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Bone and Mineral Research Program (L.L.I., G.M.L., J.B.B., J.A.E.) and Cancer Research Program (R.L.S.), Garvan Institute of Medical Research, St. Vincent’s Hospital, Sydney, New South Wales 2010, Australia
Address all correspondence and requests for reprints to: Laura L. Issa, Muscle Development Unit, Children’s Medical Research Institute, Locked Bag 23, Wentworthville, New South Wales 2145, Australia. E-mail: lissa{at}cmri.usyd.edu.au
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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heterodimers. In mammalian cells, GRIP1 augmented VDR activation of the
osteocalcin promoter, whereas RAC3 enhanced VDR activation indirectly
through RXR. These data suggest different coactivators regulate VDR
function via distinct mechanisms and support the hypothesis that the
VDR recruits different coactivators depending on specific gene and
cellular contexts. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The VDR functions as a homodimer or as a heterodimer with the retinoid X receptor (RXR) to bind vitamin D responsive elements (VDREs) in gene promoters. Recent studies have shown that activation of transcription by the VDR involves direct or indirect interaction with the general transcription factors, TFIIA and TFIIB, to recruit basal components to the preinitiation complex (4, 5, 6). Transcriptional activation by NHRs also requires direct interaction with coactivators that recruit and stabilize the binding of RNA polymerase II to the activated promoter. For example, the TATA-binding protein (TBP)-associated factor, TAFII135, potentiates VDR-, TR-, and RXR-mediated transcription (7).
Several other putative transcriptional coactivators for NHRs have been identified. These include SRC-1 (NCoA-1 or p160), GRIP1 (TIF2, NCoA-2 or SRC-2), and RAC3 (pCIP, ACTR, AIB1, or SRC-3; 8–16). Collectively, these molecules form a group of structurally and functionally related proteins, termed p160 coactivators. These coactivators interact with a range of NHRs in a ligand-dependent manner via the receptors’ activation function (AF-1/AF-2) domains.
Members of the p160 coactivator family share a series of LxxLL motifs, the so-called nuclear receptor (NR) boxes, which are required for interaction with NHRs (17, 18, 19). Specific receptors exhibit both coactivator and NR box binding preference, which allow for multiple interactions of a few coactivators with a diverse family of NHRs (20, 21). Transcriptional coactivators act as coregulators by integrating multiple signal transduction pathways with NHRs. For instance, SRC-1 interacts with TBP, TFIIB, and the CREB binding proteins, CBP/p300 and p/CAF in addition to NHRs (22, 23). Furthermore, SRC-1, CBP/p300 and RAC3 possess histone acetyltransferase activity, which is thought to modify chromatin structure and enhance access of NHR-cofactor complexes to DNA (15, 24).
Whether p160 coactivators participate in forming a transcriptionally active complex with the VDR and precisely how they modulate its cell-type-specific genomic actions is not known. Coactivators shown to interact with the VDR include GRIP1, RAC3, SUG1, SRC-1, RIP140, and NCoA-62 (11, 12, 14, 25, 26, 27, 28, 29). Previous studies investigating VDR interactions with GRIP1 and RAC3 do not address the functional role of these coactivators in VDR regulation of a natural VDRE particularly in the context of RXR, the major heterodimeric partner of VDR, and the ligand for RXR, 9-cis RA. Consequently, the present study investigated whether GRIP1 and RAC3 are bona fide transcriptional coactivators for the VDR in the absence and presence of RXR, and whether they modulate VDR function via similar or distinct mechanisms. In addition to examining ligand-dependent interactions between the VDR, GRIP1, and RAC3 in vitro, and formation of ternary complexes with VDR homodimers and VDR:RXR heterodimers, these interactions were examined using electromobility shift assays and activation of the osteocalcin gene promoter VDRE in a mammalian cell system.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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cDNA fragment from
pGST-RXR was cloned into pACTIIß (31). pGAD424-GRIP1
containing nucleotides 204-4878 of GRIP1 cDNA was kindly provided by
Dr. M. Stallcup (11). pGAD-RAC3.1 containing nucleotides
1289–3698 (amino acids 401-1204) of RAC3 was a gift from Dr. D. Chen
(14). The yeast LexA-DNA binding domain expression
construct, pEG202 was provided by Dr. R. Brent. hVDR cDNA from
pAS2–1-VDR was subcloned into BamHI linearized pEG202.
hRXR cDNA from pACTIIß-RXR was cloned into
EcoRI/BamHI-linearized pEG202. GRIP1 cDNA from
pGAD424-GRIP1 was subcloned into EcoRI cut pEG202.
BglII RAC3 cDNA from pGAD-RAC3.1 was inserted in
BamHI linearized pEG202.
Yeast constructs expressing truncated hVDR were generated using
pPH13-VDR as a template. pAS2–1-VDR
1–191 (VDR:LBD) was constructed
by PCR amplification of nucleotides 574-1281. pAS2–1-VDR365–427
(VDRAF-2) was generated by PstI digestion followed by
ligation of pAS2–1-VDR. pAS2–1-VDR1–280 was constructed by
removing the first 954 nucleotides by NcoI digestion and
religation of pAS2–1-VDR. pAS2–1-VDR1–89 was generated by PCR
amplification of nucleotides 116–382 of pPH13-VDR which were
subsequently cloned into the BamHI site of pAS2–1.
For transient transfections of mammalian cells, hVDR cDNA was PCR
amplified from SaoS-2 cellular RNA and cloned into
HindIII/ApaI linearized pRC-CMV
(Invitrogen, Carlsbad, CA). To clone the EcoRI
GRIP1 cDNA fragment in frame with the EcoRI site of pCMV2
(Eastman Kodak Co., Rochester, NY) the vector was first
modified by Klenow fill-in of the HindIII site to create
pCMV2c. EcoRI/BglII RXR cDNA fragment was
subsequently cloned in pCMV2c. pCMV2-RAC3 was generated by PCR
amplification of the RAC3 cDNA using pGAD-RAC3.1 as a template with
primers containing BglII 5' overhangs. The human osteocalcin
gene promoter-luciferase reporter construct, pOSLUC2, containing 1.34
kb of promoter sequence, was created by exchanging the
XhoI/Vsp1 fragment (corresponding to the CAT
cDNA) of pOSCAT2 (32) with the
XhoI/Vsp1 luciferase cDNA fragment of pGL-3basic
(Promega Corp., Madison, WI). The Renilla
luciferase gene reporter, pRL-TK (Promega Corp.), was used
to determine transfection efficiency.
VDR and RXR cDNA fragments from pAS2–1-VDR and
pACTIIß-RXR were subcloned into pSG5 (Promega Corp.)
and used for in vitro coupled transcription and translation.
A modified pSG5 vector, designated pSG5ß, was created by
replacing the EcoRI/BglII polylinker fragment of
pSG5 with a
double-stranded-oligonucleotide-(5'-AATTACGCTACAACGCCATGGGCGAATTCAGGATCCGCATCGATTAGCTGAATAGTA-3').
EcoRI GRIP1 cDNA fragment was inserted in EcoRI
linearized pSG5ß. BglII RAC3 cDNA from pGAD-RAC3.1 was
cloned in BamHI linearized pSG5ß. PCR primer sequences are
available on request.
Northern blot analysis
Total RNA was isolated from subconfluent cells by denaturation
in guanidinium followed by purification using a cesium chloride step
gradient. Total RNA (15 µg) was subjected to Northern analysis. cDNA
probes for hVDR, hRXR, mRXRß, GRIP1, RAC3, and SRC-1 were labeled
with [32P]dCTP by random priming using
Klenow DNA polymerase (Promega Corp.) and purified through
a Nick column (Amersham Pharmacia Biotech, Uppsala,
Sweden). Northern blots were prehybridized for 4 h at 50 C
in prehybridization buffer (10x Denhardt’s solution; 5x SSC
(1x SSC is 0.15 M NaCl, 0.015 M sodium
citrate); 50 mM Tris-HCl, pH 7.5; 1% SDS; 0.2x
SSPE [1x SSPE is 0.18 M NaCl, 10 mM
NaPO4, 1 mM EDTA, pH 7.7), and 180
µg/ml denatured salmon sperm DNA]. Blots were hybridized with
labeled probes overnight at 50 C in hybridization mixture (1x
Denhardt’s solution; 0.8x SSC; 50 mM Tris-HCl, pH 7.5;
1% SDS; 0.04x SSPE; 9.6% dextran sulfate; 50% deionized formamide,
and 3.6 µg denatured salmon sperm DNA). Blots were washed twice in
2x SSC and 0.1% SDS at 63 C for 30 min, followed by two washes in
0.2x SSC and 0.1% SDS at 63 C for 30 min then autoradiographed at
-80 C for 4–24 h.
Coimmunoprecipitation
Radiolabeled ([35S]-Methionine;
NEN Life Science Products, Boston, MA) VDR, RXR, GRIP1,
and RAC3 were expressed from pSG5 vectors in vitro using the
rabbit reticulocyte lysate coupled transcription-translation kit (TNT;
Promega Corp.). Protein lysates (10 µl) were incubated
for 30 min at 22 C with 30 ng of recombinant hVDR (Affinity BioReagents, Inc., Neshanic Station, NJ), in the presence of 100
nM calcitriol or vehicle. Binding reactions were
performed in a binding buffer containing 50 mM
HEPES (pH 7.5), 150 mM NaCl, 10% glycerol, 1%
Triton X-100, 1.5 mM MgCl2,
1 mM EGTA, 10 mM
Na-pyrophosphate, 20 mM NaF, 1
mM phenylmethylsulfonyl fluoride, 200
µM Na-orthovanadate, and 10 µg/ml of each of
aprotinin and leupeptin. Rat anti-VDR antibody (100 ng of 9A7;
Affinity BioReagents, Inc.) was added and the mixture
incubated 30 min at 4 C. Rabbit antirat IgG2a (100 ng; Zymed Laboratories, Inc., South San Francisco, CA) was added for 45
min at 4 C. The protein-immunocomplexes were immobilized on protein
A-Sepharose 4B conjugate (Zymed Laboratories, Inc.)
following incubation for 30 min at 4 C and extensive washes in binding
buffer. Protein-immunocomplexes were resuspended in 3x Laemmli buffer
and resolved by SDS-PAGE. The gels were dried and autoradiographed
using Kodak Biomax film (Eastman Kodak Co.,
Rochester, NY).
Yeast two-hybrid assay
A two-hybrid mating assay was used to examine interactions
between gal4DBD-hVDR and various gal4AD-cofactor fusion proteins. The
yeast reporter strain CG-1945 (CLONTECH Laboratories, Inc.) was transformed with 1 µg of pAS2–1 or pAS2–1hVDR
using an alkali cation method (BIO 101, Inc., La Jolla, CA).
Transformants were selected on complete synthetic media (CSM; BIO 101)
plates lacking tryptophan. Y187 cells (CLONTECH Laboratories, Inc.) were transformed with 1 µg of either pACTII, pACTII-VDR,
pACTIIß-RXR, pGAD424-GRIP1, or pGAD10-RAC3.1 and selected on CSM
plates lacking leucine. Transformed CG-1945 cells were mated with Y187
cells in microtiter plates containing YEPD medium for 16 h at 30
C. Diploid cells were selected, grown to saturation then used to
inoculate (OD600 of 0.1) test cultures. These
were grown for 4 h then treated with calcitriol or vehicle and
grown for 16 h at 30 C. Yeast cells were harvested in 100 µl
Breaking Buffer (100 mM Tris pH 8; 20% glycerol; 5
mM phenylmethylsulfonyl fluoride; 2 mM
dithiothreitol) and lysed by vortexing with glass beads. Protein
concentration was determined by the Bradford method (Bio-Rad Laboratories, Inc., Sydney, New South Wales, Australia). Protein
extracts (10–20 µg) were assayed for ß-galactosidase activity at
room temperature for 1 h using the Galactolight reporter assay kit
(Tropix, Bedford, MA) and quantitated with a Berthold AutoLumat LB953
(Bad Wildbad, Germany).
For yeast experiments in which cofactor interactions with hVDR dimers
were investigated, SFY526 cells (CLONTECH Laboratories, Inc.) were transformed with pAS2–1 or pAS2–1hVDR. SFY526
transformants were mated with the Y187 strains described above. These
diploid master strains were subsequently transformed with pEG202,
pEG202-hVDR, pEG202-RXR, pEG202-GRIP1, and pEG202-RAC3 and selected
on CSM plates lacking tryptophan, leucine, and histidine. Test cultures
were grown and assayed as for the yeast two-hybrid assay.
Western blot analysis
For determination of gal4 fusion protein expression in yeast
cells, cells were grown to saturation, harvested, and the protein
extracted by a TCA precipitation method. Uniform expression of
gal4DBD:VDR across different yeast diploid strains was confirmed by
immunoblotting for hVDR. An antibody for the gal4DBD was used to
determine expression of truncated gal4DBD:VDR proteins. Protein lysates
(20–30 µg) were loaded onto a 10 or 12% SDS-PAGE gel. Gels were
electroblotted onto nitrocellulose membrane and blocked with
Tris-buffer saline containing 0.1% Triton-X 100 (TBS-T) containing 5%
skim milk powder and 1% BSA for 16 h at 4 C. Membranes were
exposed to rat anti-VDR (1 µg/ml; Affinity BioReagents, Inc.) or mouse anti-gal4DBD antibody (0.5 µg/ml; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) in TBS-T + 1% BSA for
2 h at 22 C, followed by rabbit antirat (1/36,000) or sheep
antimouse HRP-conjugated secondary antibody (1/2000; Amersham International, Buckinghamshire, UK) for a further 1–2 h at 22
C. Immunoblotted bands were detected by a chemiluminecent method (ECL;
Amersham Pharmacia Biotech).
Electromobility shift assay
VDR, RXR, GRIP1, and RAC3 were expressed in vitro
using the TNT kit (Promega Corp.). Oligonucleotides
corresponding to nucleotides -521 to -450 of the human osteocalcin
gene promoter were labeled with [32P]dCTP
using Klenow DNA polymerase (Promega Corp.), isolated by
electrophoresis through a 15% polyacrylamide gel, eluted in TE buffer,
pH 8, and purified through glasswool. Receptor lystates (8 µl) were
incubated with 100 nM calcitriol or/and 1
µM 9-cis RA or vehicle in a binding buffer (10
mM Tris HCl/HEPES; 200 mM
KCl; 0.5 mM EDTA; 2 mM
MgCl2; 1 mM dithiothreitol)
for 20 min at 22 C. Labeled probe (45,000 cpm) was added and the
reactions incubated a further 20 min at 22 C. Protein-DNA reactions
were separated by electrophoresis in TBE buffer (pH 7.4) through a 4%
nondenaturing polyacrylamide (29, 1) gel containing
1.6% glycerol. Gels were dried and autoradiographed for 4–8 h.
Mammalian cell transient transfections
Cells were transfected using the nonliposomal FuGENE reagent
(Roche Molecular Biochemicals, Sydney, New South
Wales, Australia). Approximately 5 x 105
CV-1 monkey kidney cells, maintained in DMEM (Life Technologies, Inc., Gaithersburg, MD) supplemented with 10% FCS, were seeded
in 10 cm2 dishes. After 4 h in DMEM
containing 2% charcoal-stripped serum, cells were transfected with a
total of 10 µg plasmid DNA containing 2 µg of pOSLUC2 or pOSLUC1
and 0.1 µg of pRL-TK. Sixteen hours post transfection, the cells were
trypsinized and seeded into 12-well plates. Test cultures were treated
with appropriate ligand for a further 16 h before being harvested,
lysed in 1x lysis buffer (Promega Corp.), and tested for
Firefly and Renilla luciferase.
Firefly luciferase data were normalized to
Renilla luciferase data to correct for transfection
efficiency. For transient transfections of P19 mouse embryonal
carcinoma cells (maintained in -MEM supplemented with 5% serum),
106 cells were transfected as above.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Although not apparent by Northern
blot analysis, low levels of VDR protein expression in CV-1 cells has
been confirmed by responsiveness to calcitriol in reporter gene
transfections. RXR was ubiquitously expressed but at differential
levels; with the highest expression in COS1, mOB, NIH3T3, ROS17/2.8,
and ROS24 cells, and the lowest expression in CV-1 and P19 cells.
RXRß was also widely expressed, with the highest expression in NIH3T3
and P19 cells. Similarly, GRIP1 was highly expressed in NIH3T3, P19,
and the bone cell lines. RAC3 messenger RNA (mRNA) expression followed
an inverse pattern to that of GRIP1 with the highest expression in the
Green Monkey kidney cell lines, COS1 and CV-1, and very low levels in
other cell lines. SRC-1 was widely expressed at differential levels,
with high expression levels in COS1 and ROS24 cells and low levels in
mOB cells.
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as an experimental control. GRIP1 and RAC3 were
coimmunoprecipitated with recombinant VDR only in the presence of
calcitriol (Fig. 2).
Coimmunoprecipitation of RXR with VDR was observed in the absence of
calcitriol; however, this was augmented by addition of calcitriol.
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interacted with
gal4DBD:VDR in the absence of ligand (Fig. 3). However, calcitriol increased the
response almost 5-fold. The gal4DBD:VDR fusion protein
interacted with gal4AD:GRIP1 or gal4AD:RAC3
but only in the presence of calcitriol. When fused to the gal4AD
neither VDR, RXR, GRIP1, nor RAC3 activated reporter gene expression
nonspecifically, i.e. when coexpressed with gal4DBD
alone.
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, GRIP1, and
RAC3 were examined by deletion analysis (Fig. 4A). Comparable levels of expression of
these VDR mutants were confirmed by Western blotting with an
anti-gal4DBD antibody (Fig. 4B). RXR interacted with full-length
VDR, but not with any of the deletion mutants (Fig. 5A). Similarly, GRIP1 displayed
ligand-dependent interaction only with the wild-type receptor and
failed to interact with either the N-terminal deletion mutant, VDR-LBD,
or the AF-2 deletion mutant, VDR-AF-2 (Fig. 5B). In contrast, RAC3
displayed ligand-dependent interaction with the full-length wild-type
VDR and with the VDR-LBD mutant but did not interact with either the
VDR-AF-2 mutant or the VDR-1–280 (Fig. 5C).
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). Excess GRIP1 did not interfere with
this interaction, whereas RAC3 expression potentiated
calcitriol-dependent interaction between gal4DBD:VDR and
gal4AD:GRIP1 by 2- to 3-fold (Fig. 6A). Interaction
between gal4DBD:VDR and gal4AD:RAC3 was
largely unaffected by coexpression of VDR, RXR or RAC3. However,
coexpression of GRIP1 induced a 2- to 3-fold enhancement of
calcitriol-dependent gal4DBD:VDR/gal4AD:RAC3
interaction (Fig. 6B). These results demonstrate that GRIP1 and RAC3
can coassemble with the VDR, possibly by using alternative contact
sites.
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heterodimer complexes
by GRIP1 and RAC3). Similarly, the coactivators enhanced
transactivation by heterodimeric interaction between the
gal4DBD:VDR and gal4AD:RXR fusion proteins
in a calcitriol-dependent manner (Fig. 7B). Again, the effect of RAC3
was more potent than that of GRIP1. 9-cis RA cotreatment did
not affect calcitriol-induced interaction between
gal4DBD:VDR and gal4AD:RXR in the presence
of GRIP1 or RAC3; hence, the coactivators interact with the heterodimer
via ligand-activated VDR.
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heterodimeric activity, relative to empty
vector. RAC3 potentiated transactivation by VDR:RXR heterodimers by
60 (±25; n = 3)-fold compared with 15 (±5)-fold for VDR
homodimers. Overexpression of VDR or RXR did not compete with the
gal4-VDR fusion protein for interaction in the VDR homodimer strain
[1.2 (±0.2) and 0.9 (±0.2), respectively]. However, RXR
displaced interaction between gal4DBD:VDR and
gal4AD:RXR [0.5 (±0.1)], but not between
gal4DBD:VDR and gal4AD:VDR [0.9 (±0.4)].
The control reactions, in which the cofactors were transformed in a
yeast cell background containing gal4DBD with gal4AD, or
gal4DBD:VDR with gal4AD, showed low nonspecific activity
(Fig. 7
, C and D).
GRIP1 and RAC3 displaced VDR:RXR heterodimer binding to DNA in
vitro
The above data provided evidence that the cofactors retain ability
to interact with the VDR when it is dimerized or heterodimerized with
RXR. Consequently, ternary interactions between GRIP1 and RAC3 with VDR
dimers were examined in the context of a VDRE by an electromobility
shift assay. The VDR bound the osteocalcin VDRE probe (-521 to
-450) as a heterodimer with RXR and in a ligand-dependent
manner (Fig. 8A). Neither VDR alone nor
RXR alone bound the VDRE probe in the absence or presence of their
cognate ligands. Coincubation with GRIP1 displaced the VDR:RXR-DNA
complex induced by calcitriol, in the absence and presence of
9-cis RA, by almost 50%. These effects were reproducible
both qualitatively and quantitatively in multiple experiments (n =
2–5). Similarly, addition of RAC3 displaced VDR:RXR heterodimeric
binding to DNA. To test whether coactivator displacement of the
heterodimer-DNA complex was due to competition for RXR, RXR was
titrated against a fixed amount of VDR and GRIP1 (Fig. 8B). At a ratio
of VDR:RXR:GRIP1 of 1:2:4, the intensity of the ligandinduced
heterodimeric-DNA complex was restored to the same level as that seen
in the absence of GRIP1. At a ratio of VDR:RXR:GRIP1 of 1:4:4 the
heterodimeric-DNA complex induced by calcitriol and 9-cis RA
cotreatment was inhibited by 50%, whereas the calcitriol-induced
complex was unaffected. These results suggest that VDR:RXR
heterodimeric binding to a VDRE precludes interaction with GRIP1 and
RAC3.
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). Overexpression of GRIP1 with VDR and
RXR potentiated calcitriol-mediated transactivation by 3.6
(±1.4)-fold compared with the 1.5 (±0.7)-fold induction seen in cells
transfected with only VDR and RXR. GRIP1 also increased the
9-cis RA-induced response by 4.0 (±1.5)-fold in the absence
of overexpressed RXR, and by 37 (±7)-fold in the presence of
RXR, compared with the 17 (±2.2)-fold increase seen in the VDR and
RXR transfected cells. Cotransfection of GRIP1 with VDR and RXR
had an additive effect on the transcriptional response to calcitriol
plus 9-cis RA cotreatment, inducing 10.4 (±3.3)-fold
increase in reporter activity, compared with 3.1 (±1.3)-fold in VDR
and GRIP1 transfected cells and 4.2 (±1.0)-fold in VDR and RXR
transfected cells.
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).
However, coexpression of RAC3 with VDR and RXR enhanced
9-cis RA-induced transcriptional response by 89 (±28;
n = 4)-fold compared with 17 (±2.2)-fold in VDR and RXR
transfected cells. Coexpression of RAC3 with VDR and RXR also
enhanced induction following calcitriol plus 9-cis RA
cotreatment by 23 (±10)-fold, compared with 4.2 (±1.0)-fold in the
VDR and RXR transfected cells.
In CV-1 cells, which have abundant endogenous RAC3 but low GRIP1
expression, overexpression of RAC3 did not significantly affect
transactivation of pOSLUC2 (not shown). Coexpression of GRIP1 with VDR,
in this cell line, resulted in a modest 2.1 (±0.6; n = 5)-fold
induction of pOSLUC2 activity by calcitriol (Fig. 9B). This effect was
attenuated in cells transfected with GRIP1, VDR, and RXR.
Coexpression of GRIP1 with VDR also increased the transcriptional
response to 9-cis RA treatment by 4.5 (±1.1)-fold, and the
response to calcitriol plus 9-cis RA cotreatment by 3.8
(±1.4)-fold, relative to cells transfected with VDR alone.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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GRIP1 and RAC3 were coexpressed with the VDR in cell lines
representative of vitamin D-responsive tissues and
participated in direct contact with ligand-activated VDR in
vitro. In yeast cells, VDR-coactivator interactions were both
ligand and AF-2 domain-dependent. GRIP1 and RAC3 appeared to interact
simultaneously with the VDR through distinct regions of the VDR.
Interaction studies in yeast cells suggest that the coactivators form
ternary complexes with VDR:RXR heterodimers. However, in
vitro gel shift studies showed DNA binding by VDR:RXR
heterodimers was disrupted by GRIP1 and RAC3 apparently via competition
for RXR. In mammalian cells, GRIP1 potentiated VDR-mediated
transactivation of the osteocalcin VDRE, directly through
ligand-activated VDR, and, indirectly through ligand-activated RXR.
Hence GRIP1 may function to integrate cooperative signaling between VDR
and RXR. In contrast, RAC3 did not potentiate VDR-mediated
transactivation directly but facilitated transactivation by VDR:RXR
heterodimers through ligand-activated RXR.
The presence of three structurally similar cofactors that can serve as coactivators for NHRs, notably GRIP1 and RAC3 as well as SRC-1, suggests that these coactivators may play different roles in different cells (9, 11, 14). Consequently, mRNA expression of these cofactors was examined across a panel of cell lines representative of vitamin D target tissues. This revealed that GRIP1 and RAC3 are expressed differentially and, more importantly, inversely relative to each other. SRC-1, however, is ubiquitously expressed. Possibly, the VDR uses different coactivators to activate transcription depending on the cell line or target promoter. In support of this concept, the bone cell lines, mOB, ROS17/2.8 and ROS24, expressed abundant GRIP1 mRNA with low or undetectable levels of RAC3. Consistent with a possible role in modulating VDR function in bone, GRIP1 potentiated transactivation of the osteocalcin gene promoter, which is expressed only in osteoblasts. The cell lines CV-1 and COS1 are derived from primate kidney, a tissue involved in calcium and phosphate homeostasis and vitamin D activation. These cells had high levels of RAC3 expression but low GRIP1 expression. It remains to be shown whether RAC3 is involved in regulating vitamin D-responsive genes in kidney-derived cell lines. Coexpression of SRC-1 with RAC3 in the kidney cell lines, and with GRIP1 in the bone cell lines, suggests that coactivators may substitute one another depending on the target promoter, or more than one coactivator can assemble with the VDR at an active promoter. The latter is consistent with data presented here showing GRIP1 and RAC3 interacted with the VDR simultaneously. Thus, different combinations of coactivators could fine-tune VDR function and its cross-talk with other cis-acting transcription factors, such as RXR.
The current study confirms, by in vitro immunoprecipitation
experiments, that the VDR interacts directly with GRIP1 and RAC3 in a
ligand-dependent manner. Hence, a ligand-induced conformational change
to the VDR is required to expose coactivator interactive domains
(36, 37, 38). Further, ligand-dependency implicates a role for
GRIP1 and RAC3 in ligand-mediated gene transactivation via the AF-2
domain. Consistent with this, neither GRIP1 or RAC3 interacted with a
VDR AF-2 deletion mutant. Whether different coactivator molecules
interact with the same or different sites on the VDR is unknown, hence
the ability of GRIP1 and RAC3 to interact with a panel of VDR
truncation mutants was investigated. These results show RAC3 interacted
with the VDR-LBD (1–190) but not the VDR1–280 mutant, and
therefore amino acids 191–280 are crucial for generating the
appropriate functional conformation necessary for efficient binding.
Amino acids 191–280 encompass -helix 3 (225–246) of
the ligand binding domain that interacts with the AF-2 domain of
-helix 12 following ligand-induced intramolecular folding (27, 36, 37). In contrast, GRIP1 did not interact with the
VDR1–190 mutant, requiring both the N-terminal and C-terminal
regions of the VDR. Recently, Chen et al. (25)
identified specific amino acids within -helices 3, 5, and 12 of the
VDR required for interaction with GRIP1 but overlooked the importance
of the N terminus. Although further VDR deletion and point mutation
analyses are required to identify the amino acids involved in
distinguishing GRIP1 from RAC3 binding, these results have led to some
novel observations. Firstly, different coactivators bind different
sites on the VDR and, secondly, the VDR appears to interact
simultaneously with more than one coactivator molecule by using
alternative binding sites (Fig. 10A).
In support of these conclusions, transactivation by the VDR:GRIP1
complex in yeast cells was markedly enhanced when RAC3 was coexpressed
by formation of a ternary complex. Similarly, GRIP1 potentiated
VDR:RAC3 interaction.
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heterodimers, suggestive of tripartite
interactions. Although GRIP1 did not appear to differentiate between
the VDR dimeric forms, RAC3 was a more potent coactivator of VDR:RXR
heterodimers. These data are consistent with the VDR deletion
experiments that suggest the coactivators require different regions of
the VDR for interaction. Possibly, the conformational changes following
heterodimerization facilitate stronger binding between VDR and RAC3.
Coexpression of RXR disrupted interaction between
gal4DBD:VDR and gal4AD:GRIP1 but not between
gal4DBD:VDR and gal4AD:RAC3. These observed
differences are consistent with independent binding sites on the VDR
for these cofactors.
The osteocalcin gene promoter, a typical vitamin Dresponsive gene,
provides a good model for investigating the interplay between VDR, RXR,
and the coactivators. The osteocalcin promoter has both a
calcitriol-responsive VDRE and a 9-cis RA-responsive element
(RXRE), which can function synergistically (40). Because
GRIP1 has been shown to interact with RXR in yeast cells and in
vitro, GRIP1 may coactivate both the VDR heterodimers at the VDRE
and RXR homodimers at the RXRE (41). In P19 cells, GRIP1
overexpression with VDR enhanced induction of the osteocalcin gene
promoter by calcitriol 2- to 4-fold. When GRIP1 was coexpressed with
VDR and RXR, there was an additive effect on the transcriptional
response to calcitriol plus 9-cis RA costimulation, relative
to either hormone alone. Thus, GRIP1 potentiates both calcitriol- and
9-cis RA-mediated transactivation of the osteocalcin
promoter. It is possible that GRIP1 oscillates between interaction with
the VDR and with RXR in the presence of both hormones. For instance, in
CV-1 cells, which have low endogenous RXR, coexpression of RXR with
GRIP1 attenuated GRIP1 coactivation of the VDR, suggesting that VDR:RXR
heterodimers and RXR:RXR homodimers were competing for GRIP1. Hence,
the VDRE and RXRE, which can function synergistically through
allosteric interaction, can also be antagonistic through competition
for coactivators, depending on the cell line. Another possibility is
that GRIP1 preferentially binds RXR. This is supported by the
electromobility shift data where, using a region of the osteocalcin
gene promoter containing the VDRE but not the RXRE, GRIP1 displaced RXR
from heterodimerization with VDR and disrupted the receptor-DNA
complex. Addition of excess RXR restored calcitriol-induced
VDR:RXR heterodimerization. However, in the presence of
9-cis RA, GRIP1 competition was further augmented,
indicating GRIP1 was preferentially interacting with ligand-activated
RXR. Similarly, RAC3 displaced VDR:RXR-DNA complexes, which may
reflect allosteric inhibition by the RXR AF-2 domain, as has been
previously suggested (26).
Collectively, these results indicate GRIP1 functions as a coactivator
for both the VDR and for RXR. The direction in which the multipartite
complex forms is dependent on both the abundance of individual NHRs and
coactivators in the cell and the activating hormone (Fig. 10B).
Possibly, GRIP1 may function to coordinate dimerization between VDR and
RXR in response to hormonal signals. In contrast, RAC3 interacted with
the VDR but did not appear to act as a direct coactivator of VDR in the
context of the osteocalcin promoter. Although, others have observed
that RAC3 potentiates VDR induction of a synthetic consensus VDRE
weakly in CV-1 cells, they did not address 9-cis
RA-dependent activation (26). RAC3 did not potentiate
VDR-mediated transcription in CV-1 cells under our conditions. However,
overexpression of RAC3 in P19 cells potentiated 9-cis
RA-dependent but not calcitriol-dependent transactivation, and thus
RAC3 may regulate the VDR indirectly through RXR (Fig. 10C).
In summary, this study examined two functionally and structurally
related coactivators in relation to VDR function. GRIP1 and RAC3 played
distinct roles in regulating VDR function. They both interacted with
the VDR in a ligand- and AF-2-dependent manner but differed in their
sites of interaction, their ability to form ternary complexes with VDR
homodimers and VDR:RXR heterodimers, and in their effects on the
interplay between vitamin D and retinoid signaling pathways. Further,
GRIP1 and RAC3 exhibited differential expression across cell lines
representative of vitamin Dresponsive tissues. These data lead us
to hypothesize a model whereby the cell- and promoter-specific
functions of the VDR may be mediated through differential recruitment
of coactivators. The interplay between VDR, RXR, and the
coactivators is complex and dependent on the relative abundance of
cofactors and activating hormones. This functional complexity may be
the basis for transcriptional switches controlling diverse and complex
programs of gene expression in response to hormonal signals.
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Acknowledgments |
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Footnotes |
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2 Recipient of an Australian Postgraduate Award.
3 Recipient of an NH & MRC Postgraduate Medical Scholarship.
Received July 14, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ligand-binding domain bound to all-trans retinoic acid.
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M (eds) 10th Workshop on Vitamin D. Chemistry, biology and clinical
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) and presence of
10 nM calcitriol (
). Data are the mean ±
SD of three separate yeast cultures and are representative
of three to five independent experiments.
). Data are the
mean ± SD of triplicate cultures and are
representative of three independent experiments. Note the activity data
in (A) and (B) are RLU x 105 and in (C) and (D) as
RLU x 104. Also note the broken scales in (A) and
(B).
), 10
nM calcitriol (
), 1 µM
9-cis RA (