From the Laboratoire de Biologie Cellulaire
Hématopoïétique, Equipe Mixte Inserm
00-03, Institut Universitaire d'Hématologie, Hôpital
Saint-Louis, 1 Avenue Claude Vellefaux, Paris 75010 and the
¶ Institut de Génétique et de Biologie
Moléculaire et Cellulaire, Unité Mixte de
Recherches 7104, 67404 Illkirch, Cedex
France
Received for publication, October 18, 2002, and in revised form, December 6, 2002
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ABSTRACT |
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Ligand-induced transcription activation of
retinoic acid (RA) target genes by nuclear receptors (retinoic acid
(RAR) and retinoid X (RXR) receptors) depends on the recruitment of
coactivators. We have previously demonstrated that the small
15-kDa cellular RA-binding protein II (CRABPII) is a coactivator
present in the RA-dependent nuclear complex. As identifying
cell-specific partners of CRABPII might help to understand the novel
control of RA signaling, we performed a yeast two-hybrid screen of a
hematopoietic HL-60 cDNA library using human CRABPII as bait and
have subsequently identified human cyclin D3 as a partner of CRABPII.
Cyclin D3 interacted with CRABPII in a ligand-independent manner and
equally bound RAR Retinoic acid (RA)1
plays a pivotal role in the development and homeostasis of vertebrates
through its ability to directly control the transcription of target
genes involved in the control of cell proliferation, differentiation,
and survival. Retinoids mediate transcription through two
classes of nuclear receptors, the retinoic acid receptors (RAR Ligand binding induces conformational changes in the LBD involving
helix H12, which encompasses the core of AF-2 of the nuclear receptor,
resulting in the creation of a new surface for the recruitment of
coactivators, such as proteins of the p160 family (SRC-1
(steroid receptor
coactivator-1)/NCoA1, TIF2/GRIP-1/SRC-2, and
p/CIP/RAC3/ACTR/AIB-1/TRAM-1) and CBP (cAMP-response
element-binding protein-binding protein)/p300 (2-4). These coactivators are also associated with other large histone
acetyltransferase complexes (such as the p/CAF complex) that lead to
chromatin decondensation (5, 6). Nuclear receptors further recruit an
additional complex variously termed TRAP, DRIP, SMCC, or Mediator,
which establishes contacts with RNA polymerase II and the general
transcription factors (7, 8).
We previously showed that a small protein (15 kDa) belonging to the
family of intracellular lipid-binding proteins that bind small
hydrophobic molecules such as retinoids and fatty acids (9), cellular
retinoic acid-binding protein II (CRABPII), also acts as a coactivator
of nuclear retinoid receptors (10). Indeed, CRABPII can be found in the
nucleus (10, 11) and more specifically in the RA-dependent
nuclear complex (10). Moreover, we (10) and others (12-14) have shown
that overexpression of CRABPII enhances transactivation of RA target
genes by RXR·RAR heterodimers either in transfected cells or in human
hematopoietic cells. This coactivator effect results from a physical
interaction of CRABPII with the LBD of RAR As identifying cell-specific partners of CRABPII in the
RA-dependent nuclear complex may help to understand the
novel control of RA signaling, we performed a yeast two-hybrid screen
of a hematopoietic HL-60 cDNA library using human CRABPII as bait
and have subsequently identified human cyclin D3 as a novel partner of
CRABPII. We found that cyclin D3 interacted with CRABPII in a
ligand-independent manner. No interaction was noted with other D-type
cyclins (D1 or D2). Interestingly, cyclin D3 was also found to interact
with RAR Antibodies and Reagents--
Mouse monoclonal antibodies
directed against CRABPII (5CRA3B3) and rabbit polyclonal antibodies
directed against the F region of RAR Plasmids--
The expression vectors for RAR Yeast Strains and Transformation--
The yeast reporter strain
Y190 (HIS3, lacZ, trp1,
leu2, cyhr2) was previously described by
Harper et al. (20). Transformation was carried out using the
lithium acetate procedure (21), and expression of the fusion proteins
was checked by immunoblotting. Standard media
(Clontech) were used for yeast growth according to
the manufacturer's protocols.
Two-hybrid Screening--
An HL-60 Gal4AAD fusion library (a
gift from J.-M. Garnier and P. Chambon) was inserted into the pACT2
vector, which contains the LEU2 marker and a cassette
expressing nuclear localized Gal4AAD preceding a polylinker with
cloning sites, and introduced into the Y190 reporter strain expressing
hCRABPII from the pAS2-1 vector. The cells were then spread on
Trp Protein-Protein Interaction Using the Yeast Two-hybrid
System--
The yeast strain Y190 was co-transformed with pACT2
expressing cyclin D3 and with either hCRABPII or hRAR Immunoblot Analysis--
Protein extracts were quantified by the
BCA protein assay (Pierce) and by Coomassie staining after separation
by SDS-PAGE. The proteins were transferred onto a nitrocellulose
membrane and incubated with a 1:200 dilution of anti-cyclin D1, D2, or
D3 antibody; a 1:1000 dilution of anti-RAR antibody; a 1:500 dilution
of anti-CRABPII antibody; or a 1:4000 dilution of anti-actin antibody.
The proteins were identified by chemiluminescence.
In Vitro GST Pull-down Assays--
GST and GST fusion proteins
(GST-hRAR Immunoprecipitation--
COS-6 cells were transiently
transfected in the presence or absence of 1 µM of
all-trans-RA using the calcium phosphate precipitation technique as described (22). Whole cell extracts were prepared (10) and
immunoprecipitated as follows. The extracts were first incubated with
goat anti-cyclin D3 or D2 antibody for 1 h and then with protein
G-Sepharose (Amersham Biosciences) for an additional hour. The
immunocomplexes were recovered by centrifugation, washed, and resolved
by SDS-PAGE. The immunoprecipitated proteins were revealed by
immunoblotting and chemiluminescence.
HL-60 Cell Transfection--
HL-60 cells were routinely
maintained in RPMI 1640 medium (Invitrogen) supplemented with 15%
fetal bovine serum, 2 mM glutamine, and 100 µg/ml
penicillin/streptomycin (all from BioWhittaker Europe, Verviers,
Belgium). HL-60 cells were electroporated, as previously described
(10), with the luciferase reporter gene (hRAR Differentiation of HL-60 Cells--
Differentiation of HL-60
cells into granulocytes was induced by treatment with 1 µM all-trans-RA or 9-cis-RA or
1.3% Me2SO. Nuclear extracts of control or treated cells
were prepared after 1, 3, or 6 days; resolved by 12% SDS-PAGE; and
tested for D-type cyclin and actin protein expression by
immunoblotting. Cell differentiation was assayed by scoring the
percentage of nitro blue tetrazolium-positive cells in treated cells
versus untreated controls at 6 days after treatment. Cell
growth was determined by counting the number of viable cells at the
different time intervals with the trypan blue exclusion dye test.
Identification of Cyclin D3 as a CRABPII-binding Protein in Yeast
Two-hybrid Screening--
The yeast two-hybrid system was used to
identify proteins that interact with hCRABPII. Gal4DBD fused
amino-terminally to hCRABPII in the pAS2-1 vector was expressed in
yeast strain Y190, which contains two reporter genes: a HIS3
reporter gene and a lacZ reporter gene, both under the
control of Gal4-binding sites. No growth was observed on plates lacking
histidine (either in the absence or presence of 3-AT, a competitive
inhibitor of the HIS3 gene product), indicating that
hCRABPII on its own does not transactivate the HIS3 gene
(data not shown). An HL-60 cDNA library was constructed in the
pACT2 vector that directs synthesis of polypeptides fused to Gal4AAD.
Y190 yeast cells expressing hCRABPII were transformed with this library.
Approximately 1.0 × 106 Y190 yeast transformants were
spread on His Cyclin D3 Interacts Specifically with CRABPII and the DEF Regions
of RAR
We also tested whether cyclin D3 is able to interact with the DEF
regions of RAR Cyclin D3 Interacts with CRABPII in the Presence or Absence of RA
in Vitro--
To study further the data obtained with the yeast
two-hybrid system, binding assays with human cyclin D3 and hCRABPII
were performed in vitro with recombinant proteins.
35S-Labeled cyclin D3 produced by in vitro
translation in rabbit reticulocytes was incubated with the GST-hCRABPII
fusion protein attached to glutathione-Sepharose beads. Bound cyclin D3
was revealed by autoradiography after SDS-PAGE. In agreement with the
two-hybrid data, human cyclin D3 interacted with hCRABPII (Fig.
2). Beads loaded with the control GST
protein did not retain cyclin D3 (Fig. 2, lane 2). This
interaction occurred either in the presence (data not shown) or absence
(Fig. 2, lane 3) of RA. No interactions were identified with
various hCRABPII deletion mutants fused to GST corresponding to the
nuclear receptor-interacting domains and the LBD of hCRABPII (15) (data
not shown), suggesting that the complete structure of hCRABPII is
essential for its interaction with cyclin D3. Interestingly, no
interactions were observed under the same conditions with cyclins D1
and D2 (Fig. 2), suggesting that CRABPII interacts with only one type
of D-type cyclin, cyclin D3.
Cyclin D3 Interacts with the DEF Regions of RAR
To identify which RAR Human Cyclin D3 Co-immunoprecipitates with Both CRABPII and RAR
Anti-cyclin D3 antibody immunoprecipitated both cyclin D3 and RAR
When both RAR Cyclin D3 Enhances the Transactivation of RA Target Genes through
CRABPII--
We previously demonstrated that CRABPII acts as a
coactivator for RA-mediated transactivation of target genes in HL-60
cells through its binding to RARs (10). Having shown herein that
RAR Cyclin D3 Stabilizes the CRABPII-RAR Expression of Cyclin D3 Is Up-regulated by Retinoids in HL-60
Cells--
To unravel the physiological relevance of the interactions
between RAR In this study, we isolated human cyclin D3 as a
CRABPII-interacting protein. A physical RA-independent interaction
between cyclin D3 and CRABPII was evidenced both in vitro
and in vivo. No interactions were observed with various
CRABPII deletion mutants, suggesting that the complete structure of
CRABPII is essential for its interaction with cyclin D3. Furthermore,
no interactions were identified between CRABPII and cyclin D1 or D2
in vitro. These data suggest that the retinoic
acid-independent CRABPII-cyclin D3 interaction is specific for only one
member of D-type cyclins.
Interestingly, we also demonstrated that cyclin D3 interacted with
RAR As we have previously identified a coactivator function of CRABPII for
the nuclear RARs (10), the interaction between cyclin D3, CRABPII, and RAR This is the first study demonstrating that cyclin D3 is involved in
nuclear receptor transcriptional regulation. Another D-type cyclin (D1) has been described as a bridging factor between the estrogen receptor and its coactivators to positively regulate nuclear
receptor-mediated transactivation. Similar functions can be found
between cyclins D1 and D3, as cyclin D1 binds estrogen receptor- Hence, the involvement of a D-type cyclin in the
transcriptional control of ligands directly implicated in cell growth
and differentiation provides a new concept in which cell cycle
regulatory proteins could play a dual function. Indeed, although
D-type cyclins are essential regulatory subunits of the
cyclin-dependent kinases (33-35) and operate in
mid-to-late G1 to allow cell progression in S phase (34,
36-38), the transactivation function of cyclin D1 was shown to be
independent of CDK4 and Rb phosphorylation (16, 26, 27). Although
cyclins D1 and D2 have been extensively studied, few reports are
available for cyclin D3. Structurally related to cyclins D1 and D2,
cyclin D3 appears nevertheless to be more ubiquitously expressed.
Furthermore, although its role in cell proliferation is clearly
evidenced in lymphocytes, it is mainly shown to accumulate in
terminally differentiated tissues (23). This characteristic was
corroborated in vitro during differentiation of rodent
myoblasts in which cyclin D3 expression was induced and reported to
play an important role in irreversible cell cycle arrest of
differentiated myocytes (39-41). In this study, we confirm and extend
previous data on HL-60 myeloid or F9 teratocarcinoma cells. Although
all three cyclins could be detected in the nuclear compartment of
different hematopoietic cells (HL-60, NB4, and U-937), only cyclin D3
expression paralleled the acquisition of differentiation features and
the arrest of cell growth, as previously noted (23, 24). Retinoids act
as regulators of cell growth, differentiation, and apoptosis and have
been shown to specifically arrest myeloid cells in the G1
phase of the cell cycle (42). In the ML-1 myeloid cell line, the cell
cycle arrest associated with all-trans-RA-induced
differentiation involves regulation of the expression of cyclin D3 and
cyclin kinase inhibitors (p18 and p21), affects the
phosphorylation status of cyclin-dependent kinases, and
ultimately triggers dephosphorylation of the Rb protein (43). Thus, in
RA-mediated myeloid differentiation, the increased levels of cyclin D3
contrast with the cells' progressive arrest in G1 and
point to a distinct effect on the establishment and/or maintenance of
the differentiated status of the cell. The former may be explained by
titration of cyclin D3 by either the cyclin D3-RAR or CRABPII-cyclin D3
interaction, whereas the latter may be related to cyclin D3
transcription activity on RA target genes involved in either
differentiation (RAR, CRABPII, CD11b, Hox, and
granulocyte colony-stimulating factor genes (G-CSF)) and/or cell cycle inhibition (p21 and p27 genes) (44).
In summary, we have identified in this study a novel level of molecular
control associating three distinct partners of RA-mediated transcription (cyclin D3, CRABPII, and RAR, but not RXR
, and only in the presence of RA.
We further show that cyclin D3 positively modulated RA-mediated
transcription through CRABPII. Therefore, cyclin D3 may be part of a
ternary complex with CRABPII and RAR. Finally, we show that cyclin D3 expression paralleled HL-60 differentiation and arrest of cell growth.
These findings led us to speculate that control of cell proliferation
during induction of differentiation may directly involve, at the
transcriptional level, nuclear receptors, coactivators, and proteins of
the cell cycle in a cell- and nuclear receptor-specific manner.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
,
RAR
, and RAR
) and the retinoid X receptors (RXR
, RXR
, and
RXR
), which bind as RXR·RAR heterodimers to RA-response elements
located in the promoter region of RA target genes. Like most nuclear
receptors, RARs and RXRs share a highly conserved structure (1), with
ligand-binding (LBD) and DNA-binding (DBD) domains. In addition, they
possess two autonomous transcription activation functions, AF-1 and
AF-2. AF-1, located at the N-terminal end (A/B region), is
ligand-independent; in contrast, AF-2, located in the C-terminal E
region, is ligand-dependent (1).
or RXR
(15), which
releases all-trans-RA from CRABPII to RAR
(13). To
explain the function of this novel ligand-binding coactivator, we
proposed the following scenario (15). First, holo-CRABPII docks to the
apo-receptors bound to their promoters in the nucleus; the docking
occurs around key structures of the ligand entrance pockets of CRABPII
and the nuclear receptor. This establishes a channel that allows the
release of RA from holo-CRABPII to apo-RAR
or apo-RXR
. Because
the CRABPII-nuclear RAR interaction does not require the
presence of RA (10), apo-CRABPII could remain bound to the
holo-receptor, preventing dissociation of RA from the nuclear receptor.
Thus, CRABPII would increase the stability of the DNA-bound RXR·RAR
complex (10), further contributing to the enhancement of RA-mediated transcription.
, but not with RXR
, and only in a
RA-dependent manner. Cyclin D3 enhanced RA-mediated
transactivation of RA target genes by increasing the interaction of
CRABPII with RAR
. Furthermore, we show that cyclin D3 expression
paralleled the induction of differentiation in HL-60 cells. Our results
identify cyclin D3 as a partner of a ternary complex with the
coactivator CRABPII and the nuclear receptor RAR
and demonstrate
another level of transcriptional control during RA-induced
differentiation and arrest of cell growth.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(RP
(F)) or the A region of
RXR
(RPRX
(A)) (a gift from P. Chambon) were as described (10).
Rabbit polyclonal antibodies directed against cyclins D1-D3 (sc-717,
sc-181, and sc-182, respectively) and goat polyclonal antibody directed
against cyclin D3 (sc-182-G) were from Santa Cruz Biotechnology (Santa
Cruz, CA). Rabbit polyclonal antibody directed against actin (A-2066)
was from Sigma (St. Quentin Fallavier, France). All-trans-RA
and 9-cis-RA were supplied by Hoffman-La Roche (Basel, Switzerland).
(pSG5-hRAR
),
RXR
(pSG5-mRXR
), and CRABPII (pTL1-mCRABPII) were provided by P. Chambon. Plasmids encoding cyclins D1-D3 (pRC/CMV-hcyclinD1,
pRC/CMV-hcyclinD2, and pRC/CMV-hcyclinD3, respectively) (16) were
provided by M. E. Ewen. The hRAR
2-luciferase reporter construct
(17) was provided by H. de Thé. Plasmids encoding GST-hRAR
,
GST-hRAR
DEF, GST-hRAR
DEF
(408-416), GST-mRAR
AB, and
GST-mRXR
(18, 19) were provided by P. Chambon. The plasmid encoding
GST-hCRABPII was previously described (15). hCRABPII was amplified by
PCR from NB4 cells and cloned into the yeast pAS2-1 plasmid
(Clontech), which contains the TRP1
marker and directs synthesis of Gal4DBD fusion proteins. The following primers were used: 5'-CCCGAATTCATGCCCAACTTCTCTGGC-3' (forward) and
5'-AGTGGATCCTCACTCTCGGACGTAGACCCT-3' (reverse). hRAR
DEF was cloned
into the same vector after PCR amplification from pSG5-hRAR
using
the following primers: 5'-CCCGAATTCATGTCCAAGGAGTCGGTG-3' and
5'-AGTGGATCCTCACGGGGAGTGGGTGGC-3'. All constructs were generated using
standard cloning procedures and verified by restriction enzyme analysis
and DNA sequencing.
/Leu
/His
plates containing
25 mM 3-amino-1,2,4-triazole (3-AT) and 1 µM all-trans-RA. Plasmids from the isolated positive clones
were recovered into Escherichia coli strain HB101
according to the protocol provided by Clontech and
subjected to restriction analysis. pACT2 plasmids containing cDNA
were identified by colony PCR using primers specific for the
LEU2 gene contained in pACT2. The inserts were then sequenced.
DEF in pAS2-1.
The cells were plated in selective medium lacking tryptophan, leucine, and histidine and supplemented with 25 mM 3-AT and 1 µM all-trans-RA. After 3 days, a qualitative
-galactosidase assay was performed.
, GST-mRAR
AB, GST-hRAR
DEF,
GST-hRAR
DEF
(408-416), GST-mRXR
, and GST-hCRABPII) were expressed in E. coli and bound to glutathione-Sepharose
beads (Amersham Biosciences). The GST proteins bound to the beads were then incubated with rabbit reticulocyte lysates containing translated 35S-labeled cyclin D3 (T7 Quick coupled
transcription/translation system, Promega, Madison, WI) or with
extracts from COS-6 cells overexpressing cyclin D3 as described (10).
Reactions were performed in the presence or absence of 1 µM all-trans-RA. Bound proteins were recovered
in SDS loading buffer, resolved by 12% SDS-PAGE, and analyzed by
immunoblotting or by autoradiography of dried gels.
2-luciferase) in the
presence or absence of the expression vectors for cyclin D or CRABPII.
All transfections also contained the
-galactosidase expression
vector (pCH110) as an internal standard and pRC/CMV as a carrier. Cells
were treated or not with 1 µM all-trans-RA for
24 h, and luciferase assays were performed according to a standard
procedure (Promega). All results are expressed as -fold induction based
on the basal activity of the reporter gene in the absence of RA and of
any expression vector.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
plates containing 25 mM 3-AT in
the absence or presence of 1 µM all-trans-RA.
We analyzed colonies that grew on selective medium in the presence of 1 µM all-trans-RA and that turned blue when tested in a
-galactosidase assay. Plasmids were recovered from the
few positive clones, amplified, subjected to restriction analysis, and
sequenced. Sequence comparison with the GenBankTM/EBI Data
Bank and the Swiss Protein Database identified one 0.651-kb cDNA
insert as the sequence encoding human cyclin D3 (accession number
NM_001760). This cDNA fragment depicted the known open reading frame for cyclin D3, but lacked the nucleotides encoding the
first 26 N-terminal amino acids and the last 33 C-terminal amino acids
(Fig. 1A).
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Fig. 1.
Cyclin D3 interacts with CRABPII and
RAR in the yeast two-hybrid system.
A, the yeast two-hybrid screen using CRABPII as bait
allowed us to isolate a clone encoded by a 0.651-kb cDNA fragment
(yellow) with 100% identity to human cyclin D3. This
cDNA fragment depicts the known open reading frame for cyclin D3.
Protein sequence alignment revealed that the identified clone lacks the
first 26 N-terminal residues as well as the last 33 C-terminal residues
(yellow). B, cyclin D3 interacts with CRABPII and
the DEF regions of RAR
in yeast. Individual colonies were streaked
onto medium containing histidine (panel a) or lacking
histidine (panels b and c) to characterize the
plasmid combinations resulting in activation of the
HIS3 gene. In panel c,
-galactosidase
activity was tested to measure the activation of the lacZ
reporter gene. Area 1, pAS2-1RAR
DEF plus pACT2-cyclinD3;
area 2, pAS2-1 plus pACT2-cyclinD3; area 3,
pAS2-1CRABPII plus pACT2-cyclinD3. Specific interactions were observed
only with the following hybrid pairs: RAR
DEF plus cyclin D3
(area 2) and CRABPII plus cyclin D3 (area
3).
in Yeast--
To confirm the interaction of cyclin D3 with
CRABPII, the Gal4AAD-cyclin D3 hybrid protein was expressed in the Y190
yeast strain in combination with pAS2-1CRABPII or the empty vector. The
transformants were spread on His
plates containing 25 mM 3-AT and 1 µM all-trans-RA.
Both proteins interacted as evidenced by growth of colonies on
histidine-deficient medium and activation of
-galactosidase activity
(Fig. 1B, area 3). No clones were obtained with
the unrelated bait vector pAS2-1 (Fig. 1B, area
2). These findings demonstrate that cyclin D3 interacts with
CRABPII.
. The Gal4DBD-RAR
DEF hybrid protein was expressed
in the Y190 yeast strain either alone or in combination with the
Gal4AAD-cyclin D3 hybrid protein. Both proteins interacted as evidenced
by growth of colonies on histidine-deficient medium and activation of
-galactosidase activity (Fig. 1B). No clones were
obtained when Y190 yeast cells were transformed with the empty vector
(pACT2) and either pAS2-1CRABPII or pAS2-1RAR
DEF (data not shown).
Together, these results indicate that cyclin D3 can interact with both
CRABPII and the DEF regions of RAR
in yeast.
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Fig. 2.
CRABPII interacts with cyclin D3 in
vitro. 35S-Labeled cyclins D1-D3 produced
by in vitro transcription/translation in a rabbit
reticulocytes lysate were incubated with GST (lane 2) and
GST-CRABPII (lane 3) immobilized on glutathione-Sepharose
beads. Equal loading of the GST fusion proteins was confirmed by
Coomassie staining. Lane 1 corresponds to the input (5%).
Bound cyclins were resolved by 12% SDS-PAGE and detected by
autoradiography.
in the Presence
of RA in Vitro--
Binding assays with cyclin D3 and the DEF regions
of RAR
were also performed in vitro. Extracts from COS-6
cells overexpressing the different cyclins (D1-D3) were incubated with
the GST-RAR
fusion protein attached to glutathione-Sepharose beads
in the presence or absence of all-trans-RA (Fig.
3). Bound cyclins were revealed by
immunoblotting. No interaction was detected in the absence of RA (Fig.
3A, lane 2). However, a RA-dependent
interaction was observed between RAR
and cyclin D3 (Fig.
3A, lane 3). No interaction was detected with
cyclins D1 and D2 (data not shown).
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Fig. 3.
Cyclin D3 interacts with the DEF regions of
RAR in vitro. A,
cyclin D3 interacts with RAR
in vitro in a
RA-dependent manner. Extracts from COS-6 cells transfected
with the human cyclin D3 expression vector were incubated with GST
(lane 1), GST-RAR
(lanes 2 and 3),
or GST-RXR
(lanes 4 and 5) immobilized on
glutathione-Sepharose beads in either the absence (lanes 1,
2, and 4) or presence of all-trans-RA
(lane 3) or 9-cis-RA (lane 5). Bound
human cyclin D3 was detected by immunoblotting. The cyclin D3 input
corresponds to a doublet because anti-cyclin D3 antibody recognizes
both exogenous human cyclin D3 (upper band) and endogenous
simian cyclin D3 (lower band). B, schematic
representation of the different RAR
deletion mutants fused to the
GST protein (not drawn to scale). The gray boxes correspond
to the AF-2 domain core (amino acids 408-416) of RAR
.
C, the cyclin D3-RAR
interaction involves the DEF regions
of RAR
, but not the AF-2 activation domain core. Extracts
from COS-6 cells transfected with the cyclin D3 expression vector were
incubated with GST (lane 2) or the various GST-RAR
fusion
proteins immobilized on glutathione-Sepharose beads: GST-RAR
(lanes 3 and 4), GST-RAR
AB (lanes 5 and 6), GST-RAR
DEF (lanes 7 and 8),
or GST-RAR
DEF(
408-416) (lanes 9 and 10) in
the absence (lanes 3, 5, 7, and
9) or presence (lanes 4, 6,
8, and 10) of all-trans-RA. Bound
cyclin D3 was detected by immunoblotting.
domain is involved in the interaction with
cyclin D3, several GST fusion proteins carrying deletions of RAR
were tested in the presence or absence of RA. Compared with full-length
RAR
, RAR
with its DBD and LBD deleted (RAR
AB) (Fig.
3B) was impaired in its ability to interact with cyclin D3
(Fig. 3C, lanes 5 and 6). In contrast,
the LBD of RAR
(RAR
DEF) retained its capacity to bind cyclin D3
in the presence of RA (Fig. 3C, lanes 7 and
8). Similarly, a RAR
DEF mutant bearing an internal
deletion of the core motif (RAR
DEF
(408-416)) (Fig. 3B) involved in the recruitment of coactivators retained the
RA-dependent interaction with cyclin D3 (Fig.
3C, lanes 9 and 10). Altogether, these
results suggest that the interaction with cyclin D3 involves a motif
located in the LBD, distinct from the AF-2 domain core, but
whose accessibility depends on RA binding. It must be noted that, under
the same conditions, cyclin D3 did not interact with GST-RXR
even in
the presence of 9-cis-RA (Fig. 3A, lanes
4 and 5), indicating that cyclin D3 interacts in
vitro specifically with RAR
.
in Vivo--
The interaction of cyclin D3 with CRABPII and RAR
was
further investigated in co-immunoprecipitation experiments using COS-6 cells overexpressing cyclin D3 either alone or in combination with
RAR
or CRABPII and treated or not with RA. Extracts were immunoprecipitated with anti-cyclin D3 antibody and analyzed by SDS-PAGE/immunoblotting (Fig. 4).
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Fig. 4.
Cyclin D3 co-immunoprecipitates with both
CRABPII and RAR in vivo.
A, COS-6 cells were cotransfected with the RAR
and cyclin
D3 expression vectors and treated or not with RA. Whole cell extracts
were incubated with goat anti-cyclin D3 antibody and then with protein
G-Sepharose beads (lanes 3 and 5). Control
immunoprecipitations (IP) were performed with anti-cyclin D2
antibody (lanes 2 and 4). The immunocomplexes
were resolved by SDS-12% PAGE, transferred to nitrocellulose
membranes, and immunoblotted with anti-cyclin D3 or anti-RAR
antibody. B, COS-6 cells were cotransfected with the
CRABPII, RAR
, and cyclin D3 expression vectors and treated or not
with all-trans-RA as indicated. Whole cell extracts were
immunoprecipitated as described for A and immunoblotted with
anti-cyclin D3, anti-RAR
, or anti-CRABPII antibody.
from extracts of COS-6 cells cotransfected with the corresponding expression vectors and treated with RA (Fig. 4A). These
proteins were not revealed in control immunoprecipitations. Similarly, anti-cyclin D3 antibody co-immunoprecipitated CRABPII when both proteins were coexpressed in COS-6 cells (data not shown). Such a
co-immunoprecipitation occurred whether the cells were treated or not
with RA, corroborating the results obtained in two-hybrid and GST
pull-down assays.
and CRABPII expression vectors were cotransfected with
cyclin D3 in COS-6 cells, anti-cyclin D3 antibody co-immunoprecipitated the three proteins (Fig. 4B). It must be noted that this
co-immunoprecipitation was observed whether or not the cells were
treated with RA. Such a ligand-independent co-immunoprecipitation of
RAR
with cyclin D3 and CRABPII may reflect the RA-independent
interaction of the receptor with CRABPII (10, 15). Thus, one can
hypothesize that, in vivo in the absence of RA, CRABPII
forms a bridge between RAR
and cyclin D3.
forms a complex not only with CRABPII, but also with cyclin D3, we studied the effects of cyclin D3 on RAR
transactivation in either
the absence or presence of CRABPII. HL-60 cells were transiently transfected with the hRAR
2-luciferase reporter construct in the absence or presence of the CRABPII and/or cyclin D3 expression vectors
and treated with RA (Fig. 5).
Surprisingly, the RA-induced luciferase activity was not affected upon
overexpression of cyclin D3 (Fig. 5A). Similar results were
obtained with different amounts of cyclin D3 expression vector (Fig.
5B). However, cyclin D3 significantly enhanced the increase
in luciferase activity induced by CRABPII (Fig. 5). This effect was
proportional to the amount of cyclin D3 expression vector (Fig.
5B). Cyclin D2 had no such effect (Fig. 5A).
Altogether, these data indicate that cyclin D3 modulates the
coactivator effect of CRABPII on the transactivation of RA target
genes.
View larger version (15K):
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Fig. 5.
Cyclin D3 enhances RA-mediated
transactivation through CRABPII. A, HL-60 cells were
electroporated with the RAR 2-luciferase reporter gene in association
or not with the cyclin D3, cyclin D2, or CRABPII expression vector as
indicated and treated or not with all-trans-RA (0.1 or 1 µM) for 24 h. All transfections also contained the
-galactosidase expression vector (pCH110) as an internal standard
and pRC/CMV as a carrier. Extracts were tested for luciferase activity.
Results are expressed as -fold induction relative to the luciferase
activity displayed in the absence of RA and in the absence of cyclins
and CRABPII. All experiments were normalized to
-galactosidase. The
data are representative of the average of three similar experiments
done in triplicates. Means ± S.D. are shown. B, HL-60
cells were transfected with the luciferase reporter gene in association
or not with the expression vectors for CRABPII (5 µg) and cyclin D3
(5 or 10 ng). Cells were treated with 1 µM
all-trans-RA, and extracts were analyzed for luciferase
activity. Results are expressed as described for A.
Interaction--
To
investigate further how cyclin D3 enhances the transactivation of RA
target genes through CRABPII, in vitro GST pull-down assays
were performed. 35S-Labeled RAR
produced by in
vitro translation in rabbit reticulocytes was incubated with the
GST-hCRABPII fusion protein immobilized on glutathione-Sepharose beads
in the presence or absence of COS-6 extracts overexpressing cyclin D3
(Fig. 6). As expected, in the absence of
cyclin D3, RAR
interacted with CRABPII, independently of RA (Fig. 6,
lanes 2 and 3) (10). However, higher amounts of RAR
were retained in the presence of cyclin D3 in a
RA-independent manner (Fig. 6, lanes 4 and 5).
Therefore, cyclin D3 appears to increase the efficiency of the
formation of the CRABPII·RAR complexes in vitro. These
data confirm the results from the in vivo assay showing the
formation of the ternary complex involving RAR, CRABPII, and cyclin D3
(Fig. 4B). Such a process might be required for the increase
in the transactivation of RA target genes observed with cyclin D3 in
the presence of CRABPII.
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Fig. 6.
Cyclin D3 stabilizes the
CRABPII-RAR interaction in
vitro. 35S-Labeled RAR
produced by
in vitro translation in reticulocytes lysates was incubated
with GST (lane 1) and GST-CRABPII immobilized on
glutathione-Sepharose beads in the absence (lanes 2 and
3) or presence (lanes 4 and 5) of
cyclin D3 overexpressed in COS-6 cells. Incubations were performed in
the absence (lanes 2 and 4) or presence
(lanes 3 and 5) of all-trans-RA. Bound
RAR
was detected by autoradiography.
, CRABPII, and cyclin D3, the nuclear expression of cyclin D3 was examined in several RA-sensitive hematopoietic cell lines: myeloblastic HL-60, promyelocytic NB4, and monoblastic U-937
(Fig. 7A). As shown by Western
blot analysis, cyclin D3 was expressed in the nuclear compartment of
all three cell lines, with the highest amounts being detected in U-937
cells (Fig. 7A). Cyclin D2 and, to a lesser extent, cyclin
D1 were also expressed in the three cell lines (Fig. 7A).
Because the induction of expression of cyclin D3 had previously been
observed during differentiation of HL-60 cells by
12-O-tetradecanoylphorbol-13-acetate or Me2SO (23) and in F9 teratocarcinoma cells treated with different retinoids
(24), we studied its expression in HL-60 cells after treatment with
all-trans-RA (1 µM), 9-cis-RA (1 µM), or Me2SO (1.3%). Differentiation and
cell growth were analyzed after 6 days of culture. Nuclear extracts of
control and treated cells were prepared after 1, 3, or 6 days and
immunoblotted with anti-cyclin D3 antibody (Fig. 7B). Both
retinoids and Me2SO differentiated HL-60 cells as evidenced
by the acquisition of redox function (nitro blue tetrazolium test). A
weaker efficacy for all-trans-RA compared with
9-cis-RA was noted, as previously reported (25). Arrest of
proliferation was achieved after 6 days of treatment (data not shown).
Me2SO increased the expression of cyclin D3 from days 1 to
6 (Fig. 7B, lanes 4, 8, and
12). Cyclin D3 expression was modulated by both retinoids,
with higher levels achieved with 9-cis-RA compared with
all-trans-RA from day 3 (Fig. 7B,
lanes 7 and 11 and lanes 6 and
10, respectively). Likewise, the cyclin D3 up-regulation
correlated with all-trans-RA or 9-cis-RA
differentiation efficiency (Fig. 7B, lanes 10 and
11, respectively) and cell proliferation inhibition (data
not shown).
View larger version (33K):
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Fig. 7.
Cyclin D3 expression in RA-treated
hematopoietic cells. A, cyclins D1-D3 were detected by
immunoblotting in nuclear extracts from exponentially growing
hematopoietic cells: HL-60 (lane 1), NB4 (lane
2), and U-937 (lane 3). B, HL-60 cells were
treated with all-trans-RA (1 µM; lanes
2, 6, and 10), 9-cis-RA
(1 µM; lanes 3, 7, and
11), or Me2SO (DMSO; 1.3%;
lanes 4, 8, and 12). Under these
conditions, these cells differentiated along the granulocytic lineage.
At different times, day 1 (lanes 1-4), day 3 (lanes
5-8), and day 6 (lanes 9-12), nuclear extracts were
immunoblotted with anti-cyclin D3, anti-D1, or anti-D2 antibody.
Lanes 1, 5, and 9 correspond to
control mock-treated cells. HL-60 differentiation was assayed by
scoring the percentage of nitro blue tetrazolium
(NBT)-positive cells in treated cells versus
untreated controls at 6 days after treatment.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, but not with RXR
. Once again, cyclin D3 was the only
D-type cyclin that could interact with RAR
. This
interaction involved the DEF region of RAR
and was observed in
vitro as well as in yeast cells. Although the CRABPII-cyclin D3
interaction was RA-independent, the cyclin D3-RAR
interaction
required the presence of RA both in vitro and in
vivo. Although cyclin D3 interacted physically with RAR
in a
RA-dependent manner, this interaction did not require the
AF-2 "core" harboring the LLXXXL motif of RAR
generally implicated in the ligand-dependent interaction between the nuclear receptors and the LXXLL motifs of their
coactivators. RA may, however, be required to induce conformational
changes around the AF-2 domain to facilitate the cyclin D3-RAR
interaction. Surprisingly, the presence of CRABPII allowed cyclin D3
and RAR
to interact in the absence of RA. Because CRABPII interacted
with both RAR
and cyclin D3 in a RA-independent manner, we
hypothesize that CRABPII facilitates the recruitment of cyclin D3 on
RAR
both in the absence and presence of RA.
led us to investigate the role of cyclin D3 in
RA-mediated transactivation. On its own, cyclin D3 had no effect on
RA-mediated transactivation. By contrast, in the presence of CRABPII,
cyclin D3 positively modulated transcription activation. This may be
due to an increase in the recruitment of CRABPII on RAR
or an
increase in the stability of the CRABPII-RAR
interaction. Indeed,
cyclin D3 appeared to increase the efficiency of the formation of the
CRABPII·RAR complexes in vitro. Thus, cyclin D3 belongs to
a ternary complex with RAR
and CRABPII and enhances the CRABPII coactivator transactivation activity.
in
the absence of ligand and enhances estrogen-dependent transactivation (16, 26, 27) and binds the estrogen receptor and one of
its coactivators, SRC-1 (28). Cyclin D1 may also promote or stabilize
the association between the estrogen receptor and p/CAF, thereby
increasing the histone acetyltransferase activity of the
transcriptional machinery (29). Conversely, cyclin D1 has also been
reported to bind the androgen receptor through the N-terminal domain
and to inhibit transactivation by directly competing for p/CAF binding
(30, 31). Apart from being specific for RAR
, cyclin D3 has other
distinct features. In contrast to the interaction between cyclin D1 and
SRC-1, which involves the LLXXXL motif of cyclin D1 and the
LXXLL motif of SRC-1 (32), the interaction between cyclin D3
and CRABPII is different, as cyclin D3 does not harbor any
LLXXXL motif and thus will not bind potential or candidate
coactivators through their LXXLL motifs. Interestingly, CRABPII does not have a LXXLL motif, strongly suggesting
that, for its interaction with cyclin D3, other recognition motifs are involved.
) that connects three key
cellular processes: the cell cycle, ligand bioavailability, and gene
expression. From our results and other published data (10, 15, 45), we
may establish a physiological model (Fig. 8). After binding to RA, CRABPII
translocates from the cytoplasm to the nucleus (45). Holo-CRABPII then
docks to the apo-nuclear receptors bound to their promoters. Cyclin D3
forms a ternary complex and allows the stability of the CRABPII-RAR-RA
interaction to increase and thus to enhance the transactivation
activity of the complex.
View larger version (62K):
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Fig. 8.
Model for the interconnection between cell
cycle (cyclin D3), vitamin A metabolism and RA transport (CRABPII), and
RAR-mediated transactivation of RA target genes. Cyclin D3
enhances transactivation of RA target genes through its interaction
with CRABPII associated with RAR . RARE, RA-response
element.
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ACKNOWLEDGEMENTS |
---|
We gratefully acknowledge H. de Thé and M. E. Ewen for providing plasmids, P. Chambon for providing plasmids and antibodies, and J.-M. Garnier for the HL-60 cDNA library. We also thank R. Losson for helpful technical assistance and advice on the yeast two-hybrid screen system. We thank B. Papp for reading and criticism of the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported in part by INSERM.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.
§ Supported by the Ministry of Research of France and the Fondation pour la Recherche Médicale.
To whom correspondence should be addressed. Tel.:
33-1-4249-4234; Fax: 33-1-4200-0160; E-mail:
lbch@chu-stlouis.fr.
Published, JBC Papers in Press, December 12, 2002, DOI 10.1074/jbc.M210697200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: RA, retinoic acid; RAR, retinoic acid receptor; RXR, retinoid X receptor; h, human; m, mouse; LBD, ligand-binding domain; DBD, DNA-binding domain; CRABPII, cellular retinoic acid-binding protein II; GST, glutathione S-transferase; AAD, acidic activation domain; 3-AT, 3-amino-1,2,4-triazole.
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