Repression of FasL expression by retinoic acid involves a novelmechanism of inhibition of transactivation function of the nuclear factors of activated T-cells Mi-Ock Lee1,*, Hyo-Jin Kang
Trang 1Repression 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 Lee1,*, Hyo-Jin Kang1,*, Young Mi Kim1, Ji-Hyun Oum2and Jungchan Park2
1
Department of Bioscience and Biotechnology, Institute of Bioscience, Sejong University, Seoul, Korea;2Department of Bioscience and Biotechnology, Hankuk University of Foreign Studies, Kyounggi-do, Korea
Retinoids are potent immune modulators that inhibit Fas
ligand (FasL) expression and thereby repress the
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 the
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 transcriptional activity was significantly inhibited by
all-trans-RA Furthermore, all-trans-RA inhibited
activa-tion of the distal NFAT binding motif present in the inter-leukin (IL)-2 promoter, suggesting that the 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-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: retinoic 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 FasL 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, including cellular proliferation,
differ-entiation, and immune modulation All-trans-retinoic acid
(RA) and 9-cis-RA inhibit FasL expression, and thereby
suppress the activation-induced apoptosis of immature
thymocytes and T-cell hybridomas [6–9] The inhibitory
effects of RA are mediated through two classes of nuclear
receptors, retinoic acid 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 central 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
(NFATc3, NFATx), and NFAT5, which share homology within a region of the 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 the 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 the 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) or other stimulus results in an influx of calcium and induces the dephosphorylation of NFAT, and rapid trans-location of the protein into the nucleus [19,20] Dephos-phorylated NFAT binds to specific response elements and thereby activates a number of 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: molee@sejong.ac.kr
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 contributed equally to this work.
(Received 30 July 2001, revised 18 December 2001, accepted 19
December 2001)
Trang 2encoding cytokines, cell surface receptors, signaling
mole-cules, and other, as yet unidentified, targets As NFAT
dephosphorylation is mediated by the Ca2+
/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
involve-ment of NFAT in the transcriptional activation of FasL
[22–25] Therefore, we speculated that NFAT 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 results suggest the
therapeutic potential of targeting NFAT function with RA
to achieve immunosuppression
E X P E R I M E N T A L P R O C E D U R E S
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· 106 cells) were treated with a mixture of
PMA (10 ngÆmL)1) and ionomycin (0.5 lM) for 6 h with or
without a 24-h pretreatment with various concentrations of
all-trans-RA Total RNA was prepared using Qiagen
RNeasy kit (Qiagen Inc., Chatsworth, CA, USA) following
the manufacturer’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 nucleotides )2365 to )2) and a 320-bp
fragment (nucleotides)318 to )2) of genome region located
5¢ upstream of the FasL translation initiation site, and the
luciferase reporters containing mutations 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 by restricting the 2.3-kb full promoter using XhoI and NcoI/XhoI, respectively The NFAT-Luc reporter was constructed by inserting an oligonucleotide encoding the NFAT binding site of the FasL promoter (5¢-ATTGTGGGCGGAAACTTCCAG-3¢) with additional GATC motifs at the 5¢ end into the BglII 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· 107 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 plate at 5· 104 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 lM), in the presence or absence 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 stably transfected with the NFATZH reporter construct (Oum, J.-H & Park, J., unpublished results) The reporter construct contained three copies of the distal NFAT binding site in the human IL-2 promoter and a minimal IL-2 promoter, upstream of the b-gal gene [29] The Jurkat-NFAT cells (1· 105cells per well) were cultured in a 24-well plate and stimulated for 6 h with PMA (10 ngÆmL)1) and ionomycin (0.5 lM), in the presence or absence of all-trans-RA (2.0 lM) The b-gal activity was determined using the fluorogenic substrate 4-methyl-lum-bellifery-b-galactoside, and was normalized for protein content [30] A one-way analysis of variance was performed using GraphPadINSTATÒ (GraphPad Software, San Diego,
CA, USA) A value of P < 0.05 was considered statistically significant
Electrophoretic mobility shift assay (EMSA) PBMCs (7· 106cells) obtained from healthy donors were stimulated in a 100-cm2plates precoated with anti-CD3 Ig (100 lgÆmL)1) for 4 h with or without various concentra-tions of all-trans-RA pretreatment A mouse antibody against human CD3 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 previously described methods [28] Nuclear extracts (5 lg) were incubated for 20 min at 25°C with
32P-labeled oligonucleotides encoding either the NFAT or SP-1 binding sequences in a 20-lL reaction mixture containing 10 mM Tris buffer (pH 7.5), 100 mM KCl,
1 mM dithiothreitol, 1 mM EDTA, 0.2 mM phenyl-methanesulfonyl fluoride, 1 mgÆmL)1 BSA, and 5%
Trang 3glycerol 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· 106
per well) were transiently transfected with 4 lg
Flag-NFATp using LipofectaminePlusTM(Gibco BRL, Grand
Island, NY, USA) according to the manufacturer’s
instruc-tions The transfected HeLa cells were cultured for 24 h on
poly L-lysine-coated 11-mm coverslips The cells were
stimulated with PMA (10 ngÆmL)1) and ionomycin
(0.5 lM), in the presence or absence of all-trans-RA
(1.0 lM) 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 M2) Ig (Upstate
Biotech., Lake Placid, NY, USA) at a concentration of
1 lgÆmL)1 in NaCl/Pi and 1% bovine serum albumin,
followed by a biotin-labeled, anti-(mouse Ig) Ig (1 : 1000,
Vector Laboratories, Inc., Burlingame, CA, USA), and
streptavidin–fluorescein isothiocyanate (1 : 200, Vector
Laboratories) Fluorescent cells were washed with NaCl/Pi
and visualized by confocal microscopy (Nikon, Japan)
R E S U L T S
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 repressed 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 lM, and was almost completely abolished at
1.0 lM 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 ionomycin treatment,
a finding that was consistent with previously reported results
[22] Approximately 80% of the reporter gene activity was
repressed in the presence of all-trans-RA (Fig 1B) In
summary, the results from RT-PCR and reporter gene
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 the molecular
mechanism through which RA represses FasL expression
First, we tested the responsiveness to all-trans-RA of four
reporter constructs containing 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 and ionomycin treatment These results suggested that the putative RA-responsive elements were located within the nucleotides)318 to )2 region of the FasL promoter The FasL promoter (nucleotides)318 to )2) contains several potential cis-acting regulatory elements, including binding sites for NFAT and SP-1 [22–25] However, there are no consensus retinoid-responsive elements present 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 mediated through transcriptional modulation by other nuclear transcriptional factors, such as NFAT and SP-1 To test this hypothesis, we employed reporters encoding mutated DNA-binding sequences for NFAT or 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 1B) Co-treatment with all-trans-RA of cells carrying either of these reporter constructs repressed the PMA and ionomycin-induced reporter gene activity by approximately 80% (Fig 3B) In contrast, neither PMA and ionomycin nor all-trans-RA treatment meaningfully modulated the transcriptional activity of a reporter gene containing 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 of all-trans-RA for 24 h and then treated with PMA (10 ngÆmL)1) and ionomycin (0.5 l M ) for 6 h The expression of b-actin was monitored as a control (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 procedures Transfected cells were treated with PMA (10 ngÆmL)1) and ionomycin (0.5 l M ) in the 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 ± SE of three independent measurements.
Trang 4mutated NFAT sequence These results indicated that
all-trans-RA repressed the FasL promoter, mainly through the
inhibition of NFAT activity To further confirm the
involvement of NFAT, we generated a reporter construct,
NFAT(FasL)-Luc, in which an NFAT binding site from the
FasL promoter was subcloned upstream of a heterologous
SV40 promoter and luciferase When this construct was
transfected into Jurkat cells, the reporter gene activity was
increased about threefold by PMA and ionomycin
treat-ment; approximately 60% and 70% of the PMA 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 by NFAT binding
sequences present in other NFAT target genes For 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 3D), 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 presence of NFATp Although all-trans-RA did not induce a significant repres-sion of the reporter gene activity in the absence of cotransfection with the retinoid receptor plasmid, repression was greater when plasmids containing RARa, RARb,
Fig 2 Delineation of all-trans-RA-responsive cis-acting elements in the
FasL promoter (A) Schematic representation of the deletions in the
5¢ terminus of the FasL promoter that were cloned upstream of a
luciferase reporter gene The 3¢ end of the FasL promoter contains
nucleotide )2, counted from the translation initiation site, and
tran-scription starts from nucleotide )181 [22] (B) Each reporter construct
was transiently transfected into Jurkat cells Transfected cells were
stimulated with PMA (25 ngÆmL)1) and ionomycin (0.5 l M ) in the
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) Schematic 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- binding sites and of mutations in these sites are shown (B) The indicated reporter constructs together with a b-gal expression vector were transiently transfected into Jurkat cells as described in the Experimental procedures Transfected cells were treated with PMA (10 ngÆmL)1) and ionomycin (0.5 l M ) in the pres-ence or abspres-ence of RA (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) and ionomycin (0.5 l M ) in the absence or presence
of all-trans-RA (1.0 l M ) Luciferase activity was measured and nor-malized by b-gal activity (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 ) for 6 h, as indicated 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 independent measurements.
Trang 5and/or RXRa were cotransfected (Fig 4) Interstingly,
ligand-dependent repression was observed when RXRa or
RARa/RXRa was cotransfected, implicating that RXR
plays an important role in repressing NFAT activity
However, RARc expression did not induce a significant
change in the reporter gene activity These results indicated
that the inhibition of FasL expression by RA involves a
novel mechanism of NFAT blockage that is mediated by a
subset of the retinoid receptors
All-trans-RA inhibits the DNA binding activity of NFAT
To understand the molecular mechanism of RA-induced
inhibition of NFAT activity, we investigated whether the
DNA-binding activity of NFAT was changed by RA
treatment When PBMCs were stimulated with anti-CD3
Ig, binding to the NFAT binding sequence from the FasL
promoter was significantly increased (Fig 5A) However,
the induced NFAT binding activity was significantly
inhibited by all-trans-RA treatment, whereas binding to
the consensus SP-1 binding sequence was unchanged A
100-fold excess of unlabeled probe or of an unlabeled
oligonucleotide encompassing the NFAT binding sequence
from the IL-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 binding by all-trans-RA was dose-dependent; the
repression was observed with all-trans-RA concentrations
as low as 0.1 lM, and NFAT binding was abolished in the
presence of 1.0 lMall-trans-RA In contrast, SP-1 binding
was similar at all concentrations of all-trans-RA tested
All-trans-RA blocks NFAT translocation to the nucleus
Activation via the T-cell receptor (TCR) or stimuli such as
ionomycin results in the rapid dephosphorylation of
NFAT and its translocation into the nucleus [19,20]
Therefore, we speculated that the observed decrease in
NFAT–DNA binding might be due to a decrease in 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 shuttling of NFATp We performed immunocytochemistry on HeLa cells that had been transiently transfected with Flag-tagged recombinant NFATp The Flag-Flag-tagged NFATp was found in the cytoplasm of unstimulated cells, and 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 nucleus in the 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 receptors repress 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 were 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 ) or 9-cis-RA (1.0 l M ) Luciferase activity was
measured and normalized by b-gal activity Data are shown as the
mean ± SE of three independent 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-cm2plate 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 ) for 4 h, in the presence or absence of a 24-h pretreatment with all-trans-RA, as indicated The reaction mixture containing 5 lg nuclear extract was incubated with 32 P-labeled oligonucleotide and analyzed by gel shift assay, as described 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.
Trang 6D I S C U S S I O N
Although it has been convincingly documented 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 was mediated
through the inhibition of NFAT function Both reporter
gene analyses and DNA binding assays indicated that
all-trans-RA mediated this repression 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 biological functions of RA are mainly mediated by
the ligand-dependent transcriptional factors RAR and
RXR, which belong to the steroid/thyroid receptor
super-family [7–9] Several studies indicate 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-jB or IjBb, resulting in
the repression of IL-12 production in macrophages or
altered LPS responses, respectively [31,32] Furthermore, it
has been shown that PPARc, another member of the
steroid/thyroid receptor superfamily, interacts with NFAT
at the protein level in T-lymphocytes, resulting in decreased
IL-2 production [33] Another potential mechanism involves
competition for DNA binding at the NFAT site in the FasL
promoter In this regard, RXR was reported to play a
crucial role in immunosuppression induced by 1a, 25
(OH)2D3, the active metabolite of vitamin D, by forming
heterodimers with the vitamin D receptor (VDR), which
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 in 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-RA was greater in the presence of retinoid receptors (Fig 4) Therefore, further investigations into each of these potential mechanisms are warranted, in order 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 RARa repressed NFAT function, while RARc did not (Fig 4) Thus, balanced 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 induces calcium influx and leads to the 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 stimulus or treating cells with a calcineurin inhibitor such as CsA, whereby it is 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 be targeted by RA
in order to 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 by inducing a pro-longed activation of the c-Jun N-terminal kinase [44] Our 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 ngÆmL)1) and ionomycin (0.5 l M ); E, CsA (1 lgÆmL)1) with PMA (10 ngÆmL)1) and ionomycin (0.5 l M ).
Trang 7mechanism for RA-induced repression of nuclear
translo-cation of NFAT (Kang, H.-J & Lee, M.-O., unpublished
results) Therefore, further studies are required to establish
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 and 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 3D) Currently, CsA and FK506 are the most
powerful immunosuppressive drugs available that target
calcineurin function However, their clinical use is limited
because of the toxic side-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, or 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 than those of 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 family members might
control cellular differentiation programs in these organ
systems Indeed, recent evidence suggests that NFAT
may participate in adipogenesis and myogenesis [49,50]
Interestingly, retinoid receptor expression has been
implicated in cardiomyopathy and congestive heart
failure [51–53], suggesting 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 inhibition of NFAT
func-tion by retinoids may be a critical factor in
NFAT-mediated biological signaling
A C K N O W L E D G E M E N T S
We thank Dr Carlos V Paya (The Mayo Clinic, Rochester, MN, USA)
for the luciferase reporter constructs We also thank Dr Crabtree
(Stanford University, Stanford, CA, USA) for Flag-NFATp and
NFATZH This work was supported by a grant
(KRF-99–015-DP0398) from the Korea Research Foundation to M.-O L and J P.
R E F E R E N C E S
1 Pinkoski, M.J & Green, D.R (1999) Fas ligand, death gene Cell
Death Differ 6, 1174–1181.
2 Nagata, S (1999) Fas ligand-induced apoptosis Annu Rev Genet.
33, 29–55.
3 Dhein, J., Walczak, H., Baumier, C., Debatin, K.-M & Krammer,
P.H (1995) Autocrine T-cell suicide mediated by APO-1/ (Fas/
CD95) Nature 373, 438–441.
4 Brunner, T., Mogil, R.J., LaFace, D., Yoo, N.J., Mahboubl, A.,
Echeverri, F., Martin, S.J., Force, W.R., Lynch, D.H., Ware, C.F.
& Green, D.R (1995) Cell-autonomous Fas (CD95) /Fas–ligand interaction mediates activation-induced apoptosis in T-cell hybri-domas Nature 373, 441–444.
5 Ju, S.-T., Panka, D.J., Cui, H., Ettinger, R., El-Khatib, M., Sherr, D.H., Stanger, B.Z & Marshak-Rothstein, A (1995) Fas (CD95) /FasL interactions required for programmed cell death after T-cell activation Nature 373, 444–448.
6 Iwata, M., Mukai, M., Nakai, Y & Iseki, R (1992) Retinoic acids inhibit activation-induced apoptosis in T cell hybridomas and thymocytes J Immunol 149, 3302–3308.
7 Szondy, Z., Reichert, U., Bernardon, J.-M., Michel, S., To´th, R., Kara´szi, E & Fe´su¨s, L (1998) Inhibition of activation-induced apoptosis of thymocytes by all-trans- and 9-cis-retinoic acid
is mediated via retinoic acid receptor b Biochem J 331, 767– 774.
8 Yang, Y., Minucci, S., Ozato, K., Heyman, R.A & Ashwell, J.D (1995) Efficient inhibition of activation-induced fas ligand up-regulation and T cell apoptosis by retinoids requires occupancy
of both retinoid X receptors and retinoic acid receptors J Biol Chem 270, 18672–18677.
9 Bissonnette, R.P., Brunner, T., Lazarchik, S.B., Yoo, N.J., Boehm, M.F., Green, D.R & Heyman, R.A (1995) 9-cis Retinoic acid inhibition of activation-induced apoptosis is mediated via regulation of fas ligand and requires retinoic acid receptor and retinoid X receptor activation Mol Cell Biol 15, 5576– 5585.
10 Yang, Y., Merc´ep, M., Ware, C.F & Ashwell, J.D (1995) Fas and activation-induced Fas ligand mediate apoptosis of T cell hybri-domas: Inhibition of Fas Ligand expression by retinoic acid and glucocorticoids J Exp Med 181, 1673–1682.
11 Mangelsdorf, D.J., Thummel, C., Beato, M., Herrlich, P., Schutz, G., Umesono, K., Blumberg, B., Kastner, P., Mark, M., Cham-bon, P & Evans, R.M (1995) The nuclear receptor superfamily: the second decade Cell 83, 835–839.
12 Rao, A., Luo, C & Hogan, P.G (1997) Transcription factors of the NFAT family: regulation and function Annu Rev Immunol.
15, 707–747.
13 McCaffrey, P.G., Luo, C., Kerppola, T.K., Jain, J., Badalian, T.M., Ho, A.M., Burgeon, E., Lane, W.S., Lambert, J.N., Curran, T., Verdine, G.L., Rao, A & Hogan, P.G (1993) Isolation of the cyclosporin-sensitive T cell transcription factor NFATp Science
262, 750–754.
14 Northrop, J.P., Ho, S.N., Chen, L., Thomas, D.J., Timmerman, L.A., Nolan, G.P., Admon, A & Crabtree, G.R (1994) NF-AT component define a family of transcription factors targeted in T-cell activation Nature 369, 497–502.
15 Hoey, T., Sun, Y.-L., Williamson, K & Xu, X (1995) Isolation of two new members of the NF-AT gene family and functional characterization of the NF-AT proteins Immunity 2, 461–472.
16 Masuda, E.S., Naito, Y., Tokumitsu, H., Campbell, D., Saito, F., Hannum, C., Arai, K & Arai, N (1995) NF-ATx, a novel member of the nuclear factor of activated T cells family that
is expressed predominantly in the thymus Mol Cell Biol 15, 2697–2706.
17 Lopez-Rodriguez, C., Aramburu, C.J., Rakeman, A.S & Rao, A (1999) NFAT5, a constitutively nuclear NFAT protein that does not cooperate with Fos and June Proc Natl Acad Sci USA 96, 7214–7219.
18 Kiani, A., Rao, A & Aramburu, J (2000) Manipulating immune responses with immunosuppressive agents that target NFAT Immunity 12, 359–372.
19 Beals, C.R., Clipstone, N.A., Ho, S.N & Crabtree, G.R (1997) Nuclear localization of NF-ATc by a calcineurin-dependent, cyclosporin–sensitive intramolecular interaction Genes Dev 11, 824–834.
20 Shaw, K.T.-Y., Ho, A.M., Raghavan, A., Kim, J., Jain, J., Park, J., Sharma, S., Rao, A & Hogan, A.G (1995)
Trang 8Immunosuppres-sive drugs prevent a rapid dephosphorylation of transcription
factor NFAT1 in stimulated immune cells Proc Natl Acad Sci.
USA 92, 11205–11209.
21 Ho, S., Clipstone, N., Timmermann, L., Northrop, J., Graef, I.,
Fiorentino, D., Nourse, J & Crabtree, G.R (1996) The
mecha-nism of action of cyclosporin A and FK506 Clin Immunol.
Immunopathol 80, S40–S45.
22 Holtz-Heppelmann, C.J., Algeciras, A., Badley, A.D & Paya,
C.V (1998) Transcriptional regulation of the human FasL
promoter-enhancer region J Biol Chem 273, 4416–4423.
23 Rengarajan, J., Mittelstadt, P.R., Mages, H.W., Gerth, A.J.,
Kroczek, R.A., Ashwell, J.D & Glimcher, L.H (2000) Sequential
involvement of NFAT and Egr transcription factors in FasL
regulation Immunity 12, 293–300.
24 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.
25 Latinis, K.M., Norian, L.A., Eliason, S.L & Koretzky, G.A.
(1997) Two NFAT transcription factor binding sites participate in
the regulation of CD95 (Fas) ligand expression in activated human
T cells J Biol Chem 272, 31423–31434.
26 Shin, E.-C., Shin, J.-S., Park, J.-H., Kim, H & Kim, S.-J (1999)
Expression of fas ligand in human hepatoma cell lines: role of
hepatitis-B virus X (HBx) in induction of fas ligand Int J Cancer
82, 587–591.
27 Ho, S.N., Thomas, D.J., Timmerman, L.A., Li, X., Francke, U &
Crabtree, G.R (1995) NFATc3, a lymphoid-specific NFATc
family member that is calcium-regulated and exhibits distinct
DNA binding specificity J Biol Chem 270, 19898–19907.
28 Kang, H.-J., Song, M.-R., Lee, S.-K., Shin, E.-C., Choi, Y.-H.,
Kim, S.J., Lee, J & Lee, M.-O (2000) Retinoic acid and its
receptors repress the expression and transactivation function of
Nur77: a possible mechanism for the inhibition of apoptosis by
retinoic acid Exp Cell Res 256, 545–554.
29 Mattila, P.S., Ullman, K.S., Fiering, S., Emmel, E.A.,
McCutch-eon, M., Crabtree, G.R & Herzenberg, L.A (1990) The actions of
cyclosporin A and FK506 suggest a novel step in the activation of
T lymphocytes EMBO J 9, 4425–4433.
30 Oda, Y., Kinoshota, M & Kakehi, K (1997) Fluorometric assay
of binding specificity of plant lectins to yeast cells by biotin-avidin
system and its application to the classification of yeast cells Anal.
Biochem 254, 41–48.
31 Na, S.Y., Kang, B.Y., Chung, S.W., Han, S.J., Ma, X., Trinchieri,
G., Im, S.Y., Lee, J.W & Kim, T.S (1999) Retinoids inhibit
interleukin-12 production in macrophages through physical
associations of retinoid X receptor and NFkappaB J Biol Chem.
274, 7674–7680.
32 Na, S.Y., Kim, H.J., Lee, S.K., Choi, H.S., Na, D.S., Lee, M.O.,
Chung, M., Moore, D.D & Lee, J.W (1998) IjBb interacts with
the retinoid X receptor and inhibits retinoid-dependent
transacti-vation in lipopolysaccharide-treated cells J Biol Chem 273,
3212–3215.
33 Yang, X.Y., Wang, L.H., Chen, T., Hodge, D.R., Resau, J.H.,
DaSilva, L & Farrar, W.L (2000) Activation of human T
lymphocytes is inhibited by peroxisome proliferator-activated
receptor gamma (PPARgamma) agonists PPARc
co-asso-ciation with transcription factor NFAT J Biol Chem 275, 4541–
4544.
34 Alroy, I., Towers, T.L & Freedman, L.P (1995) Transcriptional
repression of the interleukin-2 gene by vitamine D3: direct
inhib-tion of NFATp/AP-1 complex formainhib-tion by nuclear hormone
receptor Mol Cell Biol 15, 5789–5799.
35 Takeuchi, A., Reddy, G.S., Kobayashi, T., Okano, T., Park, J.C.
& Sharma, S (1998) Nuclear factor of activated T cells (NFAT)
as a molecular target for 1a,25-dihydroxyvitamine D3-mediated
effects J Immunol 160, 209–218.
36 Avots, A., Buttmann, M., Chuvpilo, S., Escher, C., Smola, U., Bannister, A.J., Rapp, U.R., Kouzarides, T & Serfling, E (1999) CBP/p300 integrates Raf/Rac-signaling pathways in the tran-scriptional induction of NFATc during T cell activation Immunity
10, 515–524.
37 Szondy, Z., Reichert, U & Fesus, L (1998) Retinoic acids regulate apoptosis of T lymphocytes through an interplay between RAR and RXR receptors Cell Death Differ 5, 4–10.
38 Szondy, Z., Reichert, U., Bernardon, J.M., Michel, S., Toth, R., Karaszi, E & Fesus, L (1998) Inhibition of activation-induced apoptosis of thymocytes by all-trans- and 9-cis-retinoic acid
is mediated via retinoic acid receptor alpha Biochem J 331, 767–774.
39 Loh, C., Shaw, K.T.-Y., Carew, J., Viola, J.P.B., Luo, C., Perrino, B.A & Rao, A (1996) Calcineurin binds the transcription factor NFAT1 and reversibly regulates its activity J Biol Chem 271, 10884–10891.
40 Beals, C.R., Sheridan, C.M., Turck, C.W., Gardner, P & Crab-tree, G.R (1997) Nuclear export of NF-Atc enhanced by glycogen synthase kinase-3 Science 28, 1930–1934.
41 Zhu, J., Shibasaki, F., Price, R., Guillemot, J.C., Yano, T., Dotsch, V., Wagner, G., Ferrar, P & McKeon, F (1998) Intracellular masking of nuclear import signal on NF-AT4 by casein kinase I and MEKK1 Cell 93, 851–861.
42 Chow, C.W., Rincon, M., Cavanagh, J., Dickens, M & Davis, R.J (1997) Nuclear accumulation of NFAT4 opposed by the JNK signal transduction pathway Science 278, 1638–1641.
43 Porter, C.M., Havens, M.A & Clipstone, N.A (2000) Identifi-cation of amino acid residues and protein kinases involved in the regulation of NFATc subcellular localization J Biol Chem 275, 3543–3551.
44 Martinez, S., Gomez, P., Armesilla, A.L., Aramburu, J., Luo, C., Rao, A & Redondo, J.M (1997) Blockade of T cell activation by dithiocarbamates involves novel mechanisms of inhibition of nuclear factor of activated T cells Mol Cell Biol 17, 6437–6447.
45 Felli, M.P., Vacca, A., Meco, D., Screpanti, I., Farina, A.R., Maroder, M., Martinotti, S., Petrangeli, E., Frati, L & Gulino, A (1991) Retinoic acid-induced down-regulation of the interleukin-2 promoter via cis-regulatory sequences containing an octamer motif Mol Cell Biol 11, 4771–4778.
46 Grazia, U., Felli, M.P., Vacca, A., Farina, A.R., Maroder, M., Cappabianca, L., Meco, D., Farina, M., Screpanti, I., Frati, L & Gulino, A (1994) Positive and negative regulation of the composite octamer motif of the interleukin 2 enhancer by AP-1, Oct-2, and retinoic acid receptor J Exp Med 180, 1485– 1497.
47 Armesilla, A.L., Lorenzo, E., Gomez, P., Martinez, S., Alfranca,
A & Redondo, J.M (1999) Vascular endotherial growth factor activates nuclear factor of activated T cells in human endothelial cells: a role for tissue factor gene expression Mol Cell Biol 19, 2032–2043.
48 Mosieniak, G., Pyrzynska, B & Kaminska, B (1998) Nuclear factor of activated T cells (NFAT) as a new component of the signal transduction pathway in glioma cells J Neurochem 71, 134–141.
49 Ho, I.C., Kim, J.H., Rooney, J.W., Spiegelman, B.M & Glimcher, L.H (1998) A potential role for the nuclear factor of activated T cells family of transcriptional regulatory proteins in adipogenesis Proc Natl Acad Sci USA 95, 15537–15541.
50 Ranger, A.M., Grusby, M.J., Hodge, M.R., Gravallese, E.M., Charles, F., Hoey, T., Mickanin, C., Baldwin, H.S & Glimcher, L.H (1998) The transcription factor NF-ATc is essential cardiac valve formation Nature 392, 186–190.
51 Colbert, M.C., Hall, D.G., Kimball, T.R., Witt, S.A., Lorenz, J.N., Kirby, M.L., Hewett, T.E., Kievitsky, R & Robbins, J (1997) Cardiac compartment-specific overexpression of a modified
Trang 9retinoic acid receptor produces dilated cardiomyopathy and
congestive heart failure in transgenic mice J Clin Invest 100,
1958–1968.
52 Zhou, M.D., Sucov, H.M., Evans, R.M & Chien, K.R (1995)
Retinoid-dependent pathways suppress myocardial cell
hypertro-phy Proc Natl Acad Sci USA 92, 7391–7395.
53 Gruber, P.J., Kubalak, S.W., Pexieder, T., Sucov, H.M., Evans, R.M & Chien, K.R (1996) RXR alpha deficiency confers genetic susceptibility for aortic sac, conotruncal, atrioventricular cushion, and ventricular muscle defects in mice J Clin Invest 98, 1332– 1343.