the reporter gene activity. These results indicated
that the i nhibition of FasL expression by RA involves a
novel mechanism of NFAT blockage that is mediated by a
subset of the r etinoid receptors.
All-
trans
-RA inhibits the DNA binding activity of NFAT
To understand the molecular m echanism of RA-induced
inhibition of NFAT activity, we investigated whether the
DNA-binding activity o f N FAT w as changed b y R A
treatment. When PBMCs were stimulated with anti-CD3
Ig, binding to the NFAT binding sequence from t he FasL

promoter was s ignificantly increased (Fig. 5A). However,
the induced NFAT binding activity was significantly
inhibited by all-trans-RA t reatment, whereas binding to
the consensus S P-1 b inding seq uence was unchanged. A
100-fold excess of unlabeled probe or of an unlabeled
oligonucleotide encompassing the NFAT binding sequence
from the I L-2 promoter, completely abolished the protein–
DNA complexes, whereas a 100-fold excess of a nonspecific
oligonucleotide had no effect, indicating that the complex
was specific. As shown in Fig. 5B, the repression of NFAT–
DNA b inding by all-trans-RA was dose-dependent; the
repression was observed with all-trans-RA concentrations
as low as 0 .1 l
M
, and NFAT binding was abolished in the
presence of 1.0 l
M
all-trans-RA. In contrast, SP-1 binding
was similar at all co ncentrations of all-trans-RA tested.
All-
trans
-RA blocks NFAT translocation to the nucleus
Activation via the T-cell receptor (TCR) or stimuli suc h as
ionomycin results in the rapid dephosphorylation of
NFAT and its translocation into t he nucleus [ 19,20].
Therefore, we spec ulated that the observed d ecrease in
NFAT–DNA binding might be due to a decrease i n the
amount of NFAT proteins translocated from the cytosol
into the nucleus. To test this hypothesis, we analyzed the
effects of all-trans-RA on the nuclear s huttling of

NFATp. We performed immunocytochemistry on HeLa
cells that had b een t ransiently transfected with Flag-
tagged recombinant NFATp. The Flag-tagged NFATp
was found in the cytoplasm of unstimulated cells, a nd
all-trans-RA treatment did not induce significant changes
in the recombinant protein localization (Fig. 6). Following
stimulation with PMA and ionomycin, NFATp was
translocated to the nucleu s in t he majority of the cells.
PMA and ionomycin-induced translocation was reduced
by approximately 70% when the cells received cotreat-
ment with all-trans-RA, and was almost completely
inhibited by the addition of CsA.
Fig. 4 . Retinoid r eceptors repres s the transcriptional activity of the
NFAT response element. The NFAT(FasL)-Luc was cotransfected,
along with the indicated retinoid receptor expression vector (25 ng)
and NFATp (25 ng), into CV-1 cells, as described in the Experimental
procedures. Transfected cells w ere incubated for 6 h with PMA
(10 ngÆmL
)1
) and ionomycin (0.5 l
M
) in the absence or presence of all-
trans-RA ( 1.0 l
M
)or9-cis -RA (1.0 l
M
). Lu ciferase activity was
measured and normalized by b-gal activity. Data are shown as the
mean ± S E of three independen t measurements.
Fig. 5 . All-trans-RA represses the DNA-binding activity of NFAT. A,

PBMCs (7 · 10
6
cells) obtained from a healthy donor were stimulated
in a 100-cm
2
plate that was precoated with anti-CD3 Ig for 4 h with or
without 1.0 l
M
all-trans-RA. B, Jurkat cells (3 · 10
6
cells) were treated
with PMA ( 10 ngÆmL
)1
) and ionomycin (0.5 l
M
)for4h,inthe
presence or a bsence of a 24 -h pretreatment with all-trans-RA, as
indicated. The reaction mixture co ntaining 5 lg nuclear extract was
incubated with
32
P-labeled oligonucleotide and analyzed by gel shift
assay, as d escribed in the Experimental procedures. The designations
for cNFAT(FasL), cNFAT(IL-2), and cSP-1 indicate a 100-fold excess
of the competing unlabeled oligonucleotides.
1166 M O. Lee et al. (Eur. J. Biochem. 269) Ó FEBS 2002
DISCUSSION
Although i t h as been convincingly d ocumented that RA
induces the repression of T-cell apoptosis and FasL gene
expression, the underlying molecular mechanism has not
been clarified. In this study, we demonstrated that the

repression of FasL transcription by RA w as mediated
through the i nhibition of NFAT f unction. Both r eporter
gene analyses and DNA binding assays indicated that all-
trans-RA mediated t his r epression through the NFAT
binding sequence in the FasL promoter. In addition, we
showed that all-trans-RA inhibited NFAT–DNA binding,
as well as NFAT entry into the nucleus from the cytosol.
Therefore, our results indicate that FasL expression inhibi-
tion by RA involves a novel mechanism of NFAT
transcription inhibition.
The biolo gical functions o f RA are mainly mediated by
the ligand-depend ent transcriptional factors RAR a nd
RXR, which belong to the steroid/thyroid receptor super-
family [7–9]. Several studies ind icate that protein–protein
interactions between nuclear retinoid receptors mediate
cellular cross-talk, thus generating diverse gene-regulatory
pathways. For instance, it was found that RXR could
physically interact with either NF-jBorIjBb, resulting in
the repression o f IL-12 production in macrophages or
altered LPS responses, respectively [31,32]. Furthermore, it
has been shown t hat PPARc, a nother member of t he
steroid/thyroid receptor superfamily, interacts with NFAT
at the protein level in T-lymphocytes, resulting in d ecreased
IL-2 production [33]. Another potential mechanism involves
competition for DNA binding at the NFAT site in the FasL
promoter. In t his regard, RXR w as reported to play a
crucial role in immunosuppression induced by 1 a,25
(OH)
2
D

3
, the active metabolite of vitamin D, by forming
heterodimers with the vitamin D r eceptor (VDR), wh ich
can compete with NFAT-AP-1 binding on the IL-2
promoter NFAT site [ 34,35]. In addition, it has recently
been reported that the activity of the inducible N-terminal
transactivation domain of NFATc was coactivated by CBP/
p300, well-characterized coactivators of RAR/RXR [36].
Therefore, competition for CBP/p300 between these tran-
scriptional factors might result i n the inhibition of the
NFAT activity. Similarly, the cross-talk between retinoid
receptors and NFAT might take place at the protein level,
as NFAT inhibition by all-trans-RAwasgreaterinthe
presence of retinoid receptors (Fig. 4). Therefore, further
investigations into each of these potential mechanisms are
warranted, in o rder to further understand the retinoid
receptor-induced inhibition of NFAT. Interestingly, Szondy
and others have shown that RARa stimulation inhibited,
whereas RARc enhanced, activation-induced apoptosis
[37,38]. Similarly, we showed that RAR a repressed NFAT
function, while RARc did not (Fig. 4). Th us, b alanced
RARa/RARc stimulation may decide whether all-trans-RA
enhances or inhibits the transcriptional activity of NFAT
and thereby FasL expression, which controls activation-
induced apoptosis.
Activation via the TCR or some other stimulus ind uces
calcium influx and leads to th e dephosphorylation and rapid
translocation into the nucleus of NFAT, where it activates a
number of target genes. The dephosphorylated NFAT may
be rephosphorylated at serine residues by either removing

the s timulus or treating cells with a c alcineurin i nhibitor
such as CsA, whereby it i s translocated back to the
cytoplasm [19,39]. While NFAT dephosphorylation is
mediated by calcineurin, rephosphorylation is catalyzed by
a variety of serine kinases, such as glycogen synthase kinase-
3, ERK, p38, casein kinase-2, and c-Jun N-terminal kinase
[40–43]. These enzymatic activities may b e targeted by RA
in order t o block NFAT dephosphorylation by repressing
calcineurin and/or activating the specific serine kinases. For
example, dithiocarbamate, a powerful inhibitor of NF-jB,
inhibited NFAT dephosphorylation b y i nducing a pro-
longed activation of the c-Jun N-terminal kinase [44]. O ur
preliminary results indicated that all-trans-RA inhibited
dephosphorylation of NFAT, which could be an important
Fig. 6 . All-trans-RA blocks nuclear translocation of NFAT. A, HeLa cells, transfected with an expression vector encoding the Flag epitope-tagged
NFATp, were cultured on poly
L
-lysine-coated coverslips for 24 h. Cells were treated with PMA and ionomycin or vehicle for 30 min in the
presence or absence of all-trans-RA or CsA. The cells were fixed and stained with anti-Flag Ig, followed by mouse-biotin and streptavidin–FITC, as
described in the Experimental procedures. (A) no treatment; (B) all-trans-RA (1.0 l
M
); (C) PMA (10 ngÆmL
)1
) and ionomycin (0.5 l
M
); (D) all-
trans-RA (1.0 l
M
) with PMA (10 n gÆmL
)1

) and ionomycin (0.5 l
M
); E, CsA (1 lgÆmL
)1
) with PMA (10 ng ÆmL
)1
) and ionomycin (0.5 l
M
).
Ó FEBS 2002 Repression of NFAT by retinoic acid (Eur. J. Biochem. 269) 1167
mechanism for RA-induced repression of nuclear translo-
cation of NFAT (Kang, H J. & Lee, M O., unpublished
results). Therefore, further studies are required to e stablish
whether RA modulates the activities of the enzymes that
affect nuclear translocation and transcriptional activity of
NFAT.
The NFAT proteins regulate the expression of FasL and
a discrete set of cytokines involved in the regulation of
immune responses, such as proliferation and differentiation,
as well as in multiple effector functions of immune cells. The
promoters of the IL-2, GM-CSF, IL-3, IL-4 a nd tumor
necrosis factor alpha genes contain different types of NFAT
binding elements that are independently active or combine
with AP-1 binding sites [12]. The previous observations that
all-trans-RA repressed IL-2 production and IL-2 gene
transcription [45,46] correlate with our present findings
(Fig. 3 D). Currently, CsA and FK506 are the most
powerful i mmunosuppressive drugs available t hat target
calcineurin function. However, their clinical use is limited
because of the toxic s ide-effects caused by inhibition of the

many biological pathways controlled by calcineurin. There -
fore, there is considerable therapeutic interest in drugs that
directly target NFAT and allow reductions in CsA/FK506
dosage. In this regard, RA, o r its more potent and receptor
subtype-selective analogues, may sub serve the role of such
agents.
Recently, the physiological importance of NFAT in
cells other t han t hose o f the immune system has been
uncovered. The widespread distribution of NFAT
mRNA and/or proteins in nonlymphoid tissues, including
the heart, testis, brain, ovary, small intestine, prostate,
colon, muscle, placenta, lung, and kidney, as well as in
skin [47–50], suggests that NFAT f amily members might
control cellular differentiation programs in these organ
systems. Indeed, recent evidence s uggests t hat NFAT
may participate in a dipogenesis and m yogenesis [49,50].
Interestingly, retinoid receptor expression has been
implicated in cardiomyopathy and congestive heart
failure [51–53], sugge sting a potential link between
RA-induced repression of NFAT and the pathophysiol-
ogy of these diseases. Given the importance of NFAT in
fundamental physiology, the i nhibition of NFAT func-
tion by retinoids may be a critical factor in NFAT-
mediated biological signaling.
ACKNOWLEDGEMENTS
We thank Dr Carlos V. Paya (The Mayo Clinic, Rochester, MN, USA)
for t he luciferase reporter constructs. We also thank Dr Crabtree
(Stanford University, Stan ford, CA, USA) for F lag-NFATp and
NFATZH. This work w as supported by a grant (KRF-99–015-
DP0398) from the Korea Research Foundation to M O. L . and J. P.

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