From the Cell Biology Program, Memorial Sloan-Kettering Cancer Center, Sloan-Kettering Division, Cornell University Graduate School of Medical Sciences, New York, New York 10021
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ABSTRACT |
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The primary function of activated T lymphocytes is to produce various cytokines necessary to elicit an immune response; these cytokines include interleukin-2 (IL-2), interleukin-4, and granulocyte-macrophage colony-stimulating factor (GMCSF). Steroid hormones and vitamin A and D3 metabolites act to repress the expression of cytokines. 1,25-Dihydroxyvitamin D3 (1,25-(OH)2D3) down-modulates activated IL-2 expression at the level transcription, through direct antagonism of the transactivating complex NFAT-1/AP-1 by the vitamin D3 receptor (VDR). We report here that GMCSF transcription in Jurkat T cells is also directly repressed by 1,25-(OH)2D3 and VDR. Among four NFAT/AP-1 elements in the GMCSF enhancer, we have focused on one such element that when multimerized, is sufficient in mediating both activation by NFAT-1 and AP-1 and repression in response to 1,25-(OH)2D3. Although this element does not contain any recognizable vitamin D response elements (VDREs), high affinity DNA binding by recombinant VDR is observed. In contrast to VDR interactions with positive VDREs, this binding is independent of VDR's heterodimeric partner, the retinoid X receptor. Moreover, VDR appears to bind the GMCSF element as an apparent monomer in vitro. Protease digestion patterns of bound VDR, and receptor mutations affecting DNA binding and dimerization, demonstrate that the receptor binds to the negative site in a distinct conformation relative to a positive VDRE, suggesting that the DNA element itself acts as an allosteric effector of VDR function. This altered conformation may account for VDR's action as a repressing rather than activating factor at this locus.
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INTRODUCTION |
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The classical effects of the secosteroid 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3)1 include the regulation of calcium absorption in the intestine, maintenance of mineral homeostasis in the kidney, and regulation of bone formation and remodeling (1-3). More recently, the scope of vitamin D action has broadened with the detection of the nuclear receptor for this ligand, vitamin D3 receptor (VDR), in tissues such as skin, testis, pancreas, colon, muscle, breast, prostate, thymus, and bone marrow. Generally, 1,25-(OH)2D3 acts as a growth inhibitor in many of these cell types. For example, the ligand is a potent inducer of differentiation of promyelocytic leukemia cells (4-6). The identification of the cyclin-dependent kinase inhibitor gene product, p21Waf1,Cip1, as a direct target of 1,25-(OH)2D3 action in myeloid cells (7) provides a direct link between the general actions of this ligand and growth control/differentiation in these cells. Transcription of the p21 gene is directly enhanced in response to 1,25-(OH)2D3 through the binding of VDR to specific regulatory sites in the p21 promoter. VDR, as is typical of many members of the nuclear hormone receptor superfamily, binds to DNA and activates transcription as a heterodimeric complex with the retinoid X receptor (RXR) (8-11). VDR homodimers (12, 13) as well as monomers (9) are also capable of binding some vitamin D response elements (VDREs), such as that found in the mouse osteopontin gene promoter (14), but are probably not capable of transactivating (15). The architecture of this consensus positive VDRE is two directly repeating hexameric half-sites consisting of the sequence PuGG/TTCA spaced by three nucleotides (DR3) (13, 16). Transactivation by VDR from a DR3 is strictly ligand-dependent, where the ligand stabilizes VDR-RXR formation (9, 10), as well as enhances interactions with components of the transcriptional preinitiation complex, such as TFIIA (15) and perhaps as yet unidentified coactivators, as has been demonstrated for several other steroid and nuclear receptors (reviewed in Ref. 17).
Whereas considerable information exists describing transactivation by
steroid/nuclear receptors, the mechanisms through which these receptors
elicit repression of activated transcription, which we call here
transrepression, are poorly understood. Several genes have been
identified as targets of transrepression by various steroid and nuclear
receptors (18-22). Genes that are down-regulated in response to
1,25-(OH)2D3 include those encoding human and
chick parathyroid hormone (PTH) (23, 24), rat 1(I) collagen (25), human atrial natriuretic (26), interleukin-2 (IL-2) (27), and the rat
bone sialoprotein (28). For the chick PTH and rat bone sialoprotein
genes, imperfect DR3 elements have been identified at promoter proximal
sites through which 1,25-(OH)2D3-mediated repression occurs. However, the promoters for the human PTH, human atrial natriuretic, and human IL-2 genes do not appear to contain canonical DR3-type VDREs that are functionally conferring
transrepression. Nevertheless, direct VDR binding has been demonstrated
within these promoters. The repressive site identified in the human PTH gene consists of an extended imperfect VDRE half-site as well as some
flanking sequence. In the IL-2 promoter, Alroy et al. (27)
found that the minimal sequence required to confer repression consists
of a sequence known to bind the T cell transcription factor NFAT-1 as
well as a weak AP-1 binding site. Included in this sequence is an
extended imperfect VDRE half-site. VDR DNA binding was shown to be
necessary but not sufficient to confer repression.
Our previous study on the mechanism of repression of activated IL-2
transcription by 1,25-(OH)2D3 in T cells
provided a molecular explanation of how this ligand acts as a modest
immunosuppressing agent. 1,25-(OH)2D3-mediated
effects in T lymphocytes were first reported as a decrease in the
proliferation of peripheral blood lymphocytes (29). This effect was
dependent on the expression of the VDR and was accompanied by a
decrease in the mRNA levels of IL-2, interferon-, and
granulocyte-macrophage colony-stimulating factor (GMCSF) (30-32).
Interestingly, VDR is only expressed following activation in T cells
(33), and therefore the direct transcriptional repression of a primary
response cytokine such as IL-2 and secondary response factors such as
GMCSF could lead to a progressive deactivation of the immune
response.
GMCSF is a glycoprotein that signals through a cell surface receptor of the hematopoietin receptor family. This signaling molecule is synthesized in activated T cells, activated macrophages, endothelial cells, and fibroblasts (34). GMCSF stimulates the proliferation and subsequent differentiation of leukocyte precursors, but its primary physiological function is to respond to immune activation and to promote the activation of the inflammatory response. An additional role for GMCSF activity was observed in lung tissue of GMCSF knockout mice (35). These mice displayed defects in the level of lung surfactant produced, and consequently developed respiratory pathologies. Just as the lack of GMCSF expression can promote disease, aberrant expression of GMCSF also has severe implications in human disease. Various leukemias, such as acute myeloid leukemia (36) and juvenile chronic myeloid leukemia (37), display a dysregulation of GMCSF. Constitutive expression of GMCSF is also evident in chronic inflammatory conditions such as rheumatoid arthritis and asthma. Down-regulation of GMCSF gene expression may facilitate the gradual diminution of the inflammatory response necessary for a return to the homeostatic state in damaged tissue.
We demonstrate here that the down-regulation of GMCSF mRNA observed in response to 1,25-(OH)2D3 in peripheral blood lymphocytes is occurring at the level of transcription. Moreover, this repression is both VDR-dependent and 1,25-(OH)2D3 dose-dependent. We have delineated a 35-bp region within the GMCSF enhancer that confers the 1,25-(OH)2D3-mediated repression. This region does not contain a consensus VDRE, but analogous to the IL-2 locus, it does include an AP-1 site as well as a NFAT-1 element. Using a series of mutant oligonucleotides, we have narrowed down the receptor binding site to seven bases that have some homology to an extended nuclear receptor half-site. In contrast to paradigmatic heterodimer binding, VDR binds the GMCSF element selectively and with high affinity as an apparent monomer, with no detectable contribution by RXR. Moreover, VDR appears to be poised on the DNA in an altered conformation when bound to the negative element as compared with a positive DR3 VDRE, suggesting that the DNA sequence itself is acting as an allosteric effector of VDR function.
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MATERIALS AND METHODS |
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Antibodies and Overexpressed Proteins--
Rat monoclonal
anti-VDR antibody was purchased from Affinity BioReagents (Golden, CO).
Polyclonal anti-VDR antibody was generously provided by Affinity
BioReagents. VDR was overexpressed in Escherichia coli using
the pET system as described previously (9). To purify VDR, induced cell
pellets were lysed in a buffer containing 50 mM Tris-HCl,
pH 7.5, 1 mM EDTA, 500 mM NaCl, 5 mM dithiothreitol, 1 mM phenylmethylsulfonyl
fluoride (TED-500), plus 0.5 mg/ml lysozyme by Dounce homogenization
and micro-tip sonication. Sodium deoxycholate was added to a final
concentration of 0.05%, and the sample was centrifuged for 1 h at
30,000 rpm in a Ti45 fixed angle rotor. The supernatant was transferred
to a fresh tube, and polymin P (Aldrich) was added to a final
concentration of 0.2% over 10 min and incubated an additional 10 min
at 4 °C. Samples were centrifuged for 1 h at 30,000 rpm, and
the supernatant was precipitated by addition of ammonium sulfate to
30%. The sample was incubated at 4 °C for 1 h, and centrifuged
for 20 min at 20,000 rpm in a Ti45 rotor. The protein pellet was
resuspended in TED-50 and purified by gel filtration analysis as
described previously (9). The VDR DNA-binding domain derivative (DBD)
was purified as described previously (38). The mutant receptors
VDR-L262G and VDR-K45A were purified exactly as described for wild-type
VDR. FLAG-RXR was overexpressed in insect cells and purified as
described previously (39).
Plasmids--
The GMCSF reporter constructs used in transient
transfection experiments were created as follows: 716GMCSF-LUC-pGL-2
was digested with SmaI and NheI, alkaline
phosphatase-treated, and gel-purified. The
PvuII/XbaI, 1.4-kilobase pair fragment insert was
derived from pHGM716CAT (provided by P. Cockerill, Hanson Center for
Research, Australia) corresponding to the GMCSF enhancer fragment from
2.6 kilobase pairs to
3.3 kilobase pairs as well as 600 bp of the GMCSF promoter. Positive clones were verified by dideoxynucleotide DNA
sequencing. N3GMCSFLUC-pGL2 basic plasmid was digested with SacI and XhoI, filled in with Klenow enzyme,
alkaline phosphatase-treated, and gel-purified. The PvuII
N3GMCSF fragment insert was derived from plasmid HGMN3CAT (provided by
P. Cockerill). This fragment consists of the NFAT/AP-1 site at
position 550 within the 716 bp of the GMCSF minimal enhancer sequence,
multimerized three times, yielding a 158-bp fragment fused to the
600-bp GMCSF promoter fragment. GMCSFLUC-pGL2 basic plasmid was
digested with SstI and XhoI, filled in by Klenow
enzyme, alkaline phosphatase-treated, and gel-purified. The
PvuII/StuI 600-bp GMCSF promoter fragment was
derived from HMGN3CAT. Expression plasmids were generated using the
cytomegalovirus-driven pRC-CMV vector (Invitrogen); CMV-VDR (40) was
constructed as described previously. VDR mutant constructs were
subcloned as described for wild-type VDR.
Oligonucleotide-directed in Vitro Mutagenesis-- Site-directed mutagenesis was carried out using pCMV-VDR as template, generating single-stranded DNA, and synthesizing the mutant strand with the mutagenic oligonucleotide. For VDR-L262G, the mutagenic oligonucleotide 5'-TGACTTCAGCCCTACGATCTGGT-3' was used to change leucine 262 to a glycine residue. VDR-K45A was generated using the mutant oligonucleotide 5'-CTGAAGAAGCCTGCGCAGCCTTC-3' to change lysine 45 to an alanine residue. Mutant pools were screened for the appropriate codon changes by dideoxynucleotide DNA sequencing. The mutated VDR was transferred to a T7 overexpression vector as an NdeI-BamHI fragment, and protein was produced and purified as described for wild-type VDR.
Electrophoretic Mobility Shift Analysis-- VDR DNA binding was assessed by gel mobility shift electrophoresis using conditions described previously (11). The VDRE from the mouse osteopontin gene (DR3) was generated as complimentary oligonucleotides of the sequence 5'-GATCCACAAGGTTCACGAGGTTCACG TCCG-3' (top strand). The NFAT/AP-1 site at position 550 within the 716 bp of the GMCSF enhancer (GM550) was synthesized as complimentary oligonucleotides of the sequence 5'-GATCTCTTATTATGACTCTTGCTTTCCTCCTTTCA-3' (top strand). The sequence of the NFAT-IL2 oligonucleotide top strand is 5' CTAGCAGAAAGGAGGAAAAACTGTTT CATACAGAAGGCGTT-3'. Mutant oligonucleotides are listed in Fig. 3A. Equimolar amounts of complimentary strands were annealed and 32P-end-labeled as described previously (38). Overexpressed, purified VDR was preincubated with 12 fmol of the indicated oligonucleotide duplex for 20 min at room temperature together with 50 µg of poly(dI-dC)/ml in binding buffer (20 mM Tris-HCl, pH 7.9, 1 mM EDTA, 50 mM KCl, 10% glycerol, 0.05% Nonidet P-40, and 1 mM dithiothreitol). Protein-DNA complexes were resolved by electrophoresis on 10% nondenaturing acrylamide gels run in 0.5× Tris borate-EDTA, at 250 V, constant voltage at 4 °C. Gels were dried and subjected to autoradiography. For supershift analyses, receptors were preincubated with preimmune sera or specific antibody for 10 min at room temperature. DNA was then added, and incubation proceeded for an additional 20 min at room temperature. Electrophoretic mobility shift assay (EMSA) was performed as described above.
Proteolytic Clipping Assay-- EMSAs were carried out as described above with the following modifications. 100 ng of recombinant VDR was incubated with 20 fmol of the indicated 32P-end-labeled oligonucleotide duplex for 20 min at room temperature. Trypsin protease (Life Technologies, Inc.) was made up in 137 mM NaCl at a stock concentration of 25 µg/µl. Trypsin protease was then added directly to the binding reactions to the indicated final concentration for 10 min at room temperature. For the time-course experiment, 10 ng/µl trypsin was added directly to the binding reactions for the indicated times. Complexes were resolved by electrophoresis on 10% nondenaturing acrylamide gels as described above, dried, and subjected to autoradiography.
Cell Transfection and Reporter Assays--
The T cell line
Jurkat was transfected by electroporation method using BTX (San Diego)
0.2 cuvettes. Cells were grown in RPMI medium containing sodium
pyruvate, glutamine, and penicillin-streptomycin to a final
concentration of 100 µg/ml. Fetal calf serum was added to 10%, and
cells were maintained at a density of approximately 8 × 105 cells/ml. For transfection, cells were washed in RPMI
medium and resuspended in this medium to a density of 3 × 107 cells/200 µl. Each transfection reaction contained 5 µg of reporter plasmid, 1.25 µg of internal control plasmid, 500 ng
of producer plasmid, and 5 × 106 cells. All reactions
are done in triplicate and with two treatments. Therefore, six
reactions were transfected per BTX cuvettes in a final volume of 200 µl. BTX settings were as follows; T = 500 V,
C = 1700 microfarads, R = 72 ohms,
S = 126 V. After electroporation, cells were incubated
for 30 min and the contents of each cuvette was then added to 6 ml of
RPMI containing sodium pyruvate, glutamine, penicillin-streptomycin,
and charcoal-stripped fetal calf serum to 10%. Cells were plated as 1 ml of transfection mix added to 14 ml of the same stripped serum
containing medium and allowed to incubate 24 h (5%
CO2, 37 °C). At 24 h after transfection, cells were
treated in one of the following ways for 9 h: (a) no treatment, (b) addition of activating agents phorbol
myristate acetate (PMA, 50 ng/ml; Sigma) and phytohemagglutinin (PHA, 2 µg/ml; Sigma), (c) addition of 5 × 108
M 1,25-(OH)2D3 (Biomol), or
(d) addition of activating agents and
1,25-(OH)2D3. Experiments were harvested and
normalized to protein concentration as well as to
-galactosidase
activity produced off the internal control plasmid CMV-
-gal included
in each transfection. Equal amounts of total cell extract were added to
luciferase assays, and results were quantitated as relative light units
using a luminometer.
RNase Protection Assay--
Jurkat cells were transiently
transfected as described previously with the reporter construct
N3GMCSFLUC, as well as the producer plasmids pRC-CMV or pCMV-VDR.
Transfected cells were treated with the activating agents PMA (50 ng/ml) and PHA (2 µg/ml) alone or in the presence of 1 × 108 M 1,25-(OH)2D3.
All cells were treated with 10 mM cycloheximide for the
duration of the experiment. Cells were harvested after 6 h of
treatment, and total RNA was isolated using Trizol reagent (Life
Technologies, Inc.). The antisense luciferase probe was generated as
described previously (27). 3 µg of RNA was ethanol-precipitated with
1 × 105 cpm of either luciferase antisense probe or
an antisense
-actin probe. The reaction was then resuspended in
hybridization buffer (Ambion RPA II kit), heated to 90 °C, and
vortexed thoroughly. Hybridization reaction was incubated overnight at
45 °C. Digestion of unprotected RNA was performed by the addition of
a 1:100 dilution of an RNase A/RNase T1 mixture, and allowed to
incubate at 37 °C for 30 min. Protected fragments were ethanol
precipitated, resuspended in a glycerol loading buffer and heated to
90 °C for 4 min. Reactions were analyzed on a 6% denaturing
polyacrylamide gel run at 400 V at room temperature.
Immunoblotting-- Transfected Jurkat cells were harvested and resuspended in 250 mM Tris buffer, pH 8.0. Whole cell extracts were prepared by repeated freeze/thaw lysis, and centrifuged at 14,000 rpm at 4 °C for 15 min in a microcentrifuge. Supernatants were retained, and protein concentration was determined by the Bradford method (Bio-Rad). 30 µg of whole cell extract was analyzed by SDS-polyacrylamide gel electrophoresis. Protein was transferred to polyvinylidene difluoride membrane (NEN Life Science Products) at 140 mA for 30 min in 25 mM Tris, 192 mM glycine, and 20% methanol. The membrane was blocked in 5% nonfat dry milk (Carnation) in phosphate-buffered saline overnight at room temperature. Monoclonal rat anti-VDR (Affinity Bioreagents) was diluted 1:6000 in phosphate-buffered saline, 1% nonfat dry milk, 0.1% Tween 20 and incubated for 3 h. The membrane was washed extensively in phosphate-buffered saline, 0.1% Tween 20. The membrane was then incubated in a secondary antibody solution consisting of a 1:2000 dilution of horseradish peroxidase-conjugated sheep anti-rat IgG (Amersham Pharmacia Biotech) in phosphate-buffered saline, 1% nonfat dry milk, 0.1% Tween 20 for 45 min. Membrane was washed as previously described and developed by using enhanced chemiluminescence (Amersham Pharmacia Biotech).
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RESULTS |
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VDR Directly Mediates Repression of the GMCSF Locus by Acting
through a NFAT/AP-1 Site in the GMCSF Enhancer--
A group of genes
encoding the cytokines IL-2, GMCSF, and interferon- that are
activated in response to T cell stimulation are also repressed by the
secosteroid 1,25-(OH)2D3. We previously demonstrated that 1,25-(OH)2D3-mediated
repression of IL-2 transcription is a direct, VDR-dependent
effect (40). To determine if the down-regulation of GMCSF mRNA
levels observed in response to 1,25-(OH)2D3 is
also occurring at the level of transcription, a transient transfection experiment was performed with the promoter constructs depicted in Fig.
1A. 716 bp of the GMCSF
enhancer located from position
2600 bp to
3316 bp from the start
site of transcription were fused to the GMCSF promoter and subcloned
into a luciferase reporter backbone construct yielding the construct
716GMCSF-LUC. This construct was used to transiently transfect, with
or without a VDR producer plasmid, Jurkat cells, a transformed T cell
leukemia cell line. In each transfection series, cells were
(a) left untreated, (b) treated for 9 h with
the activating agents PMA and PHA, (c) treated with
1,25-(OH)2D3 alone, or (d) treated
with activating agents and 1,25-(OH)2D3.
Activation levels were reduced by only 14% upon the addition of
1,25-(OH)2D3 in the absence of overexpressed VDR (Fig. 1B). It has been reported that VDR is not
expressed in resting peripheral blood lymphocytes, and is only
detectable following activation (33). We have been able to detect VDR
expression in non-activated Jurkat cells but at very low levels (for
example, see Fig. 7A). However, upon transient
overexpression of VDR, activation levels were reduced by nearly 60%
following addition of 1,25-(OH)2D3 (Fig.
1B). This repression was also ligand
dose-dependent, with maximal repression of 98% occurring
at 1 × 10
6 M
1,25-(OH)2D3 (data not shown). The increase in
repression in response to VDR levels and ligand concentration indicates
that the effects are both receptor- and ligand-dependent.
The 1,25-(OH)2D3 repression is independent of
de novo protein synthesis since the observed down-regulation
of GMCSF mRNA as detected in ribonuclease protection assays was
resistant to the presence of 10 mM cycloheximide (Fig.
1C).
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VDR Is Capable of Binding to a Noncanonical Negative Element in the GMCSF Enhancer with High Affinity, Independent of RXR-- As mentioned, our previous study on the IL-2 enhancer demonstrated that the target for 1,25-(OH)2D3-transrepression was also a composite NFAT/AP-1 element (27). Although this region lacks a readily identifiable vitamin D response element, at least as defined by the consensus positive VDRE, we found that VDR and VDR·RXR were in fact able to bind this NFAT/AP-1 site specifically (see Ref. 27 and Fig. 2B). Moreover, VDR mutants that disrupted specific DNA binding to the element in vitro were incapable of conferring transrepression in vivo. We therefore asked if the NFAT/AP-1-containing region at position 550 within the GMCSF enhancer, which confers 1,25-(OH)2D3-transrepression, also binds VDR selectively. Like the IL-2 element, the GMCSF site does not contain any recognizable VDREs. A 35-bp oligonucleotide duplex containing the NFAT/AP-1 site, termed GM550, was synthesized, and DNA binding was analyzed by EMSA. An additional oligonucleotide containing a positive DR3 VDRE served as a control for in vitro DNA binding by recombinant VDR and RXR. Fig. 2A demonstrates that VDR alone, but not RXR alone, bound both the DR3 and GM550 probes (lanes 2-4 and 8-10). Surprisingly, the high affinity VDR-RXR heterodimeric complex, which is the predominant species on the DR3, was not detected on GM550 (compare lanes 5 and 6 with lanes 11 and 12). Moreover, the VDR-GM550 complex migrated with a faster mobility than that of the VDR-DR3 complex (Fig. 2A, lanes 9 and 10 versus lanes 3 and 4). The complex bound to the GM550 element is indeed VDR, since an anti-VDR monoclonal antibody directed against the C-terminal region of the VDR DNA-binding domain blocked receptor binding to both the DR3 and GM550 sites (Fig. 2C, lanes 2 and 4). but a monoclonal antibody raised against an unrelated protein, the Src family member Fyn, was unable to disrupt both complexes (lanes 7 and 10). Moreover, the VDR monoclonal antibody was unable to perturb the Jun·Fos binding complex on the negative element, indicating that the loss of a binding complex by the anti-VDR antibody was specific to VDR. In addition, the VDR-GM550 complex is competed specifically by an excess of DR3 competitor oligonucleotide but not the analogous amount of an oligonucleotide containing a glucocorticoid response element (data not shown).
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The VDR Binding Site within GM550 Is a Core of Seven Base Pairs
That Overlaps the NFAT-1 Binding Site--
To further delineate the
binding site for VDR in the GM550 element, a series of mutant
oligonucleotide duplexes, shown in Fig.
3A, were synthesized and
tested in gel mobility shift assays. As seen in Fig. 3B,
mutation of the AP-1 site (lanes 16-20) had no effect on
VDR binding. Similarly, alterations generated in the GM550 mutB
oligonucleotide (lanes 21-25), which include the last two
bases of the AP-1 site and three bases that separate the AP-1 and NFAT
sites (Fig. 3A), also had little effect on VDR's ability to
bind. However, mutations that completely change the NFAT binding site
as well as six additional bases that extend into the AP-1 binding site
(GM550 mutA; Fig. 3A) completely abolished VDR binding (Fig.
3B, lanes 6-10). Taking into account that the GM550(AP1) mutant oligonucleotide confers normal VDR affinity and
mutA abolishes binding, we reasoned that the NFAT binding site may be
critical for VDR recognition. We therefore generated GM550(
NFAT),
where only the NFAT-1 binding site was mutated (Fig. 3A). As
seen in Fig. 3B (lanes 11-15), VDR binding to
this oligonucleotide is severely reduced. Thus, the lack of VDR binding
to GM550 mutA and
NFAT mutant probes defines the sequence GCTTTCC,
which superficially resembles an extended VDRE half-site, as the
minimal requirement for VDR binding to the GM550 negative element.
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The GM550 Negative Element Acts as an Allosteric Effector of
VDR--
The faster mobility profile exhibited by VDR when bound to
the negative GM550 versus the positive DR3 observed in Fig.
2 suggested that VDR binding to the negative element was unique. To
address this, several approaches were taken to compare VDR binding to each element. First, a gel mobility supershift assay was carried out.
Fig. 4A illustrates that when
VDR was prebound to the DR3, a polyclonal antibody raised against the
receptor (distinct from that shown in Fig. 2B) was able to
specifically supershift the receptor-DNA complex (lane 6).
However, when VDR was preincubated with GM550, neither the preimmune
serum nor this VDR-specific antibody supershifted the complex
(lanes 11 and 12). As was observed in Fig. 2, the
VDR·GM550 complex migrated with a faster mobility when compared with
VDR bound to the DR3. Additional evidence that VDR utilizes an
alternative strategy in binding the negative element was derived from a
VDR mutant generated by site-directed mutagenesis termed VDR-K45A. This
lysine, at residue 45, is absolutely conserved among all the known
members of the steroid and nuclear receptor superfamily and lies within
the specificity -helix of the receptor DBD, immediately proceeding
the first zinc finger. This lysine residue has been shown in several
crystal structures to be making direct side-chain contacts on positive
hormone response elements (44). As expected, DNA binding of the K45A
mutant to the DR3 VDRE was completely abolished in the presence or
absence of RXR (Fig. 4B, lanes 11-15). However,
VDR-K45A was capable of binding to the negative GM550 element, albeit
with a slightly lower affinity than the wild-type receptor (lanes
16-20). As with wild-type VDR, addition of RXR had no effect on
the binding. This result suggests that a specific contact within the
receptor DNA-binding domain essential for interaction with a positive
response element is not making an equivalent contact on the negative
GM550 site. Transient transfection of cells with the VDR-K45A mutant
correlates with the in vitro DNA binding data. The K45A
mutant receptor was unable to activate transcription from a
DR3-regulated reporter, but repressed from the GM550 element (although
repression is reduced to a similar extent as VDR-DNA binding is
decreased; data not shown). Taken together, the data reinforce the
importance of DNA binding by VDR on the GM550 element but infer that
VDR is associating with this element in a conformation that is distinct
from how it binds a positive VDRE. This difference in VDR conformation
would presumably be imposed by the DNA element itself.
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VDR Binds to the GM550 Element as a Monomer-- We have demonstrated that the high affinity DNA binding species on GM550 does not include RXR (Figs. 2 and 4B), indicating that the repressing VDR species is not a heterodimer. Cheskis and Freedman (9) demonstrated that, in addition to the usual heterodimeric binding, VDR is also capable of binding to the mouse osteopontin positive VDRE as both homodimers and monomers, whereby preformed homodimers on DNA are dissociated to monomers upon addition of 1,25-(OH)2D3. To address whether VDR is binding to GM550 as a homodimer or a monomer, we took advantage of a truncated version of VDR constituting only the DBD. In the absence of the strong dimerization interface that co-localizes to the C-terminal ligand-binding domain, the VDR DBD cannot form dimers in solution, and as such resolves as both monomers and cooperative dimers on a DR3 in gel shift assays (13). When gel mobility shifts were carried out comparing VDR DBD binding to the positive versus negative element, as shown in Fig. 6A, the VDR DBD yielded two bound species with the DR3 (lanes 1-6), representing monomeric and cooperative dimeric species (dbd1 and dbd2, respectively), but resolved only one bound band with the GM550 probe, running with the same mobility as the faster of the two bound species seen with the DR3 probe (lanes 7-12). This is consistent with a VDR monomer associated with the GM550 element. As a control for monomeric binding, we used an oligonucleotide duplex containing only one half-site from the DR3 element (DR1/2); this probe restricted binding to predominately a single bound species that is presumably a VDR monomer (lanes 13-18). To further demonstrate this, a mixing experiment was carried out in which full-length VDR and VDR DBD were co-incubated, bound to each probe, and resolved by EMSA (Fig. 6B). Although an intermediate species consisting of a VDR·DBD heterodimer bound to the positive DR3 element was readily apparent (lanes 6 and 7), no such species was detected on the negative GM550 element, even at high concentrations of both receptors (lanes 11 and 12). These data strongly suggest that the DNA binding species on the GM550 element is a VDR monomer.
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DISCUSSION |
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The paradigm for vitamin D3 receptor binding to DNA elements that confer transcriptional activation in response to 1,25-(OH)2D3 is a dimeric receptor species comprising one molecule of VDR and one molecule of RXR bound to two directly repeating hexameric half-sites (consensus: PuGG/TTCA), which are spaced by three nucleotides, and termed DR3 (reviewed in Ref. 47). Each monomeric subunit makes a series of asymmetric interactions at the two DNA-binding domains, and symmetrical contacts within each ligand-binding domain. The heterodimeric complex can then presumably interact directly with the preinitiation complex or indirectly through a number of coactivators (15, 48-53), whose identities and functions are just now being defined, yielding the net result of transcriptional activation. This of course is all contingent on ligand binding, and the role of ligand in all of this appears to be manifold. Ligand binding increases the affinity of VDR for RXR (9, 10, 54) and therefore for the cognate DNA binding element, it induces conformational changes in the tertiary structure of the receptor (55-57), and it enhances interactions with components of the transcriptional machinery, presumably resulting in the stabilization of the preinitiation complex so that productive initiation can occur.
Since transcriptional repression is essentially the antagonism of all of these events, it is not surprising that the mechanism of transrepression by a nuclear receptor as we currently understand it is fundamentally different from activation. A number of genes have been described that are targets of repression by steroid and nuclear receptors, and a variety of mechanisms ranging from DNA-independent inhibition of positive factor (22, 58) to competition for common binding sites on a promoter have been proposed (18, 19, 21, 24, 28, 40, 59-61). However, what has not been adequately accounted for is how the same trans-factor, i.e. a steroid/nuclear receptor, can activate some target genes and repress others. An attractive hypothesis proposed by Yamamoto and others (62) is that in scenarios involving direct DNA binding, it is the DNA element itself that acts as an allosteric effector of receptor structure/function. This is feasible since in those cases where negative elements have been delineated, they rarely resemble positive elements (24, 26, 28, 61). Thus, the interaction of the receptor with a noncanonical recognition sequence might induce an alternative structure that would then be incapable of activating transcription, but has also gained a repressive function, possibly by precluding the binding of another, unrelated positive factor, such as Fos and Jun.
Four key pieces of data presented in this work are consistent with DNA
acting as an allosteric effector as an explanation for how VDR
represses the activated transcription of the GMCSF gene. First, we
clearly observe specific receptor binding to an element we have defined
to 7 bp at 2758 to
2765 consisting of the sequence GCTTTCC in the
GMCSF enhancer. Second, VDR binding to this element induces a
conformation in the receptor that is distinct from its apparent
conformation when it binds a positive DR3 VDRE, as deduced from both
limited protease digestion and from DNA binding and dimerization
mutants that are unable to bind and activate from the DR3 but can bind
and repress from the GMCSF negative element with near or equal affinity
and potency as wild-type receptor. Third, whereas VDR binds to and
activates transcription from the DR3 as a VDR·RXR heterodimer, RXR is
completely excluded from the VDR binding species at the negative
element. Fourth, VDR appears to bind the negative element as a monomer.
The last two observations are consistent with what we have previously
demonstrated concerning how 1,25-(OH)2D3
affects the dimerization state of VDR (9). In the presence of RXR,
1,25-(OH)2D3 promotes heterodimerization with
VDR, but in the absence of RXR, the ligand actually inhibits homodimerization and can in fact dissociate a preformed VDR homodimer to monomers. Thus, as depicted in Fig. 8,
we propose that the GM550 element allosterically induces VDR into a
conformation that precludes dimerization with RXR. Preformed VDR-RXR
heterodimers would not be able to bind this element, as we have shown
in vitro, or the element itself would actually induce the
dimeric complex to dissociate to monomers.
1,25-(OH)2D3 would enhance DNA binding by
preventing homodimerization and stabilizing monomers, since it is the
monomeric species that is the high affinity binding form on the GM550
negative element.
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As stated earlier, the GM550 negative element does not contain a canonical VDRE. It does, however, include binding sites for the activating factors NFAT-1 and AP-1 family members. Cockerill et al. (42) showed previously that occupancy at both of these sites is absolutely required for maximal activation of the GMCSF gene, in addition to occupancy at three other NFAT/AP-1 sites within the defined enhancer region. Since the negative element we have described here at position 550 overlaps an NFAT binding site, one possible mechanism for VDR-mediated transrepression is simple steric hindrance, in which a transcriptionally inert VDR monomer blocks the association of NFAT-1 protein to one half of the NFAT-1/AP-1 site. We are also interested in addressing the possibility that a similar antagonistic effect on NFAT binding to the additional NFAT/AP-1 sites within this enhancer may also contribute to the 1,25-(OH)2D3 effect on this gene. The overall inability of NFAT-1 to bind one or more of these sites would effectively block transcriptional activation of GMCSF (42). Targeting NFAT is also a strategy important immunosuppressive drugs such as cyclosporin A utilize, but at the level of NFAT dephosphorylation and nuclear localization (63-66). Moreover, the apparent allosteric changes the negative element is impinging on VDR may be translated into the creation of new protein surfaces. New surfaces normally inaccessible could then recruit a distinct set of interacting factors. Such factors might also be necessary for antagonizing the positive effects of AP-1 proteins and/or NFAT-1. Co-occupancy of VDR and the positive transactivating factors on the GM550 site is also a possible mechanism we cannot rule out. However, in this model, VDR would somehow have to lock NFAT-1 or AP-1 into an "off" state such that they could no longer transactivate, perhaps by directly interacting with one or both and precluding a productive interaction with a coactivator or basal factor. In this case, the simultaneous binding of VDR and NFAT proteins at the GM500 site is unlikely since their binding sites appear to overlap. Nonetheless, this model must be considered along with others; we are currently addressing the nature of VDR's interaction with AP-1 and NFAT proteins at the GM550 site to determine the molecular mechanism of this repression. Preliminary data from nuclear extracts prepared from Jurkat cells treated with activating agents and 1,25-(OH)2D3 show a disruption of a PMA/PHA-inducible DNA binding complex. However, we have yet to show occupancy of this element by VDR from these extracts and are currently working to address this.
Previous work from our laboratory identified the IL-2 gene as a
direct target of 1,25-(OH)2D3-mediated
repression. We believe this begins to explain the molecular basis of
the immunosuppressive effects of 1,25-(OH)2D3
at the level of the activated T cell, but not completely. The present
study extends this work by identifying the GMCSF locus as an additional
direct target of 1,25-(OH)2D3, and suggests a
more diverse role of vitamin D action in the immune system. The primary
role of GMCSF is to activate mature granulocytes and macrophages,
thereby eliciting the body's response to infection and initiating
inflammatory responses (67, 68). This response clearly must be tightly
regulated such that activation occurs only at appropriate times, and
that a gradual down-modulation of the response is initiated. We believe
that the data presented here is consistent with the role of
1,25-(OH)2D3 contributing directly to this
gradual decline of the inflammatory response at the level of repressing
the transcription of GMCSF in activated T cells. This would lead to a
decrease in the levels of GMCSF protein in the tissue periphery. It is
also likely that a similar regulatory process is occurring directly in
macrophages, although we have yet to test this. In fact, the role of
1,25-(OH)2D3 at the level of GMCSF and in
macrophages is further linked by the inference of a
1,25-(OH)2D3 feedback loop in this cell type.
Activated macrophages have been shown to produce the enzyme
1--hydroxylase (69, 70). This enzyme is the critical regulatory
enzyme required to convert the inactive vitamin D metabolite
25-(OH)D3 to the biologically active ligand
1,25-(OH)2D3. The ability of activated macrophages to promote an increase in
1,25-(OH)2D3 levels would in essence lead to
the deactivation of macrophages via repression of GMCSF expression.
There are potentially important clinical implications to this
autoregulatory loop. Dysregulation of GMCSF leads to pathological
conditions in diseases such as juvenile chronic myeloid leukemia (37),
acute myeloid leukemia (36), and rheumatoid arthritis. In these
scenarios, it is conceivable that the regulatory actions of
1,25-(OH)2D3 may not be operating normally, or
the feedback loop may be inactive. The close association of
1,25-(OH)2D3 and GMCSF as demonstrated here
suggests that the role of this ligand be evaluated carefully in these
and other diseases.
Acknowledgments-- We thank Peter Cockerill for original GMCSF plasmids, Amy Wolven for providing the FYN monoclonal antibody, and Ben Luisi and Mercy Devasahayam for critically reading this manuscript. We are also grateful to Christophe Rachez, Bryan D. Lemon, and Robert Benezra for helpful discussions and insights.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grants DK454460 (to L. P. F.) and CA08748 (to Sloan-Kettering).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Sloan-Kettering Institute Rudin Scholar.
§ Scholar of the Leukemia Society of America. To whom correspondence should be addressed: Cell Biology Program, Memorial Sloan-Kettering Cancer Center, Cornell University Graduate School of Medical Sciences, 1275 York Ave., New York, NY 10021. Tel.: 212-639-2976; Fax: 212-717-3298; E-mail: l-freedman{at}ski.mskcc.org.
1 The abbreviations used are: 1,25-(OH)2D3, 1,25-dihydroxyvitamin D3; VDR, vitamin D3 receptor; VDRE, vitamin D response element; RXR, retinoid X receptor; PTH, parathyroid hormone; IL-2, interleukin-2; bp, base pair(s); PMA, phorbol myristate acetate; PHA, phytohemagglutinin; GMCSF, granulocyte-macrophage colony-stimulating factor; EMSA, electrophoretic mobility shift assay; DBD, DNA-binding domain; CMV, cytomegalovirus.
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