From the
To determine which factors may regulate the DNA binding and
transcriptional properties of retinoic acid receptors (RARs and RXRs),
we investigated the sensitivity of reporter genes bearing various
retinoic acid response elements (RAREs) to protein phosphatases
(PPases) inhibition. PPases inhibition by okadaic acid led to an
increase of the reporter genes activity in a RARE-dependent and
ligand-independent manner and was dependent on the type of response
element used. Overexpression of protein phosphatases 2A and 1 (PP2A and
PP1) decreased the inducibility of the reporter genes tested. Nuclear
extracts from okadaic acid-treated COS cells displayed an
2-5-fold increased level of receptor binding to RAREs in
vitro, suggesting that PPases inhibition increased the DNA binding
activity of retinoid receptors. Treatment of receptors extracted from
COS cells by alkaline phosphatase and partially purified PP1 and PP2A
decreased their DNA binding activity, but heterodimers bound to DNA
were not sensitive to phosphatase treatment. Reconstitution experiments
showed that phosphorylation of both receptors increased the DNA binding
activity of RXR/RAR heterodimers. Taken together, these data show that
the modulation of the phosphorylation state of RARs and RXRs represents
an other level of regulation of the retinoid signaling pathway.
All- trans-retinoic acid (ATRA)
An
additional level of control of the transcriptional activity of these
receptors may also be provided by extracellular signals, as reported
for the progesterone receptor
(9) and the estrogen receptor
(10) . Most of the nuclear receptors have been shown to be
phosphoproteins
(11) , including RAR
The phosphorylation state of a given protein is the result of both
phosphorylation and dephosphorylation reactions. The tumor promoter
okadaic acid (OA) is a complex fatty acid polyketal that specifically
inhibits protein phosphatases PP1 and PP2A, both in vitro and
in cultured cells
(17) . PP2A is responsible for a significant
protein phosphatase activity in various tissues and has a broad
spectrum of substrates in vitro. Significant amounts of PP1
and PP2A are located in the nucleus
(18, 19) . PP1 has a
5-fold higher specific activity in the nuclear compartment than in the
cytosolic fraction
(19) , and is associated with chromatin (see
(18) and references therein). Inhibition of PP1 and PP2A by OA
has also been shown to cause hormone-independent activation of
progesterone
(9, 20) and glucocorticoid-regulated
reporter genes
(21) . However, the strong effect on
transcription could not be correlated with detectable alterations of
the phosphorylation state of PR
(22) and GR
(21) , and
was, in the latter case, attributed to a post-translational
modification of a putative GR-associated factor.
We therefore used
OA to oppose the activity of protein kinases constitutively active in
COS cells, which were transfected with different RA-responsive reporter
genes and expression vectors coding for RAR
Sf9 cells were infected with a recombinant
baculovirus encoding for the hRAR
A similar
analysis was performed with the TREpal-CAT construct, which is not
inducible in the absence of overexpressed RAR or RXRs
(Fig. 1 B). Overexpression of RAR, RXR, or both receptors
conferred a significant inducibility by retinoic acid on this promoter,
indicating that the observed activation is due solely to transfected
receptors. This result is in agreement with previously reported results
and reflects the low concentrations of RAR and RXR in nontransfected
COS cells
(37) . 9- cis-RA was a better activator than
ATRA, showing that RXR activation increases the promoter activity. In
the absence of transfected receptors, OA displayed virtually no effect
on the TREpal-CAT activity. On the contrary, OA caused a clear increase
in TREpal-CAT expression in response to ATRA (2-fold) and
9- cis-RA (1.3-fold) in the presence of RAR
The
rCRBPII-RXRE-CAT construct, containing five repeats of the sequence
AGGTCA spaced by one nucleotide, is poorly activated in the absence of
transfected receptors or in the presence of RAR alone. OA was
nevertheless able to increase the CRBPII promoter activity in the
absence of receptors or in the presence of RAR, and it increased the
basal activity to a level similar to the one observed with ATRA or
9- cis-RA. This effect was noticeable, although the CAT
activity was much lower than that seen in the presence of RXR. Indeed,
this reporter gene became highly inducible in the presence of
transfected RXR, to reach a 25-30-fold higher activity in the
presence of both ATRA and 9- cis-RA (Fig. 1 C).
Initially described as a RXRE, the rCRBPII-RARE is, in our experimental
conditions, activated by a RAR-specific ligand, indicating that RAR is
a component of the activation complex. The activation of a
CRBPII-driven promoter by ATRA was also reported, although this could
be due, in the reported conditions, to a metabolic conversion of ATRA
to 9- cis-RA
(32) . Thus our results suggest that this
particular response element can be activated by hRAR
DR2 response elements have been shown, by random selection
of binding sites for RAR/RXR heterodimers, to bind heterodimers with a
lower affinity than a DR5
(40) . However, the mCRABPII DR2
conferred a significant inducibility to the thymidine kinase promoter
in response to ATRA and 9- cis-RA in the absence of transfected
receptors (Fig. 1 D). 9- cis-RA was in all cases
a better inducer than ATRA in the absence of OA, and RAR overexpression
yielded a higher level of CAT activity than RXR overexpression.
Coexpression of RAR and RXR did not significantly increase the
CRABPII-CAT promoter activity. In that promoter context, OA increased
the basal level of CAT activity, which reached values similar to that
obtained in the presence of ligand alone when RXR was overexpressed. At
the specified concentrations, ligand and OA effects on the promoter
activity were cumulative. This result is analogous to that of
TREpal-CAT (see Fig. 1 B).
This set of experiments
demonstrates several interesting features of retinoid-induced
transcription in response to OA treatment in COS cells: (i) the
observed effects are specific for RARE-containing promoters since the
OA effect was not detected when the parental reporter gene
Molecular mechanisms controlling cellular fate determination
and proliferation are subject to various levels of regulation. These
processes are either triggered by molecules binding to membrane
receptors or by liposolubles hormones (vitamin A and derivatives,
vitamin D, and steroid hormones) that bind to intracellular receptors.
Both types of signals affect, directly or indirectly, the expression
rate of key regulatory genes. An understanding of interactions
occurring between these two signaling pathways is therefore required to
decipher cellular responses at the nuclear level in response to
mitogenic or differentiating signals.
We present here evidence that
alteration of the intracellular equilibrium between phosphorylation and
dephosphorylation processes leads to the modulation of the activity of
different RA-responsive reporter genes in COS cells. The effects of OA
(Fig. 1) and that of PP1 and PP2A (Fig. 2) were variable
depending on the response element used. In contrast to the high
affinity
Reports from various laboratories
identified RXRs in mammalian cells
(34, 38, 45, 46) as the primary dimerization partners of
all- trans-retinoic acid receptors. Our in vitro DNA-binding experiments showed that (i) OA treatment increased the
DNA binding activity of RAR/RXR dimers and (ii) that RAR and RXR
dephosphorylation is detrimental to RAR/RXR heterodimers binding to
RAREs, lowering the heterodimer
OA is a powerful pharmacological
tool which has been used to demonstrate the involvement of
phospho/dephosphorylation processes in the regulation of the
transactivating potential of transcription factors. Initial reports
from B. O'Malley's
(9, 20) laboratory
established the importance of phosphorylation processes in PR-mediated
transcription using this compound and other modulators of kinases. OA
was able to induce ligand-free activation of the PR, and to potentiate
the ligand-inducible transcription by GR
(21, 48) .
Because of the lack of correlation between OA treatment and the
phosphorylation state of GR, it has been proposed that phosphorylation
of coactivators involved in GR-mediated transcription could be
responsible for this potentiation. Alternatively, processes such as the
nuclear/cytoplasmic shuttling of receptors could be modified as well
(49) . The difficulty to establish a clear role for
phosphorylation of steroid receptors is undoubtedly linked to the
multiplicity of the experimental systems used, as well as technical
limitations. For example, PP1 and PP2A have recently been reported to
be histidine phosphatases
(50) . Phosphohistidine residues are
present in proteins in quantities comparable to that of phosphoserine
and phosphothreonine. However, phosphohistidine residues are acid
labile and thus not detected by the standard procedures of phosphoamino
acid analysis. Indeed, multiple phosphorylation sites have been
identified and mutated in the GR, without strongly altering its
transactivating potential
(51) . On the contrary, critical
serine or threonine residues have been identified in v-erb-A
(52) and the estrogen receptor
(53) . Lin and colleagues
(54) have also demonstrated a correlation between OA-induced
hyperphosphorylation of hT
Our
observations thus show that phosphorylation of RARs and RXRs provides
another level of regulation of the function of these receptors, in
addition to that already provided by multiple receptors isoforms,
multiple dimerization partners, distinct response elements, diverse
promoter contexts, and ligand variety.
We are indebted to Dr. J. Grippo (Hoffman-Laroche) for
9- cis-RA, to Dr. B. Sablonniere who supplied us with
anti-RAR
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)
receptors
,
, and
(RAR
, RAR
, and
RAR
) and 9- cis-retinoic acid receptors (RXR
,
,
and
) are ligand-inducible transcription factors that belong to
the nuclear receptor superfamily
(1, 2) . The
heterodimerization properties of RARs and RXRs, as well as their
relative affinity for retinoids, can account for the multiple effects
of retinoids observed in vivo (reviewed in
(3) ).
Binding of RAR-containing heterodimers to cognate DNA binding sequences
(retinoic acid-responsive elements or RAREs) is required to observe
transcriptional activation of ATRA-controlled promoters. The
recognition code is less stringent than for steroid hormone receptors,
since natural RAREs have a half-site spacing which ranges from 2 to 5
bases
(4) , and RAR/RXR heterodimers can bind to half-sites
arranged into palindromes, inverted palindromes
(5) , and direct
or inverted repeats. In addition, their cis-acting properties vary
according to the promoter context
(4, 6) and
transcriptional activation may require additional
``bridging'' factors like E1A
(7, 8) .
, RAR
, and
RAR
(12, 13, 14) . Treatment of COS cells
with retinoic acid did not induce detectable changes in the
phosphorylation state of overexpressed mRAR
, mRAR
, and
mRAR
2. On the contrary, mRAR
1 and mRAR
3 are strongly
phosphorylated upon agonist treatment
(12) , like steroid
receptors
(11) . In addition, we have recently shown that the
protein kinase C pathway is involved in the regulation of
retinoid-induced transcription
(15) , and Huggenvik et al.(16) reported that the cAMP-dependent protein kinase
pathway alters the transcriptional response to ATRA. Although these
observations suggest that phosphorylation processes could be regulating
retinoid receptors functions, it is not yet clear by which mechanism(s)
kinases and phosphatases modulate their trans-activating function.
and RXR
. The role
of basal phosphorylation was further tested by an in vitro approach in which RAR
or RXR
were selectively treated
with phosphatases and tested for their DNA binding and
heterodimerization activities.
Cell Culture
COS and HeLa cells were grown in
Dulbecco's modified Eagle medium (ICN-Flow Laboratories,
Scotland) supplemented with penicillin/streptomycin and 10% fetal calf
serum. Sf9 cells were propagated in suspension in Grace's insect
medium (Life Technologies, Inc.) supplemented with 10% fetal calf
serum, 1 mM glutamine, 50 mg/ml gentamicin, and 2.5 mg/ml
amphotericin B.
Nuclear Extract Preparation
Nuclear extracts from
Sf9, COS, and HeLa cells were performed as described previously
(23) with minor modifications. Nonidet P-40 was omitted from the
cell lysis buffer and phosphatases inhibitors were added to the
extraction buffer (10 mM sodium molybdate, 10 mM
sodium pyrophosphate, and 10 mM sodium vanadate). Protein
concentration was estimated by the Bradford assay
(24) and
usually found to be in the 3-5 mg/ml range.
Overexpression of hRAR
hRAR and hRXR
in Bacteria and
Sf9 Cells
and
A/B hRXR
were expressed in the
bacterial strain BL21(DE3) pLysS transformed with plasmid
pET3DRAR
and pET31-RXR
. Bacteria were grown in
chloramphenicol and ampicillin-containing LB medium and induced at
mid-log phase (OD
= 0.6) by addition of
isopropylthio-
-D-galactoside to 0.5 mM final.
After 4 h of induction, cells were pelleted and resuspended in 50
mM Tris-HCl, pH 8.0, 1 mM EDTA, 10% glycerol, 5
mM dithiothreitrol, 0.4 M NaCl, 10 µg/ml
aprotinin and pepstatin A, and 1 mM phenylmethylsulfonyl
fluoride
(25) . Phosphatases inhibitors were also included: 10
mM sodium molybdate, 10 mM sodium pyrophosphate, and
10 mM sodium vanadate. Lysozyme was added to 150 µg/ml,
and cells were finally lysed by addition of deoxycholate to a final
concentration of 0.05%. Cells debris were pelleted at 60,000
g, 4 °C for 2 h. Supernatant usually contained 2-5
mg/ml protein.
. Nuclear extracts from infected
cells were prepared as described above. Purified
A/B RXR
and
A/B RAR
were a kind gift from H. Gronemeyer.
Phosphatase Treatment
Extracts were treated with
either agarose-immobilized calf intestine alkaline phosphatase (CIP) or
soluble CIP (Sigma). When agarose immobilized-CIP was used (Figs. 5 and
6), 75 µg of COS cell extracts were incubated for 15 min at 37
°C with 22 units of enzyme in 150 µl of EMSA buffer. Beads were
spun down and supernatants brought to 20 mM di-sodium hydrogen
phosphate (NaHPO
) and 5 mM
NaVO
. When soluble CIP was used (Figs. 4 and 7), 25 µl
of extracts (
75 µl) were diluted 4-fold in EMSA buffer and
incubated 20 min at 30 °C with 50 units of the enzyme. The reaction
was stopped by transferring samples at 4 °C and bringing the mix to
20 mM Na
MoO
, 20 mM
NaVO
, and 20 mM Na
PO
.
These conditions were found to reduce CIP activity by more than 95% as
assayed by p-nitrophenylphosphate hydrolysis.
EMSA
Oligonucleotides containing the various
response elements (see below) were end-filled with the Klenow fragment
of DNA polymerase. Typically, 40 µg of protein was incubated with
20 fmol of the labeled probe, in the presence of 2.5 µg of salmon
sperm DNA and a binding buffer giving a final concentration of 20
mM HEPES, pH 7.4, 1 mM EDTA, 150 mM NaCl, 1
mM dithiothreitrol, and 10% glycerol. When purified
Escherichia coli RAR or RXR were used, 0.05-0.1 µg
of purified receptor was added to 4 µg of CIP-treated or control
extracts and incubated 15 min on ice prior to the binding reaction.
Control experiments were performed with heat-inactivated phosphatase (5
min at 100 °C) according to the same protocol. This treatment
allowed for a complete inactivation of CIP as judged by its lack of
activity on 5`-labeled probes (data not shown). DNA binding reactions
were for 30 min on ice or 15 min at 20 °C, in a final volume of 20
or 40 µl. When required, 1 µl of mouse monoclonal ascites fluid
Ab9()F directed against RAR
(13) or 4RX-1D12 directed
against each type of RXR, was added for a further 15-min incubation as
described previously
(13) . Protein
DNA complexes were then
resolved on a 5% nondenaturing polyacrylamide gel run at 150 V for 3 h
at 4 °C. The running buffer was 0.5
TAE (1
TAE is
40 mM Tris acetate, pH 7.5, 2 mM EDTA) (Figs. 3, 4,
7, and 8). Alternatively, gels were run in 0.5
TBE at room
temperature (45 mM Tris base, 45 mM boric acid, ans 2
mM EDTA) (Figs. 5 and 6).
Transient Transfections,
COS cells were transfected by the calcium phosphate
precipitation method as follows: 10-Galactosidase, and CAT
Assays
cells were plated per
35-mm dish. The following day, cells were fed with 1 ml of fresh
medium, and calcium-DNA coprecipitate was added 4 h later. The mixture
contained 1 µg pSV-
gal plasmid (pCH110, Pharmacia), 1 µg
of the reporter gene, 1 µg of pSG5-hRAR
(26) and
pSG5-mRXR
expression vector
(6) . The DNA concentration was
adjusted to 10 µg final with carrier DNA. Incubation with the
coprecipitate was for 16 h, after which cells were glycerol-shocked.
Cells were incubated for 10 h in complete medium and then submitted to
various treatments as indicated in the text.
-Galactosidase and
CAT assays were performed as previously reported
(15, 27) .
Western Blotting Procedure and
Antibodies
Immunoblotting of RAR was performed using the
IBI Enzygraphic Web system
(15) or
I-protein A
(13) . The anti-hRAR
polyclonal antibody IS39 was raised
against synthetic peptides from the F domain of hRAR
. AntiRAR
monoclonal antibody Ab9
(F) and antiRXR monoclonal antibodies
4RX-1D12 and 1RX-6G12 are described elsewhere
(13, 28) .
Protein Phosphatase Purification
PP1 and PP2A were
partially purified from rat livers. Purification steps were performed
as described by Silberman et al.(29) . Fractions
eluting from a DEAE-Sepharose column (6 10 cm) between 0.2 and
0.35 M NaCl were pooled, concentrated on a Centricon 10
filter, and dialyzed against buffer PPB (50 mM Tris-HCl, pH
7.4, 1 mM EDTA, 20% glycerol, 5 mM DTT, and 100
mM NaCl). The catalytic activity of the preparation was
estimated by its alkaline phosphatase activity with
p-nitrophenyl phosphate as a substrate. The specific activity
of the preparation used in the presented experiments was 85 units/mg
protein, and protein concentration was around 12 mg/ml. Inhibition by
okadaic acid was used to identify the phosphatase activity as PP1 and
PP2A.
Oligonucleotides and Plasmids
The following
oligonucleotides and their complements, flanked by BamHI
sites, were synthesized: (i) a DR5 retinoic acid response element from
the promoter P2 of the RAR- gene
(30) , (ii) a TREpal
thyroid response element
(31) , (iii) a RXRE from the rat CRBPII
gene promoter
(32) , and (iv) a DR2 from the mouse CRABPII gene
promoter
(4) . Sequences of these oligonucleotides are as
follows: (i)
-RARE (DR5), gatcGGGTAGGGTTCACCGAAAGTTCACTCG; (ii)
TREpal, gatcTTCAGGTCATGACCTGAA; (iii) rCRBPII-RXRE,
gatcTGAACTGTGACCTGTGACCTCTGACCTGTGACAGCA; and (iv) mCRBPII-DR2,
gatcGTACAGGTCATCAGGTCAAG. These response elements were cloned into
pBLCAT2
(33) , and this plasmid is referred to as
RAREtk-CAT in the text. p(RARE)
tk CAT was obtained by
inserting two copies of the DR5 oligonucleotide into the BamHI
site of
RAREtk-CAT. A similar procedure was used to clone the
TREpal and DR2 sequences as tandem copies into the same reporter gene,
whereas the rCRBPII-RXRE was cloned as a single element between the
BamHI and HindIII sites. The SV40-based expression
vectors pSG5-RAR
and pSG5-RXR
are described elsewhere
(6, 26, 34) . pCMV5-PP2A was kindly provided by
Dr. M. Mumby (University of Texas, Southwestern Medical Center, Dallas,
TX). pCMV5-PP1 was created by inserting the PP1
cDNA as an
EcoRI- BamHI fragment in pCMV5. The PP1 cDNA was
amplified from pRSET-PP1
(35) to generate a 1000-base pair DNA
using the following primers: 5`-CCGGAATTCGCCACCATGTCCGACAGC-3`
(upstream primer) and 5`-CGCGGATCCCTATTTCTTGGCTTTGGC-3` (downstream
primer).
Inhibition of PP1 and PP2A Alters Retinoic Acid-induced
Transcription
The effect of OA, an inhibitor of phosphatases PP1
and PP2A
(18) on retinoid-induced transcription, was
investigated in a cotransfection assay (Fig. 1). Various
RA-inducible reporter genes were introduced in COS cells, in the
presence or absence of expression vectors coding for RAR and
RXR
. We have used deliberately high amounts of DNA (1 µg) in
these experiments. Although this does not allow for a clear detection
of a synergy between RAR and RXR, it allowed the detection of reporter
gene activity in the presence of only one overexpressed receptor or
both, thereby reflecting preferentially the transcriptional activity of
homodimers or heterodimers. At lower DNA concentrations (25 ng), a
synergy was observed.
(
)
In preliminary
experiments, we noted that the OA effect increased with concentration
up to 100 nM and became cytotoxic above 150 nM. We
therefore used OA at a concentration of 100 nM to selectively
block PP1 and PP2A activities in transfected cells. Moreover, this
concentration did not affect the activity of the parental vector
pBLCAT2, designed
RAREtkCAT thereafter (Fig. 1). Cells, with
or without OA treatment, were treated with 50 nM ATRA or 50
nM 9- cis-RA. 50 nM ATRA exclusively
activates RARs while 9- cis-RA activates both RARs and RXRs,
due to its high affinity for both receptors
(36) .
Figure 1:
Effect of okadaic acid on RAR and
RXR-mediated transcription. COS cells were cotransfected with 0.5
µg of the indicated reporter gene and with or without 1 µg of
pSG5hRAR and pSG5RXR
. Cells were treated with 100 nM
OA for 16 h 24 h after transfection, either with vehicle
(Me
SO, empty bars), 50 nM ATRA
( dotted bars), or 50 nM 9- cis-RA. CAT
activity was assayed and normalized to
-galactosidase activity as
described under ``Materials and Methods.'' CAT activity is
expressed as a percentage of the ATRA-induced transcription level for
cells in the absence of overexpressed receptors, except for the
TREpal-CAT construct. In this latter case, 100% CAT activity is the
level of enzymatic activity detected in cells transfected with
pSG5-hRAR alone and treated with 50 nM ATRA. Values represent
the mean of at least three independent experiments performed with
triplicate assays, and standard deviations did not exceed 15% of the
mean values. Values are indicated in CAT activity normalized to
-galactosidase activity .
We first
examined the effect of inhibition of protein phosphatases on the
RARE
tkCAT (DR5) reporter gene activity. This reporter
gene is bearing a DR5 response element that can be activated by the low
level of endogenous RARs and RXRs present in COS cells
(Fig. 1 A). Overexpression of RAR
did not
significantly increase the promoter activity in response to ATRA or
9- cis-RA, whereas overexpression of RXR
increased the
-RARE CAT activity by 3-4-fold. Coexpression of both
receptors did not further increase the level of activation of the
promoter in response to 9- cis-RA treatment, although we noted
that ATRA yielded a lower CAT activity than in the presence of RXR
alone. This could suggest that RAR has a moderate inhibitory effect
under these conditions, a result comparable to the one obtained with
the rCRBPII-RXRE construct (Fig. 1 C). In no case did
addition of 100 nM OA significantly modify the detected CAT
activity either in the presence or the absence of ligand.
. More
strikingly, overexpression of RXR
alone caused a
ligand-independent activation by OA of the reporter gene to a level
similar to that achieved in the presence of 50 nM ATRA or
9- cis-RA alone. The ligand-induced transcription, in the
presence of OA, was boosted to a similar extent (3-4-fold).
Coexpression of RAR and RXR increased the inducibility of the
TREpal-CAT promoter, when compared to the level reached upon
overexpression of a single receptor, as previously reported
(38) . Phosphatases inhibition caused a further increase in CAT
activity similar to that observed with RXR alone.
, in
opposition to a ``true'' DR1
(39) . Coexpression of
RAR and RXR lowered the rCRBPII-CAT promoter activity, in agreement
with the proposed inhibitory role of RXR transactivation by RAR
(32) . Phosphatases inhibition by OA increased CRBPII-CAT
activity in the presence of overexpressed RXR, albeit to a lower
extent, and counteracted the inhibitory activity of RAR in the presence
of RXR.
RAREtkCAT was used or when the TREpal-CAT plasmid was used in the
absence of overexpressed receptors, indicating that the thymidine
kinase promoter activity is not significantly altered upon phosphatases
inhibition. Furthermore, we
(
)
and others
(9, 20, 21, 22) did not detect any
effect of OA on Rous sarcoma virus or SV40 promoter-controlled genes.
(ii) OA did not increase the activity of the
-RARE CAT construct,
whatever combination of receptors and ligands was used. (iii) The
TREpal and DR2-driven promoters, which can be considered to be equally
activated by agonists in the presence of overexpressed RAR or RXR, and
which have a lower affinity for RAR/RXR heterodimers in vitro than the
-RARE, are strongly activated by OA. Remarkably,
phosphatases inhibition was able to bring transcription, in the absence
of ligand, to a level similar to that induced by retinoids in the
presence of overexpressed RXR. (iv) The CRBPII-RXRE-CAT construct is
highly inducible upon expression of RXR. The OA effect was less marked
in the presence of overexpressed RXR than in the presence of
overexpressed RAR, and the inhibitory effect of RAR on RXR-mediated
transcription could be relieved by OA treatment of transfected cells.
These results suggest that phosphatases inhibition is able to activate
low affinity RARE-driven promoters to a maximum activity in a
ligand-independent manner.
Overexpression of PP2A and PP1 Inhibits Retinoic
Acid-induced Transcription
Although okadaic acid is a valuable
tool to study the role of PP1 and PP2A in various cellular processes,
its use has some potential drawbacks such as its cellular toxicity
(41) . Thus we analyzed the ability of each enzyme to modulate
retinoic acid-induced transcription from each type of reporter gene
(Fig. 2). Exponentially growing COS cells were transfected with a
RARE-containing reporter plasmid or the parental reporter gene
RARE tkCAT, expression vectors coding for both RAR and RXR and
increasing amounts of PP2A or PP1 expression vectors. As it could be
expected, PP2A and PP1 overexpression markedly and specifically reduced
the inducibility by ATRA of RARE-driven reporter genes in a
dose-dependent manner. PP2A overexpression inhibited the ATRA-induced
CAT activity of all reporter genes, although the RXRE-CAT construct
appeared consistently less sensitive. The basal level of CAT activity
for the DR2 and RXRE-driven reporter genes was also lowered upon PP2A
overexpression, although low levels of enzymatic activity made
quantitation of the results difficult for the
-RARE and TREpal
constructs. PP1 also inhibited the activity of all reporter genes
except that of the
RARE-tk CAT reporter gene. Furthermore,
RARE-driven reporter genes displayed a differential sensitivity to each
catalytic subunit of these enzymes (compare left and right columns, for
the 5 µg of plasmid concentration). Thus the retinoic acid
inducibility of each reporter gene was differentially affected by the
type of protein phosphatase used in this assay.
Figure 2:
PP2A and PP1 overexpression decrease the
transcriptional activity of RARE-driven promoters. COS cells were
transfected with 0.5 µg of the indicated reporter gene and 1 µg
of each expression vector coding for RAR and RXR, with increasing
amounts of CMV-PP2A ( left column) or CMV-PP1 ( right
column) plasmids. Cells were then treated with vehicle ( open
circles) or with 1 µM ATRA ( filled circles).
Levels of CAT activity as a percentage of the CAT activity detected in
COS cells transfected without PP1 or PP2A expression vectors and
treated with 1 µM ATRA. Graphic data are averaged from
four independent experiments.
OA Treatment Increases the DNA Binding Affinity of
RAR/RXR Heterodimers
Nuclear extracts from COS cells, treated or
untreated with OA and transfected with both RAR and RXR expression
vectors, were used to perform in vitro RARE-binding assays
(Fig. 3). As shown by Western blot analysis, OA treatment did not
modify the receptor content of the extracts (Fig. 3 B).
However, specific binding to each response elemen t tested was
found to be increased upon OA treatment of COS cells. This increase in
DNA binding activity was especially apparent for the TREpal and DR2
probes (3-4-fold increase), but less obvious for the -RARE
(DR5) and RXRE probes (1.5-2-fold). The latter probe yielded two
specific complexes. The upper band migrated with a mobility similar to
RXR homodimers, whereas the fastest species migrated as RAR/RXR
heterodimers.
Since RAR and RXR have been shown to be the
only RARE-binding proteins in COS cells extracts
(34) , we
conclude that phosphatases inhibition led to an increased DNA binding
activity of RAR and RXR in vitro. This is in agreement with
transient transfection results which showed a clear increase of the
DR2-CAT and TREpal-CAT expression in the presence of 100 nM OA
(see Fig. 1). This observation prompted us to further investigate
the role of basal phosphorylation in the in vitro DNA-binding
properties of RAR/RXR heterodimers.
Figure 3:
OA treatment increases the affinity of
retinoic acid receptors for various retinoic acid response elements.
A, nuclear extracts were prepared from untreated or OA-treated
COS cells transfected with RAR and RXR expression vectors. Their
ability to form specific complexes on a -RARE, TRE-pal, RXRE, or
DR2 response element was tested by electrophoretic mobility shift
assay. Nuclear extracts (20 µg of protein) from untreated cells
were used for competition experiments in which a 50-fold excess of the
same oligonucleotide ( Spec. 1, lanes 2), of the
-RARE probe ( Spec. 2, lanes 3) or the DR2 probe
when the
-RARE was the labeled probe, and of a palindromic
glucocorticoid response element ( Non Spec., lanes 4)
were used. Increasing amounts of nuclear extracts from untreated cells
( lanes 5-8) or OA-treated cells ( lanes
9-12) were incubated with the indicated probe and resolved
on a 5% nondenaturing acrylamide gel. B, Western blot analysis
of RAR and RXR in control and OA-treated COS cell nuclear extracts. 100
µg of protein was resolved on a 12% SDS-PAGE and immunodetected
with the anti-RAR
polyclonal antibody IS39 or the anti-RXR
monoclonal antibody 1RX-6G12.
In Vitro Phosphatase Treatment of Nuclear Extracts
Prevents Specific Binding to a Retinoic Acid Response
Element
Crude extracts containing overexpressed hRAR in
E. coli, Sf9 cells or RAR
and RXR
in COS cells were
submitted to a DNA-binding assay before and after treatment with calf
intestine alkaline phosphatase ( CIP, Fig. 4). Since
alkaline phosphatase has a broad substrate specificity, its use can be
compared favorably with that of PP1 and PP2A. No
-RARE-specific
binding activity was detected in mock transformed bacteria, infected
Sf9 cells, or in nuclear extracts from native COS cells (Fig. 4,
upper panel, lanes 2, 8, and
14MDRV). Upon introduction of a vector coding for hRAR
in
E. coli and Sf9 cells or vectors coding for RAR
and
RXR
in COS cells, protein
DNA complexes were formed
specifically on the
-RARE oligonucleotide ( lanes 3,
9, and 15). The specific DNA binding activity
detected in bacterial extracts, resulting from RAR homodimer formation
onto the
-RARE ( lane 3) was not sensitive to phosphatase
treatment ( lane 6). Western blot analysis ( lower
panel) of native or phosphatase-treated E. coli extracts
( lanes 3 and 6, respectively) identified a single
immunoreactive species revealed by a polyclonal anti-hRAR
antibody, with a molecular mass of 52 kDa. No difference in SDS-PAGE
electrophoretic mobility was detected after CIP treatment, indicating
that the E. coli-expressed hRAR
is not phosphorylated.
(
)
Figure 4:
Phosphatase treatment of crude nuclear
extracts inactivates DNA binding activity of hRAR synthesized in
eukaryotic cells. E. coli, Sf9 cells, or COS cells nuclear
extracts were tested for their DNA binding activity by EMSA. Samples
from nuclear extracts containing 20 µg of protein were incubated
with 20 fmol of labeled
-RARE oligonucleotide ( lanes C),
with a 50-fold excess of cold
-RARE ( lanes S), or with a
100-fold excess of a nonspecific oligonucleotide ( lanes NS).
The same amount of extract was either treated with 50 units of native
calf intestine alkaline phosphatase ( CIP) or with CIP in the
presence of inhibitors ( CIP, i). F (free)
lane, DNA alone; M (mock) lanes: DNA probe
incubated with nontransformed ( E. coli), noninfected Sf9
cells, or nontransfected COS cells extracts. Lower panel,
mock, control or phosphatase-treated extracts were analyzed in parallel
for their content in hRAR
. Forty µg of nuclear extract from
E. coli, Sf9 cells, or COS cells nuclear extracts was resolved
on a 8% SDS-PAGE and blotted onto a nitrocellulose membrane.
Immunodetection was performed using the antiRAR
polyclonal
antibody IS39. Molecular masses (in kDa) are indicated on the
right.
In contrast, the DNA binding activity of hRAR
synthesized in eukaryotic cells (Sf9 and COS) appeared to be sensitive
to phosphatase treatment (compare lane 9 to lane 12 and lane 15 to lane 18). A 75-90% decrease
in DNA binding activity was consistently observed which was concomitant
with an increase of the electrophoretic mobility of RAR
in
SDS-PAGE of the phosphatase-treated sample ( lower panel). This
increased mobility is indicative of the removal of several phosphate
groups from the RAR
molecule. We showed previously that treatment
of
P-labeled RAR
in COS cells led to a significant,
but not complete, loss of phosphate groups
(13) . Similar
results were obtained with RXRs.
The effect of phosphatase
treatment on the DNA binding activity was also observed using potato
acid phosphatase and agarose-immobilized alkaline phosphatase, and for
nuclear extracts from HEL, HL-60, and HeLa cells.
(
)
The DNA binding activity was not affected when the
phosphatase was inhibited by 10 mM inorganic phosphate, 10
mM sodium molybdate and vanadate (Fig. 4, lanes 13 and 19). Thus hRAR
is a phosphoprotein when
synthesized in eukaryotic cells, and phosphatase treatment of extracts
strongly decreased its DNA binding affinity under these conditions. The
concomitant dephosphorylation of hRAR
implies that phosphorylation
of the receptor is required for DNA binding. Alternatively, this could
mean that an inhibitory activity was unmasked after CIP treatment. This
hypothesis can be ruled out, however, since when E. coli RAR
and RXR are combined to native or CIP-treated COS cells extract, they
bind to DNA with a similar efficiency. The apparent molecular masses of
native RAR
synthesized in Sf9 and COS cells were identical. In
each case, RAR
migrated as a doublet and was detected as
54-
and 58-kDa polypeptides, and both forms appeared to be sensitive to CIP
treatment. As shown by Western blot analysis of the extracts,
overexpression of RAR
in each system yielded similar amounts of
RAR polypeptide. Receptors extracted from these cells bound ATRA with a
similar dissociation constant (
3 nM) and yielded an
equivalent amount of ATRA-binding sites (5-10 pm receptor/mg
protein, data not shown). This indicates that whatever the system used,
receptors have similar properties and stability. We cannot, however, be
sure in this system that hRAR
is the only substrate for the
alkaline phosphatase, which could also dephosphorylate other proteins
necessary for the DNA binding activity of the receptor.
Dephosphorylation of Both RAR and RXR Reduces the
Affinity of RAR/RXR Heterodimers for DNA
To further determine
the importance of the phosphorylation state of each partner in the
heterodimerization process, RAR and RXR were expressed either in E.
coli, in a nonphosphorylated form, or in COS cells in which
polypeptides are fully processed. Nuclear extracts from COS cells
containing RAR (Fig. 5 A) or RXR
(Fig. 5 B) were treated with CIP for 1 or 15 min, mixed
with purified
A/B RXR
or
A/B RAR
, respectively, and
analyzed by EMSA. Heterodimeric complexes formed on the
-RARE
probe were identified by supershifts using monoclonal anti-RAR
antibody ( 9
, lanes 2, 6, 10,
and 14), an antiRXR antibody ( 4RX, lanes 3,
7, 11, and 15), or both
( 9
+4RX, lanes 4, 8, 12,
and 16). Complexes were totally supershifted in the presence
of both antibodies ( lanes 4, 8, 12, and
18 in both panels), showing that these complexes are made of
RAR and RXR. CIP treatment of COS cell extract for 15 min caused a
strong decrease in the amount of RAR
RXR
DNA complexes,
regardless of which receptor was present in the CIP-treated extract
(compare lanes 4 to lanes 8 in panels A and
B). Interestingly, we also noted that heat inactivation of CIP
was not sufficient to prevent a partial loss of the DNA binding
activity (compare lanes 12 to lanes 16 in both
panels), in opposition to phosphatases inhibitors (sodium phosphate,
sodium molybdate, and vanadate, see Fig. 4). This is suggestive
of the presence of an endogenous phosphatases activity in COS extracts
that remains to be identified. Thus, phosphorylation of both RAR
and RXR
appeared to be important for the DNA binding activity of
RAR/RXR heterodimers. However, this post-translational modification is
not an absolute requisite for dimerization since RAR and RXR produced
in E. coli can generate similar complexes (see below),
suggesting that phosphorylation may increase the affinity of one
receptor for its dimerization partner.
Figure 5:
Alkaline phosphatase treatment impaired
the DNA binding activities of both RAR and RXR
overexpressed
in COS cells. Whole cell extracts from COS cells transfected either
with RAR ( panel A) or RXR expression vectors ( panel
B) were treated with active ( panels A and B,
lanes 1-8) or heat-inactivated CIP ( lanes
9-16) for 1 or 15 min at 37 °C. Treated COS extracts
containing RAR
( panel A) and RXR
( panel B)
were then mixed with purified
A/B RXR or
A/B RAR,
respectively, and resolved by EMSA. The protein composition of the
retarded complexes ( arrow 1) was determined by supershift
experiments using monoclonal antibodies directed against RAR
( 9
) or RXR ( 4RX). Supershifted complexes are
indicated by arrow 2. The empty arrowhead indicates
nonspecific complexes.
Bacterially Expressed RAR and RXR Are Less Efficient at
Forming Heterodimers than RAR and RXR Extracted from Eukaryotic
Cells
Receptors synthesized in eukaryotic cells and in bacteria
were used in EMSA experiments to further answer the question whether
post-translational modifications alter the ability of RAR/RXR
heterodimers to form on a RARE. To test this hypothesis, we performed
experiments comparing the ability of RAR and RXR, expressed either in
bacteria or in COS cells, to generate heterodimers on the -RARE.
The amount of RAR or RXR extracted from E. coli or COS cells
extracts necessary to generate an equivalent amount of
receptor
DNA complex in the presence of its nonphosphorylated
dimerization partner was titrated by EMSA. As shown in Fig. 6,
none of the individual components of the binding reaction bound to DNA
by itself, when used at the indicated concentrations ( lanes
13- 21). When COS or E. coli RAR
1 was added
to a constant amount of non phosphorylated ( i.e. from E.
coli extracts)
A/B RXR
(Fig. 6 A), an
identical level of heterodimer formation could be reached with both
type of extracts (compare lanes 1- 6 and lanes
7-12). However, quantification of RAR
by Western
blotting revealed that a much higher amount (8-10-fold) of the
RAR
polypeptide was present in the bacterial extract
(Fig. 6 B, compare lane 1 to lane 7),
indicating that a higher concentration of nonphosphorylated RAR is
necessary to yield an identical level of heterodimer binding to DNA.
Quantification of the retarded bands formed for each condition
(Fig. 6 C) demonstrated that E. coli or
COS-expressed RAR
yielded an equal amount of receptor
DNA
complexes, suggesting that the binding of RAR
/RXR
heterodimers to DNA is not affected by post-translational modifications
once the heterodimers are formed.
Figure 6:
RAR synthesized in bacteria forms
RAR/RXR heterodimers less efficiently than RAR
extracted from COS
cells. A, increasing amounts of whole cell extracts from COS
cells ( lanes 6 to 1) or from E. coli ( lanes 12 to 7) overexpressing RAR
were
added to a constant amount of
A/B RXR
purified from E.
coli. Protein
DNA complexes formed on the
-RARE probe
were analyzed by EMSA. The ability of each component to bind to this
probe was also assessed in a similar manner ( COS RAR,
lanes 15 to 13; E. coliRAR,
lanes 18 to 16; purified RXR, lanes 21 to
19). Protein concentration is given in µg/20 µl of
EMSA mix. Arrow 1 indicates RAR/RXR heterodimers, whereas
arrow 2 points to RAR homodimers ( lane 16). The
empty arrowhead shows nonspecific complexes. B,
quantification of RAR
in COS and E. coli extracts by
Western blot. Extracts used to run the gel retardation assay were
analyzed for their content in RAR
as described under
``Materials and Methods.'' The membrane was probed with the
polyclonal antibody Rp
(F). C, quantification of the
retarded bands shown in panel A from lanes 1 to
12. The autoradiogram was scanned using a PhosphorImager and
data plotted as a graph showing the variation of the amount of the
retarded complexes versus the protein concentration of
RAR-containing extracts.
An analogous titration experiment
was done to compare the capacity of nonpurified E. coli and
COS RXR to interact with RAR present in crude E. coli extracts (Fig. 7). In this experiment, we first estimated
the concentration of RXR present in both type of extracts by Western
blot analysis. RXR concentration in E. coli extracts was
10-fold higher in this typical experiment, since 40 µg of protein
from COS cell extracts had to be loaded to yield a signal equivalent to
that observed with 4 µg of E. coli extracts, as shown by
Western blot analysis (Fig. 7 B). However, both types of
RXR were able to form heterodimers on the
-RARE oligonucleotide,
as shown by supershift experiments (Fig. 7, lanes 9 and
11, arrow 2). COS RXR was able to generate a higher
amount of heterodimeric complexes than E. coli RXR despite its
lower concentration in COS cell extracts (Fig. 7 A,
compare lanes 1-4 to lanes 5-8).
Quantification of the results showed that COS RXR formed heterodimeric
complexes with a 10-15-fold higher efficiency than E. coli RXR. Thus it appeared from the experiments presented in Figs. 6
and 7 that nonphosphorylated RAR and RXR bound to DNA, in the presence
of their dimerization partner, with a lower efficiency (at least
10-fold) when compared to the fully processed polypeptide. This lower
efficiency could be overcome by using a higher amount of the
nonphosphorylated receptor form, which have, by all other criteria, the
same functionality. Indeed, receptors were expressed at the same rate
in E. coli and COS cells (see Fig. 4) and bound
ATRA
(
)
and DNA (Fig. 6 C) with
similar affinities. Moreover, RAR homodimer formation on the same RARE
was not compromised by the lack of post-translational modifications
(see Fig. 4), suggesting that its affinity for DNA in the
presence of its dimerization partner is decreased with respect to that
of the fully processed polypeptide in COS cells.
Figure 7:
RXR synthesized in bacteria forms RAR/RXR
heterodimers less efficiently than RXR extracted from COS cells.
A, increasing amounts (indicated in µg of proteins) of
A/B RXR
extracted from bacteria ( E. coliRXR) or full-length RXR
extracted from COS cells was
incubated in the presence of a constant amount of hRAR
and
protein
DNA complexes were analyzed by EMSA. Supershift
experiments were performed using an antiRXR monoclonal antibody in the
presence ( lane 9) or the absence ( lane 10) of nuclear
extracts. Similarly, a polyclonal antiRAR antibody was used in the
presence ( lane 11) or the absence of nuclear extract ( lane
12). In these experiments, the amount of nuclear extract was
similar to that used in lane 4. Arrows 1 indicate the
retarded complexes, whereas arrows 2 indicate complexes
supershifted by either a polyclonal antiRAR
antibody ( lane
11) or a monoclonal antiRXR antibody ( lane 9).
B, assay of the RXR
content of COS and E. coli extracts. 2.5, 5, 10, or 40 µg of COS extracts or 0.25, 0.5,
1, and 4 µg of bacterial extracts were analyzed by Western
blotting. The full-length mRXR migrated as a
54 kDa species,
whereas the
A/BRXR
migrated as a 42-kDa polypeptide. The
nitrocellulose membrane was probed with the monoclonal antiRXR antibody
1RX-6G12.
PP1 Efficiently Inhibits RAR/RXR Binding to
Transient transfection experiments showed that protein
phosphatases exert a noteworthy influence on the inducibility of
RARE-driven reporter genes. In vitro DNA binding experiments
showed that both RAR and RXR are the target for phosphatase action. To
address the question as to whether PP1 or PP2A are equally active in
abolishing the DNA binding activity of RAR/RXR heterodimers, we
partially purified PP1 and PP2A and used them as the source of
dephosphorylating enzymes. The PP1/PP2A mix turned out to be as
efficient as alkaline phosphatase (and potato acid
phosphatase-RARE in
Vitro
) to abolish the formation of RARE-receptor
complexes (Fig. 8, panel A). When OA was added at
various concentrations to selectively block PP2A and PP1, we noted that
the DNA binding activity was preserved for OA concentrations of
10-50 nM. Since PP2A and PP1 are inhibited by I
of 0.2 and 20 nM, respectively
(18) , we infer
that PP1 is, under these specific conditions, the most likely candidate
as a receptor-dephosphorylating enzyme.
Figure 8:
Okadaic acid prevents the loss of DNA
binding activity of RAR/RXR complexes treated with purified PP1 and
PP2A in vitro. A, nuclear extracts from HeLa cells were
treated with partially purified PP2A and PP1 for 1 h at 37 °C in
the presence of the indicated concentration of OA. Samples were then
analyzed by EMSA for their ability to bind to the -RARE probe.
B, decay of the RAR
RXR complexes in the presence of PP1
and PP2A. HeLa nuclear extracts were submitted to DNA-binding
conditions in the presence of labeled
-RARE, then treated for 1 h
in the presence or absence of PP1 and PP2A at 37 °C, as described
above. Samples were transferred at 4 °C, and a 200-fold excess of
cold probe was added. Samples were loaded on a 5% nondenaturing gel at
the indicated times. Results were quantified by excision of the
radioactive bands and scintillation counting. Results are expressed as
a percentage of total probe input.
The loss of binding to DNA
upon phosphatase treatment can be potentially explained by two modes of
action for these enzymes: (i) they increase the dissociation rate of
the ternary complex RARRXR
RARE, or (ii) phosphatase
treatment lowers the on-rate of the association of heterodimers with
RAREs. To test the first hypothesis, we preassembled complexes on the
-RARE, treated them with or without with PP1 and PP2A, and
followed the dissociation of RAR
RXR complexes by addition of an
excess of the same radioinert probe (Fig. 8 B). The
off-rate was similar, indicating that while RAR and RXR in solution are
highly sensitive to phosphatases action assembled heterodimers do not
display such a sensitivity.
-RARE (DR5) sequence, the TREpal and mCRABP2-CAT (DR2)
constructs were activated by OA in a ligand-independent, RXR-dependent
manner to a level equal to that reached in the presence of ATRA or
9- cis-RA. This activation was also detected for the rCRBP2
RXRE-CAT construct in the presence of overexpressed RAR, and OA
relieved the inhibitory effect of RAR upon RXR-mediated transcription.
PP2A and PP1 therefore play a role in the modulation of the activity of
RARE-driven promoters, and this control appeared to be dependent upon
the type of RARE and the ratio between the intracellular concentration
of RAR versus that of RXR. Conversely, overexpression of the
PP2A and PP1 catalytic subunit significantly lowered the inducibility
of all the reporter genes tested. The sensitivity varied for each
phosphatase in the order TREpal >
-RARE > DR2 > RXRE
(PP2A), DR2 = RXRE > TREpal
-RARE (PP1). The
lack of sensitivity of the
-RARE construct to PP1 overexpression
was unexpected considering the effect of this enzyme on the in
vitro DNA binding activity of receptors, but it has been reported
that overexpression of these enzymes yield mostly insoluble proteins
(42) . It is therefore likely that the phosphatase to receptor
ratio was not identical in these two experiments. Thus, the cis-acting
properties of a RARE could be affected by physiological conditions that
alter endogenous PP1 and PP2A expression. In that respect, we note that
the expression of PP2A is decreased upon ATRA-induced differentiation
of HL-60 cells
(43) , and we hypothesize that this
down-regulation could potentially favor the activation of RA-regulated
genes directly implicated in the differentiation process. Similarly,
PP1 and PP2A activities are regulated by insulin in rat skeletal muscle
cells in a differentiation-dependent manner
(44) . Our data
therefore suggest that, in target cells, subset(s) of RA-controlled
genes will be differentially affected by the phosphorylation state of
retinoic acid receptors.
DNA complex formation efficiency
by at least 10-fold. While this work was under review, Bhat and
colleagues
(47) reported similar values for heterodimer
formation of T
R/RXR in solution. Taken together, these data
suggest that while phosphorylation of RXR or RAR is required neither
for homodimer nor for heterodimer formation per se, it may
modulate their heterodimerization properties when one partner is
present in limiting concentrations.
R-
1 and an increase in
transcriptional activity of this receptor. This effect has been
recently attributed to a more efficient homodimerization of
hT
R-
1
(55) . Our observations further
substantiate the regulatory role of the phosphorylation state of
dimerization partners of retinoid receptors and demonstrated its role
on their transactivating potential. Given the intricacy of the retinoid
signaling pathway, which is controlled by specific ligands and
regulated by a delicate balance between heterodimer and homodimer
formation
(56) which can either potentiate (reviewed in
(3, 57) ) or inhibit
(37, 58, 59, 60, 61, 62) RAR activity, establishing a well-defined role for a given
phosphorylated amino acid from each receptor will be necessary.
polyclonal antibody IS39, and Dr. H. Gronemeyer who
provided us with purified RAR and RXR. We also acknowledge Pr. P.
Chambon for stimulating discussions and Dr. J. Clifford for critically
reading the manuscript. We are also grateful to Drs. B. Wadzinski and
L. Peruski for the gift of the PP1 cDNA and to Dr. M. Mumby who
provided us with the CMV-PP2a construct.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.