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,25-Dihydroxyvitamin D3-Mediated Effects
*
Section of Experimental Pathology, Department of Pathology, Roger Williams Medical Center, Brown University, Providence, RI 02908;
Department of Pediatrics, Women and Infants Hospital, Brown University, Providence, RI 02903; and
Department of Hygienic Sciences, Kobe Pharmaceutical University, Kobe, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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,25-dihydroxyvitamin D3
(1,25(OH)2D3) remains elusive. We
demonstrate here that 1,25(OH)2D3-mediated
suppressive effects on the inducible expression of cytokine genes in
human T cells may, in part, be due to diminished activity of the
transcription factor NFAT. The vitamin D3 receptor (VDR)
and its heterodimeric partner retinoid X receptor (RXR)
specifically bound to the distal NFAT site in the human IL-2 promoter,
and this binding was abolished by mutating unique regions in the NFAT
oligonucleotide. In vitro inhibition of NFAT complex formation was
noted when VDR-RXR heterodimers were added to DNA binding reactions
containing nuclear extracts from activated B or T cells, whereas in
vitro NF
B complex formation was not significantly influenced.
Furthermore, 1,25(OH)2D3 treatment of
activated T cells resulted in decreased formation of NFAT complexes
detected upon incubation of nuclear extracts from these cells with
32P-labeled probe. Transient expression of both VDR and
RXR, but not of a single component, was capable of inhibiting
expression of a NFAT-driven reporter gene in stimulated Jurkat cells in
a ligand-dependent manner. These results suggest that NFAT plays a
crucial role in 1,25(OH)2D3-mediated
immunosuppressive activity. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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,25-dihydroxyvitamin
D3
(1,25(OH)2D3),3
have been extensively examined in the context of calcium and bone
homeostasis (1, 2). There is now growing evidence that
1,25(OH)2D3 also plays an important role in
cellular growth, differentiation, and immunomodulation (3). This
secosteroid hormone is a unique effector of biologic functions in that
its mode of action can be controlled by both nongenomic (4, 5) and
genomic regulatory mechanisms (6, 7). For the majority of its genomic
actions, 1,25(OH)2D3 interacts with its
specific receptor, the vitamin D3 receptor (VDR), which
acts as a transcription factor through its binding to distinct
responsive elements (VDREs) (2, 6, 7). As a member of the nuclear
steroid receptor superfamily, the VDR binds to hexameric direct repeats
separated by 3 basepairs (bp) in the form of either a homodimer or a
heterodimer with the retinoid X receptor (RXR) (8, 9, 10). Similar direct
repeats separated by 4 bp or 5 bp can be occupied by heterodimers of
RXR and the thyroid hormone receptor (TR) or the retinoic acid receptor
(RAR), respectively (11, 12, 13). Notably, the diverse biologic effects of
1,25(OH)2D3 are now thought to be associated
with conformational modifications that the VDR adopts due to either the
structural changes in the ligand or the VDRE (14, 15, 16, 17). Despite these
mechanistic insights into VDR-mediated gene expression, relatively
little is known of the molecular basis of the immunosuppressive effects
of 1,25(OH)2D3.
1,25(OH)2D3 appears to exhibit a plethora of
effects on the cells of the immune system (18, 19, 20, 21). The cells of the
myeloid lineage constitutively express VDR, which can be further
enhanced by 1,25(OH)2D3 treatment with
concomitant differentiation phenotype (22, 23, 24, 25, 26). Of significance are the
observations that only activated lymphocytes express the VDR (27, 28, 29)
and that 1,25(OH)2D3 treatment of activated
T cells results in partial growth inhibition as well as in
transcriptional repression of cytokine genes, including IL-2, IFN-
,
and granulocyte-macrophage CSF (30, 31, 32, 33, 34, 35). Furthermore,
1,25(OH)2D3 and its analogues have recently
been shown to manifest, at pharmacologic doses, immunomodulatory
activities in experimental models of autoimmune diseases and
transplantation (36, 37, 38, 39). Therefore, it appears that
1,25(OH)2D3 is a potent immunomodulator and
that its T cell suppressive activity is especially noteworthy for
therapeutic considerations.
Among cytokines that are transcriptionally inhibited by
1,25(OH)2D3, IL-2 plays a central role in
regulating T cell growth and helper effector functions. Although a
number of signaling events occur upon the engagement of the T cell
receptor and costimulatory receptors, two pathways leading to
Ca2+ mobilization and protein kinase C activation are
directly associated with IL-2 production, which is an early event and
is regulated at the transcriptional level (40, 41, 42). The IL-2 promoter
represents a complex enhancer region of approximately 300 bp that
contains binding sites for several ubiquitous and inducible
transcription factors including Oct-1, AP-1, NFB, and NFAT (43, 44, 45).
Alroy et al. have recently examined the human IL-2 promoter for
1,25(OH)2D3-mediated effects on its
transcriptional activity (46). Their studies suggest that the
VDR-mediated inhibition of its activity is confined to a short 40 bp
region encompassing the distal NFAT binding site (46). On the other
hand, it has been shown that 1,25(OH)2D3
down-modulates NFB by partially inhibiting de novo synthesis of the
NFB p50 protein and its precursor, p105, in human peripheral
lymphocytes (47). Thus, the involvement of NFAT and NFB in
1,25(OH)2D3-mediated effects may be a part
of an evolved pathway by which the steroid hormones exert their effects
by targeting other transcription factors. In this regard,
glucocorticoids have recently been shown to inhibit both NFAT and
NFB (48, 49, 50).
NFAT is a member of growing family of transcription factors that
cooperatively bind with Fos and Jun family members (42, 45, 51, 52, 53, 54).
Cyclosporin A (CsA) and FK506 target NFAT activity by inhibiting
calcineurin, a Ca2+-activated serine/threonine phosphatase
necessary for nuclear translocation of cytoplasmic NFAT proteins
(55, 56, 57, 58, 59). Herein we have examined the issue of
1,25(OH)2D3-mediated inhibition of human
IL-2 promoter activity using its distal NFAT site as a target. The
distal NFAT site represents a composite site functionally occupied by
NFAT proteins and Fos/Jun heterodimers (60). We find that specific
mutations in the NFAT oligonucleotide abolish or enhance direct binding
with VDR-RXR complexes. Treatment of activated T cells with
1,25(OH)2D3 or addition of VDR-RXR
proteins in nanogram quantities to nuclear extracts from these cells
results in inhibition of NFAT complex formation. These results are
further supported by diminished NFAT-driven transcription of a reporter
gene in 1,25(OH)2D3-treated Jurkat
cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The human Burkitt’s lymphoma BJAB B cell line and Jurkat T cell
line were obtained from the American Type Culture Collection
(Rockville, MD) and routinely maintained in culture by seeding at cell
densities of 2 x 105 cells/ml in RPMI 1640 (Life
Technologies, Grand Island, NY) supplemented with glutamine (2 mM) and
10% FCS. Escherichia coli and mammalian expression plasmids
for NFATp, human VDR, and RXR were kindly provided by Drs. Anjana
Rao (Harvard Medical School, Boston, MA), J. Wesley Pike (University of
Cincinnati, Cincinnati, OH), and Ron M. Evans (The Salk Institute for
Biological Studies, La Jolla, CA) respectively. The NFAT-CAT reporter
gene plasmid, 5B3.1, a kind gift from Dr. Gerald R. Crabtree (Stanford
University, Stanford, CA), expresses chloramphenicol acetyl transferase
(CAT) driven by three human IL-2 NFAT sites upstream of a minimal
-fibrinogen promoter. 1,25(OH)2D3 was
synthesized at Hoffmann-La Roche, Nutley, NJ, and was kindly
provided by Dr. Milan R. Uskokovic. The hormone was dissolved in 100%
ethanol and was present in cell cultures in no more than 0.01%
ethanol, which had no effect on cellular growth or phenotype. CsA was a
kind gift from Sandoz, Inc. (East Hanover, NJ). Baculovirus-expressed
recombinant VDR was purchased from Pan Vera, Inc., Madison, WI. NFATp
Ab R59 and Jun/Fos proteins were kind gifts from Dr. Anjana Rao
(Harvard Medical School, Boston, MA) and Drs. Tom Curran and Tom
Kerppola (Roche Institute of Molecular Biology, Nutley, NJ),
respectively.
Isolation of tonsillar T cells
Human tonsillar T cells were isolated as previously described (29). Briefly, tonsils removed from individuals during routine tonsillectomy were subjected to fine mincing under sterile conditions and the resulting cell suspension was mixed with neuraminidase-treated SRBC. Rosetted T cells were removed from non-T cell population by Ficoll-Hypaque gradient centrifugation. T cells were further purified by lysing sheep erythrocytes, and adherent cells were removed from this preparation by incubating for 1 h in plastic petri dishes. Tonsillar T cells obtained in this manner were routinely >90% T cells, <1% B cells, and <5% monocytes.
Generation of recombinant NFATp and
glutathione-S-transferase (GST)-RXR proteins from E. coli
Murine NFATp expression vector pNFATpXS, constructed in pQE31
(Quiagen; Chatsworth, CA), contained a histidine tag at the N terminus
of NFATp, whereas RXR was expressed as a GST fusion protein.
Expression of both proteins was induced by addition of 1 mM IPTG to
E. coli cultures at OD600 0.7 to 0.9, and the
cultures were harvested after a 3-h incubation at 37°C. For NFATp,
the cells were disrupted by three cycles of freeze-thawing in 8 M urea,
5 mM 2-ME, 0.1 M sodium phosphate, and 10 mM Tris-HCl, pH 8.0. Proteins
were purified from the soluble fractions with nickle-chelate resin
(Ni-NTA-agarose; Quiagen) as previously described (61). For GST-RXR,
the cells were lysed with lysozyme in the presence of a cocktail of
protease inhibitors. The soluble fractions were subjected to
purification on glutathione Sepharose 4B columns (Pharmacia Biotech,
Inc., Piscataway, NJ) as per vendor’s instructions. The purity of
proteins was checked by SDS-PAGE, and protein concentration was
determined using the Bio-Rad (Hercules, CA) protein assay kit with BSA
as standard.
Electrophoretic mobility shift assay (EMSA)
NFAT complex formation and VDR-RXR binding reactions with the
human IL-2 NFAT and osteocalcin (OC)-VDRE were carried out essentially
as described (62). Binding reactions (16 µl) were performed with
different combinations of nuclear extracts (5 µg), NFATp (0.16
µg–0.3 µg), Jun and Fos (0.25–2 pmol), VDR (50–200 ng), and/or
GST-RXR (50–200 ng) and 300 ng of poly(dI). poly(dC), and 0.2 to
0.3 ng of radiolabeled oligonucleotides. For competition assays, 10- to
100-fold molar excess of unlabeled oligonucleotides was added to the
binding mixture. When indicated, VDR, GST-RXR, or both were
incubated for 10 min at room temperature before being added to reaction
mixtures. The products were separated on a 4% nondenaturing
polyacrylamide gel.
The NFAT and VDRE oligonucleotides are as follows: 1) human
OC-VDRE, a 45-mer containing the VDRE sequence in the promoter of human
OC gene (5'-CTAGTGCTCGGGTAGGGGTGACTCACCGGGTGAACGGGGGCATCT-3');
2) human NFAT, a 30-mer containing the distal NFAT site within the
human IL-2 promoter (5'-GGAGGAAAAACTGTTTCATACACAGAAGGCCT-3'); 3)
human NFAT mutants, the above described 30-mer containing group
mutations within the NFAT oligonucleotide (see Fig. 3
A); 4)
murine NFAT, a 33-mer containing the distal NFAT site in the murine
IL-2 promoter (5'-gatcGCCCAAAGAGGAAAATTTGTTTCATACAG-3'); 5) human
NFB, a 23-mer NFB binding site from the light chain enhancer
(5'-GATCTGAGAGGGGACTTTCCGAG-3').
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Exponentially growing Jurkat cells (1 x 107
cells) were electroporated with plasmids encoding full length VDR,
RXR-, or both (5 µg of each), 5 µg of NFAT-CAT plasmid (5B3.1),
and 2.5 µg of pTKHGH. Total plasmid amount was 25 µg, which was
adjusted with a bacterial expression plasmid, pGEM-3Z. The pTKHGH
plasmid, which expresses human growth hormone (hGH) under the control
of the herpes simplex virus thymidine kinase promoter, serves as an
internal control for the transfection efficiency. At 24 h
post-transfection, the cells were left untreated or treated with 1 µM
of ionomycin and 0.1 ng/ml of PMA in the absence or presence of 3
x 10-8 M 1,25(OH)2D3.
Following 8 h of treatment, the cells were harvested, and CAT
activity was determined as described (62). Allegro hGH kit (Nicols
Institute Diagnostics, San Juan Capistrano, CA) was used to quantitate
hGH values and transfection efficiency. The experiments were repeated
at least three times to determine SEM.
Immunoblot analysis
The presence of NFATp protein in nuclear extracts from
1,25(OH)2D3-untreated or -treated T cells
was assessed using R59 antiserum raised against NFATp, as previously
described (55). Briefly, nuclear extracts were separated on a 7%
SDS-polyacrylamide gel and transferred to a nitrocellulose membrane.
The blocking for nonspecific proteins and incubation with primary Ab
were done in TBS-T (10 mM Tris, pH 7.6, 150 mM NaCl, 0.2% Tween)
containing 5% nonfat dry milk. The blocking incubation was overnight
at 4°C and primary Ab incubation was carried out for 3 h at room
temperature using 1:3000 diluted R59 antiserum. Following primary Ab
incubation and rinsing in TBS-T, the filter was incubated with
secondary Ab, a donkey anti-rabbit IgG Ab conjugated to horseradish
peroxidase (Amersham Corp.), for 1 h at room temperature, and then
rinsed 4 to 5 times again. The membrane was further processed using the
ECL detection system (Amersham Corp., Arlington Heights, IL).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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,25(OH)2D3 inhibits NFAT complex
formation in activated normal T cells
NFAT is a multicomponent transcription factor that is assembled in
the nucleus upon activation of T cells with TCR ligands or by in vitro
treatment with PHA or ionomycin and the phorbol ester PMA; activation
with PHA or ionomycin and PMA results in nuclear translocation of
cytosolic NFAT factors and induction of Fos-Jun proteins, respectively
(42, 45, 51). Moreover, CsA or FK506 inhibits NFAT activity by inducing
phosphorylation of NFAT proteins, which leads to sequestration of NFAT
proteins in the cytoplasm and inhibition of DNA binding activity (55, 56). As a rapid and efficient means of evaluating
1,25(OH)2D3-mediated effects on NFAT, we
first performed EMSAs using a synthetic oligonucleotide corresponding
to the distal NFAT binding site in the human IL-2 promoter. As a
control, we performed EMSA using NFB oligonucleotide as a probe. To
determine the direct effect of 1,25(OH)2D3
on the inducible nature of NFAT or NFB binding activity, highly
purified human tonsillar T cells were left unstimulated or stimulated
for 24 h with 1 µM ionomycin and 2 ng/ml PMA in the absence or
presence of 5 x 10-8 M
1,25(OH)2D3. As seen in Figure 1A, nuclear extracts from
activated cells readily formed a DNA-protein complex with
32P-labeled NFAT probe, which was significantly inhibited
in nuclear extracts from 1,25(OH)2D3-treated
cells. The extent of 1,25(OH)2D3-mediated
inhibition of NFAT complex formation varied from one cell preparation
to another, possibly due to differential in vivo or in vitro cellular
activation or VDR expression (data not shown). Furthermore, diminished
NFAT binding activity in nuclear extracts from
1,25(OH)2D3-treated cells was not due to
phosphorylation of NFAT proteins or their quantitative loss, since
Western blotting with an anti-NFATp Ab revealed the presence of
nuclear dephosphorylated NFATp (data not shown). The effect on
NFAT-binding activity was specific, since NFB complex formation was
not significantly influenced by 1,25(OH)2D3
treatment of T cells under these activation conditions (Fig. 1B). Furthermore, addition of recombinant VDR and GST-RXR
fusion protein to nuclear extract from activated T cells resulted in a
significant inhibition of NFAT complex formation (Fig. 1C),
implying that the 1,25(OH)2D3-mediated
inhibition of NFAT complex formation seen in Figure 1A was
most likely VDR/RXR dependent. Thus, these data suggest that
1,25(OH)2D3 exerts a distinct molecular
effect on NFAT complex formation in activated T cells.
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heterodimers directly bind to the NFAT motif
To examine whether reduced NFAT complex formation in
1,25(OH)2D3-treated cells is a consequence
of direct binding of VDR or VDR-RXR to the NFAT site, we performed
DNA binding assays using baculovirus-expressed recombinant VDR and
GST-RXR fusion protein. Although VDR-RXR heterodimers are thought
to be required for binding to the rat or human OC-VDRE (63), it has
also been shown that E. coli-produced VDR can bind to VDREs
in the absence of RXR (64). Thus, we first evaluated DNA-binding
properties of VDR and GST-RXR preparations, using the human OC-VDRE.
When used alone even at 100 ng or 200 ng concentration, VDR or
GST-RXR failed to bind to 32P-labeled OC-VDRE probe.
However, when combined, these proteins formed a readily detectable
complex (Fig. 2A), agreeing
with earlier observations (63) that in the case of baculovirus
expressed VDR, both components are required for a complex
formation.
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heterodimers directly formed a
complex on the human IL-2 distal NFAT site. This site represents a
composite site encompassing the binding sequences for NFAT family
proteins and Jun-Fos heterodimers. As shown in Figure 2
B,
incubation of VDR and GST-RXR (100 ng of each) with
32P-labeled NFAT probe gave rise to two main retarded
bands, of which only one, labeled as VDR-RXR, was found to be specific,
as cold NFAT oligonucleotide competed with the formation of this
complex. These data are in agreement with the observations of Alroy et
al. (46) and suggest that VDR-RXR heterodimers are capable of
inhibiting the IL-2 promoter activity by interfering with NFAT complex
formation.
A unique recognition sequence(s) in the NFAT oligonucleotide is
required for binding with VDR-RXR heterodimers
Given the observations drawn from Figures 1 and 2B, it
is tempting to speculate that VDR-RXR heterodimers bind to a unique
sequence motif within the distal NFAT site, although direct
protein-protein interactions between NFAT complex proteins and VDR-RXR
complexes can not be ruled out (46). As depicted in Figure 2B, the sequence TGTTTCA in the NFAT oligonucleotide
constitutes a variant AP-1 site, and the sequence GGAAAA represents a
docking site for NFATp and other NFAT family proteins (65). To
demonstrate whether direct binding of VDR-RXR heterodimers to the
human IL-2 NFAT site (Fig. 2B) is localized to these sites
or to other unique sequences, a series of group mutants spanning the
entire NFAT oligonucleotide (Fig. 3A) was subjected to
DNA-protein complex formation in the presence of VDR, GST-RXR, or
both. In addition, we included the mouse IL-2 NFAT site, which shows
minor differences from its human counterpart, in particular 2 bp (TT)
located immediate upstream of the variant AP-1 site (Fig. 3A) (52).
As observed for the human OC-VDRE (Fig. 2A), VDR or
GST-RXR when used alone failed to form a specific complex with
32P-labeled wild-type or mutant NFAT probes (Fig. 3B), albeit a weak band is seen in all of the lanes in the
presence of VDR, which may represent a poor binding of VDR to the NFAT
site. However, VDR and GST-RXR, when combined, showed a significant
binding to the wild-type NFAT, NFAT-m5, and NFAT-m4. Notably, NFAT-m1
and NFAT-m3 mutants showed markedly diminished binding, whereas binding
of VDR-RXR heterodimers to NFAT-m2 mutant was completely abolished,
suggesting that VDR-RXR complexes require a specific docking site
within the NFAT oligonucleotide and that this interaction is influenced
by the flanking sequences. Furthermore, the murine IL-2 NFAT site
carrying a 2-bp distinction in the NFAT-m2 corresponding region of
human IL-2 NFAT also failed to significantly interact with VDR-RXR
complexes, further establishing the validity of the NFAT-m2
corresponding region as a putative recognition site for
1,25(OH)2D3-dependent transcription factors.
However, it should be pointed out that we have used human VDR and
RXR proteins. In this regard, it needs to be investigated whether
murine VDR and RXR proteins would show binding to the murine IL-2
NFAT site. A surprising observation was that the NFAT-m4 mutant
displayed, in multiple experiments, several-fold higher efficiency than
the wild-type oligonucleotide for DNA-protein interaction with
VDR-RXR complexes. The modified NFAT-m4 sequence, or its flanking
sequences, exhibited no resemblance to any of the known VDREs. This
implies that VDR-RXR heterodimers are capable of binding to a wide
array of molecularly distinct response elements, and this may not be
restricted to the known VDREs.
VDR-RXR complexes inhibit in vitro reconstitution of NFAT
complexes
Given the observations drawn from Figures 2B and 3, we
addressed the possibility that VDR and RXR inhibit NFAT complex
formation by interfering with the docking of NFAT and AP-1 proteins on
the NFAT oligonucleotide or vice versa. To address this possibility, we
performed EMSA, employing two approaches. Since activated B cells also
express NFAT binding activity (54, 55, 61) and since VDR-RXR show
direct binding to the NFAT site, we expected that exogenous VDR and
RXR would inhibit NFAT complex reconstitution in both B and T cell
extracts. Thus, in the first approach, DNA-protein binding reactions
were carried out using nuclear extracts from ionomycin- and
PMA-activated Burkitt’s lymphoma BJAB B cell line, but in the presence
of VDR, GST-RXR, or both. Second, in vitro NFAT reconstitution was
performed using recombinant NFATp and variable amounts of Jun-Fos
in the presence of VDR-RXR heterodimers. In this case, it is
possible that an efficient binding of NFATp and Jun-Fos proteins to the
NFAT oligonucleotide may preclude VDR-RXR binding.
Incubation of nuclear extracts from activated BJAB B cells with
32P-labeled NFAT probe gave rise to NFAT complexes (Fig. 4A), which is consistent with
our previous observations (54, 55, 61, 62). More striking are the
observations that VDR-RXR when added to nuclear extracts abolished
NFAT complex formation, although VDR or GST-RXR alone were also
partially effective, possibly due to low level presence of endogenous
VDR and RXR proteins (Fig. 4A). Two lines of evidence
indicate that this effect was specific. First, VDR and GST-RXR were
significantly effective only when added together, consistent with the
data on direct binding of VDR-RXR to the NFAT site or OC-VDRE site
(Fig. 2). Second, inhibition of NFAT complex formation was accompanied
by the appearance of the VDR-RXR complex.
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effect on
NFAT assembly, additional EMSAs were performed with the NFB
oligonucleotide using a similar experimental design as that employed in
Figure 4A. No significant changes were seen in the assembly
of multiple inducible NFB complexes, except partial inhibition of
faster migrating complex, possibly represented by NFB p50 homodimers
(Fig. 4B). Moreover, the more slowly migrating complexes,
possibly made up of p65-p50 heterodimers, were somewhat enhanced in the
presence of VDR-RXR proteins (Fig. 4B). Taken together,
these results support the hypothesis that
1,25(OH)2D3-mediated inhibition of NFAT
complex formation occurs primarily through the occupation of the NFAT
site by VDR-RXR complexes.
Next, we analyzed in vitro reconstitution of NFAT complexes in the
presence of VDR-RXR heterodimers, using recombinant NFATp peptide
containing DNA binding and Jun-Fos interacting domains, as well as Jun
and Fos proteins. The results show that at lower concentrations of
Jun-Fos complexes (0.25 pmol or 0.5 pmol), VDR-RXR complexes were
predominant (Fig. 5). On the other hand,
at higher concentrations of Jun-Fos proteins (1 or 2 pmol), NFAT
complexes were formed at the expense of VDR-RXR complexes (Fig. 5).
This implies that the binding of VDR-RXR heterodimers to the NFAT
site is favored under partial activation conditions, allowing
suboptimal expression of AP-1 proteins. Consistent with these
observations, cells activated in the presence of PMA exhibit a poor
response to 1,25(OH)2D3 (our unpublished
observations).
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,25(OH)2D3-dependent inhibition of
NFAT-driven transcription
Since VDR-RXR complexes were capable of interfering with NFAT
complex formation in both intact cells and in vitro reactions, we
investigated the effect of 1,25(OH)2D3 on
NFAT-driven transcription in transient transfection assays. The Jurkat
T cell line, which expresses little or no VDR (our unpublished
observations), was used in these experiments. Cells were transfected
with a construct containing the CAT reporter gene linked to three NFAT
sites upstream of a minimal -fibrinogen promoter. NFAT-driven
transcription was tested in the presence of a VDR-expressing plasmid, a
RXR-expressing plasmid, or both. Cells were grown for 24 h and
activated with ionomycin and low concentrations of PMA (0.1 ng/ml) in
the absence or presence of 3 x 10-8 M
1,25(OH)2D3 during the last 8 h. In the
control experiments, cells were left unstimulated or transfected with
control CAT plasmid.
As shown in Figure 6, treatment of cells
with ionomycin and PMA significantly induced CAT expression by
endogenous NFAT activity. Importantly, expression of both VDR and
RXR inhibited NFAT-driven CAT expression (
50%), whereas
expression of either VDR or RXR failed to exhibit any significant
affect. Two lines of evidence suggest that this effect is functionally
significant. First, inhibition of NFAT-driven transcription was
1,25(OH)2D3-dependent and required
expression of both VDR and RXR. Second,
1,25(OH)2D3 treatment of Jurkat cells in the
absence of VDR or RXR expression failed to affect NFAT functions.
These results indicate that repression of the IL-2 promoter activity,
or possibly the promoters of other
1,25(OH)2D3-sensitive cytokine genes, is
most likely due to the diminished NFAT activity.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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,25(OH)2D3-mediated effects in human T
cells. In a series of related experiments, we have shown that VDR and
GST-RXR bind to the human IL-2 NFAT site in a sequence-specific
manner. These proteins appear to block NFAT functions in intact cells
as well as in nuclear extracts isolated from activated lymphocytes.
Notably, we have found no significant effect of
1,25(OH)2D3 on NFB binding activity under
T cell activation conditions used here. Thus, although little
information is available at this time concerning the molecular
mechanism(s) underlying 1,25(OH)2D3-mediated
immunosuppression, our observations support the notion that the steroid
hormones may control expression of cytokine genes, which lack classical
responsive elements in their regulatory regions, by targeting inducible
transcription factors in immune cells (47, 48, 49, 50).
On the basis of published accounts of responsive element selection by
the VDR and ligand-dependent gene activation or repression (2, 7, 8, 9, 61, 66, 67), 1,25(OH)2D3-induced
immunosuppressive effects should be mediated through
trans-regulatory events at a putative VDRE site.
Trans-activation and -repression by VDR-RXR heterodimers
have been shown to involve interactions of these proteins with other
transcription factors (66, 68) or to involve conformational
modifications due to subtle changes in the classical VDRE sites (16) or
ligand (15). In the context of these studies, our results point to a
unique pathway by which
1,25(OH)2D3-dependent transcription factors
act as trans-repressing proteins on a non-VDRE site.
Coincidentally, the NFAT mutants NFAT-m1, NFAT-m2, and NFAT-m3,
overlapping the docking sites for NFAT proteins and Jun-Fos complexes,
fail to bind with VDR-RXR heterodimers (Fig. 3). Furthermore,
NFAT-m2 mutant, which spans the sequences AAAAC between the binding
sites for NFAT proteins and AP-1 members, shows a complete lack of
binding with VDR-RXR (Fig. 3). Curiously, the murine IL-2 NFAT site,
which shows little or no interaction with human VDR-RXR
heterodimers, differs from its human counterpart by two bp
(AAATT) in NFAT-m2 corresponding sequences (Fig. 3). Thus,
these results suggest that the region AAAAC and its flanking sequences
are most likely the VDR-RXR interaction motif. Taken together, our
results are, in part, consistent with the DNase I footprinting analysis
of the IL-2 promoter in the presence of recombinant VDR (46). Two
regions in the distal IL-2 NFAT site, which overlap the NFAT-m1 and
NFAT-m3 mutants, were shown to be footprinted; however, VDR failed to
protect the NFAT-m2 corresponding region (46). Since the footprinting
analysis was carried out in the absence of RXR proteins, it is possible
that a weak binding to the NFAT site by VDR alone may contribute to
partial footprinting. More striking are our results with the mutant
NFAT-m4, which flanks the 3' end of the variant AP-1 site (Fig. 3).
This mutant exhibited several-fold increased binding with VDR-RXR
heterodimers. Although these sequences have not been shown to be part
of the NFAT or AP-1 protein binding sites, it is tempting to speculate
that this altered sequence constitutes a novel docking site for VDR and
RXR proteins.
Despite the evidence provided here for VDR-NFAT interaction and
repression of the IL-2 promoter activity, extended studies are needed
to fully determine the overall relationship between these two
transcription factor systems. For example, structural diversity with
regard to AP-1 involvement in the NFAT sites are likely to influence
binding of VDR-RXR complexes (51). Similarly, given the existence of
multiple NFAT family proteins with varying binding affinities to the
NFAT sites (61, 69, 70, 71, 72), it is likely that VDR-RXR interaction with the
NFAT sites may also depend on the temporal involvement of a particular
NFAT member. Most importantly, T cell activation conditions will be of
critical importance in assessing the effects of
1,25(OH)2D3, since it appears that full
activation of T cells resulting in efficient Jun-Fos expression will
counteract the VDR-RXR binding (Fig. 5). Besides these concerns, our
studies also raise other general questions. Do such VDREs exist that do
not conform to the hexameric half-elements spaced by three nucleotides
or six nucleotides and that may in turn induce unusual architectural
modifications in VDR and RXR transcription factors? No direct or
palindromic repeats separated by intervening sequences are readily
apparent in the NFAT oligonucleotide, except the sequence AGGAAA at the
5' end, which resembles the rat proximal OC-VDRE half-site, AGGACA
(16). Furthermore, since the human and mouse NFAT show different
binding pattern with human VDR-RXR complexes due to a 2-bp sequence
difference (Fig. 3), the question arises whether
1,25(OH)2D3 exert its effects in mouse and
human using different regulatory mechanisms. In this regard, it is of
interest to note that subtle sequence differences within the classical
VDREs have been shown to differentially influence gene expression in
the same or distinct species. For example, Staal et al. have
demonstrated that sequence modifications in the middle of 3 half-sites
(positions 3 and 4) of the rat OC- and osteopontin-VDREs induce
distinct conformational modifications, resulting in differential
regulation of these genes (16). Importantly, Wang et al. have reported
recently that although the murine OC promoter contains rat or human
OC-VDRE-like sequences, 1,25(OH)2D3 inhibits
rather than activates the mouse OC gene expression (17). A comparative
analysis of both the murine OC-VDRE and the rat OC-VDRE revealed that
these sites differ by only 2 bp in the 5 half-site (mouse
GGGCAA; rat GGGTGA) and that the mouse OC-VDRE
exhibits a very poor affinity for VDR-RXR complexes (17). In this
regard, it is of paramount importance to determine the structural
diversity of putative VDR-RXR binding sites in the promoters of
1,25(OH)2D3-sensitive cytokine genes.
What is the significance of
1,25(OH)2D3-mediated inhibition of NFAT
functions? There has been a considerable therapeutic interest in
protein targets for the immunosuppressant CsA and FK506. However, their
clinical use is limited because of toxic side effects that arise due to
inhibition of calcineurin in the nonimmune cells. It is possible that
the drugs that directly target NFAT act as CsA/FK506 dose-reducing
agents. Indeed, our recent results (unpublished observations) suggest
that CsA and 1,25(OH)2D3 exhibit at least an
additive effect on T cell proliferation. In this regard, drugs such as
1,25(OH)2D3 or its more potent analogues,
which show in vivo immunomodulation with less pronounced calcemic
effects, may subserve the role of such agents (73, 74, 75).
|
Acknowledgments |
|---|
,25(OH)2D3, Dr. Anjana Rao for NFATp
reagents, Drs. Tom Curran and Tom Kerppola for Jun and Fos proteins,
Dr. Gerald R. Crabtree for NFAT-CAT plasmid 5B3.1, Dr. J.
Wesley Pike for VDR-expressing plasmid, and Dr. Ron Evans for
RXR-expressing plasmid. We thank Dr. Sara Peleg for critical reading
of the manuscript. We also thank Mark Sanders, Eric Wager, and Marija
Hleb of the Sharma Laboratory for their help throughout this
work. |
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Surendra Sharma, Section of Experimental Pathology, Roger Williams Medical Center, Brown University, 825 Chalkstone Avenue, Providence, RI 02908.
3 Abbreviations used in this paper: 1,25(OH)2D3, 1,25-dihydroxyvitamin D3; NFAT, nuclear factor of activated T cells; VDR, vitamin D3 receptor; VDRE, vitamin D-responsive element; bp, basepair; RXR, retinoid X receptor; CsA, cyclosporin A; CAT, chloramphenicol acetyl transferase; GST, glutathion-S-transferase; EMSA, electrophoretic mobility shift assay; OC, osteocalcin; hGH, human growth hormone.
Received for publication June 13, 1997. Accepted for publication September 15, 1997.
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