(Received for publication, November 23, 1994; and in revised form, April 28, 1995)
From the
S91 melanoma cells are growth arrested and differentiate when
treated with retinoids. These processes correlate with expression of
the retinoic acid receptor (RAR)
Retinoids, a group of chemically related molecules derived from
vitamin A (retinol), regulate a large number of biological processes in
vertebrate development, cell growth, differentiation, and homeostasis (1, 2, 3) . The actions of retinoids are
mediated by two classes of nuclear receptors: retinoic acid receptors
(RARs)
RARs and RXRs bind specific DNA elements in the
promoter region of target genes called RA response elements (RAREs).
The majority of RAREs appears to consist of two direct repeats (DR), or
half-sites, of the sequence AGGTCA spaced by five nucleotides
(DR5)(15, 16) . Numerous in vitro experiments
have shown that RARs and RXRs bind with low affinity as homodimers to
DR5, but, when mixed, RAR
An interesting aspect of
retinoids is their effect on differentiation of neoplastic cells, both in vivo and in vitro (1, 3 and references therein).
For instance, there are over 200 retinoid-sensitive tumor cell lines
that could provide a useful model system to gain insight into how
retinoids affect malignant growth(29) . In this report, we have
focused on the murine melanoma cell line S91. Upon treatment with RA,
these cells become growth arrested and display an enhanced
differentiated phenotype, exemplified by the formation of more and
longer dendritic extensions and an increase in melanin
synthesis(30, 31, 32) . The occurrence of
malignant melanoma has been steadily on the rise(33) , and
prognosis is still poor when discovered in advanced state.
Unfortunately, little is known about the molecular events of malignant
change in melanocytes, and S91 cells may be very useful for these
studies. Previous reports have shown that RAR
The
following oligonucleotides were used:
Figure 1:
RXR
Figure 5:
Chemical modification interference assay
of Complexes I and II binding to the
Figure 2:
Two different protein complexes bind
specifically, but with different kinetics, to the
First,
we used two different RAR antibodies: RARc, which cross-reacts with the
three major RAR isoforms, and a RAR
Figure 3:
Characterization of Complex I and II
binding to the
Fig. 3C shows that Complex II, on the
other hand, can only be partially supershifted with the hinge
region-epitope RXR
Our data may
also explain the disappearance of Complex I because the RXRs in that
complex may be recruited into a heterodimer complex with the induced
RAR
Figure 4:
Western blot
showing induction of RAR
Figure 6:
9-cis-RA is a more potent inducer
of the RAR
The S91 melanoma cell line is a useful model system to study
retinoid-induced growth arrest and differentiation and may provide
important clues to the transformation of melanocytes in disease.
However, the molecular basis of the retinoid-induced effects is still
largely unknown. There are two classes of retinoid receptors, and it is
likely that RARs and RXRs are at the top of the cascade of events that
lead to the arrested growth and morphological changes in these cells.
Previously, it was shown that these cells constitutively express
RAR
It was also previously observed that induction of RAR
This result is
surprising because the
However, we are unable to prove this
formally, because the N-terminal epitope, at least in RXR
RAR
It is possible
that there are very low amounts of RAR
Another interesting
aspect is that RAR
Our results suggest that a mechanism of sequential
occupation of the
We thank Dr. Pierre Chambon for providing the pSG5RAR
plasmids, Dr. Ronald Evans for the pBSRXR plasmids and the RXR
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
gene, which is induced through a
retinoic acid response element (
RARE). We wished to determine
which endogenous retinoid receptors (RARs and retinoid X receptors,
RXRs) mediate induction of the RAR
gene. We show that RXR
and
RXR
are constitutively expressed. Electrophoretic mobility shift
assays with nuclear extracts show specific binding to the
RARE
(Complex I) in untreated cells, which can be supershifted by antibodies
against RXRs but not by anti-RAR antibodies. After 48 h of treatment
with retinoic acid, Complex I is replaced by a faster migrating Complex
II, which can be supershifted by anti-RAR
and anti-RXR
antibodies. This suggests that induction of the RAR
gene is
largely mediated by RXRs only. Accordingly, we also find that 9-cis RA, which activates both RAR and RXR, is a more potent inducer of
the RAR
gene than RA, which only activates RAR. After 48 h, all
RXRs appear to be titrated by the newly synthesized RAR
into an
RAR
RXR heterodimer complex. Thus, it appears that the
RARE is sequentially occupied by RXR dimers and RAR
RXR
heterodimers.
, (
)which bind all-trans-retinoic
acid (RA) and 9-cis-retinoic acid (9-cis-RA) with
similar affinities, and retinoid X receptors (RXRs), which bind
9-cis-RA with much higher affinity than
RA(3, 4, 5, 6) . RARs and RXRs
belong to an extensive gene family of ligand-dependent transcription
factors that together form the steroid/thyroid hormone receptor
superfamily(7, 8) . There are three types of both RARs
and RXRs (
,
, and
), encoded by separate genes, that
have different spatio/temporal expression
patterns(9, 10, 11, 12, 13, 14) .
In addition, they are also subject to alternative splicing and/or
alternate promoter choice, thereby generating a large family of mainly
N-terminally different receptor isoforms(1, 5) . Thus,
it is conceivable that certain RAR
RXR isoforms may serve specific
genetic programs.
RXR heterodimers can form that bind with
much higher affinity than either homodimer. These data, together with
transfection experiments in mammalian cells and yeast, suggest that the
heterodimer is the transcriptionally active species when both partners
are present (17, 18, 19, 20, 21, 22, 23, 24, 25, 26) .
However, it was recently found that an element with a spacing of one
nucleotide (DR1) is also bound by RAR
RXR heterodimers but in a
transcriptionally inactive conformation. In contrast, in the presence
of 9-cis-RA, RXR homodimers specifically can bind and activate
transcription from promoters with this
element(27, 28) .
and RAR
are
constitutively expressed in S91 cells but that RAR
is rapidly
induced by treatment with retinoids. RAR
expression is maximal
after 24 h and is independent of de novo protein
synthesis(32, 34) . Interestingly, retinoids that fail
to induce RAR
do not cause growth arrest(32) , and
differentiation becomes phenotypically apparent only after RAR
mRNA levels have reached their highest levels(30) . One
interpretation is that a certain threshold level of RAR
is
required to facilitate differentiation of S91 cells. It is, therefore,
important to know which proteins regulate RAR
expression.
Retinoid-dependent induction of the RAR
2 gene (the major RAR
isoform) is mediated through an RARE in the RAR
2 promoter
(
RARE, a DR5-like element), which has been well characterized in in vitro studies(35, 36, 37) . In
this report, we investigated which endogenous, intracellular
RAR
RXR isoforms mediate the retinoid-dependent induction of the
RAR
gene in S91 cells. Our data provide evidence that,
surprisingly, a complex containing RXRs, but not RAR
RXR
heterodimers, largely regulates RAR
gene expression.
RNA Isolation Procedures and Northern
Blotting
Poly(A) RNA was extracted from cells
grown at about 60% confluency in 5-8 flasks (162 cm
)
for each time point, using a poly(A)
RNA isolation kit
(Stratagene, La Jolla, CA). Total RNA was isolated from 2 flasks for
each time point according to (38) . 1 µg of
poly(A)
RNA or 15 µg of total RNA was subjected to
electrophoresis through a denaturing formaldehyde-agarose gel (1%),
transferred to a nylon membrane (Duralon-UV, Stratagene, La Jolla, CA)
according to (39) , and cross-linked in a Stratagene
Stratalinker. Northern blots were hybridized with the following DNA
restriction fragments, which had been gel-purified and random-primed in
the presence of [
-
P]dCTP: RAR
, EcoRI-EagI fragment from pSG5RAR
; RXR
, AvaI fragment from pBSRXR
; RXR
, EcoRI-NheI fragment from pBSRXR
and a
cyclophilin BamHI fragment. Blots were washed in 0.1
SSC, 0.1% SDS at 55 °C and autoradiographed. Quantitation of
Northern blots was performed on a Molecular Dynamics PhosphorImager
(Sunnyvale, CA) using ImageQuant software (version 3.3).
Nuclear Extracts, EMSA, and
Oligonucleotides
Procedures for obtaining nuclear extracts from
cells (grown in 1-2 flasks (162 cm) at about 60%
confluency for each time point) and EMSA conditions were essentially
performed as described previously(40) ; typically, 1.5-3
µg of nuclear extract was used. Incubation with gel-purified
P-end-labeled oligonucleotides was performed in the
presence of 1 µg of salmon sperm DNA. The reaction mix minus probe
was incubated for 15 min at room temperature; probe (50,000 cpm,
5
fmol) was added and incubated for another 30 min, and then put on ice
for 10 min before electrophoresis on 4% polyacrylamide gel. For
antibody supershifts, 1 µl of antiserum was added at this point and
incubated at 4 °C for another 2 h before electrophoresis. The
specificity and characterization of the RXR antibodies will be
described elsewhere(54) . The RAR
antibody does not
cross-react with RAR
or RAR
. The RARc antibody reacts about
equally well with RAR
and RAR
, less well with RAR
in
immunoprecipitations, and about equally well with all three
RAR-isoforms in EMSA (data not shown).
(
)
RARE (-61/-29),
AGCTTCCGGGAAGGGTTCACCGAAAGTTCACTCGCATAAGGCCCTTCCCAAGTGGCTTTCAAGTGAGCGTATTCGA;
TK minimal promoter (-46/+1),
AGCGGTCCGAGGTCCACTTCGCATATTAAGGTGACGCGTGTGGCCTCGAACCAGGCTCCAGGTGAAGCGTATAATTCCACTGCGCACACCGGAGCTTCGA;
CTF binding site from TK promoter (-96/-62),
TCGACAGCGTCTTGTCATTGGCGAATTCGAACACGCAGATGGTCGCAGAACAGTAACCGCTTAAGCTTGTGCGTCTACAGCT;
DR1,
AGCTAGTTACTTATTGAGGTCAGAGGTCAAGTTACGTCAATGAATAACTCCAGTCTCCTGTTCAATGCTCGA.
Western Blot
10 µg of nuclear extract was
loaded on a 10% SDS slab gel and analyzed by SDS-polyacrylamide gel
electrophoresis. A 1:1000 dilution of primary antibody was used,
followed by a 1:2000 dilution of a peroxidase-coupled goat-anti-rabbit
secondary antibody. Visualization of bands was done by using an ECL
detection kit (Amersham Corp.) Exposure time was about 30 s on x-ray
film.
Cell Culture Conditions
S91 cells (ATTC CCL 53.1)
were grown in Dulbecco's modified Eagle's medium with 10%
fetal bovine serum at 37 °C in 5% CO in humidified air.
Solutions of RA and 9-cis-RA were made fresh every 24 h in
ethanol. Plates that did not receive ligand received ethanol instead.
Working concentrations were 10
or 10
M, and plates were kept in the dark as much as possible.
Chemical Modification Interference Assay
The
oligonucleotide with the RARE was cloned in the HindIII
site of pBluescript and confirmed by dideoxy sequencing. The coding
strand was labeled by cutting the plasmid with XhoI followed
by Klenow-fill-in in the presence of
[
-
P]dCTP, dTTP, and dGTP. The plasmid was
then cut with SpeI. Similarly, the noncoding strand was
labeled by digesting the plasmid with SpeI and Klenow-fill-in
in the presence of [
-
P]dCTP and dTTP
followed by digestion with XhoI. Both probes were gel-purified
and chemically modified by dimethyl sulfate or KMnO
,
essentially as described previously(41, 55) . EMSA was
performed in 6% polyacrylamide gel with 6 µg and 3 µg of the
nuclear extracts at the 0 (Complex I) and 48 (Complex II) h time
points, respectively. Free and bound probe was eluted, digested as
described(41, 55) , and run on a 8% DNA sequence gel.
Quantitation of individual bands was performed by PhosphorImager
scanning analysis, and normalized for loading differences.
S91 Cells Constitutively Express RXR
As a first step in the analysis of the and RXR
mRNA
RARE, we
decided to complete the retinoid receptor inventory in S91 cells by
analyzing the nature of the expressed RXR species. Poly(A)
RNA was isolated from cells that were untreated (0 h) or treated
with 1 µM RA for the indicated time period (Fig. 1)
and analyzed by Northern blotting. As a positive control, we probed the
blot with RAR
cDNA. In agreement with published
reports(32, 34) , levels of RAR
mRNA increase
from barely detectable in untreated cells, to readily observable after
only 2 h of treatment. Levels continue to increase largely over the
first 24 h, after which maximum levels appear to have been reached
(10-12-fold over untreated cells). The band in Fig. 1denoted as RAR
represents the product of the
RAR
2 gene, because a polymerase chain reaction-generated probe
containing the RAR
2-specific N-terminal sequence hybridizes with
the same band (not shown). Interestingly, the RXR
probe detects
three transcripts in treated and untreated cells that are weakly
up-regulated (about 2-fold) by RA. Of these, only the smallest
transcript (
5 kb) appears to have the size reported by Mangelsdorf et al.(14) for RXR
. The larger transcripts may
be due to deregulated expression and/or chromosomal rearrangements in
these cells. RXR
expression is also observed in untreated cells
and likewise is only slightly up-regulated by RA. The size of the
(single) transcript is as expected (14) . In contrast, RXR
could not be detected (data not shown). Thus, in the absence of high
doses of RA, S91 cells contain mRNAs for RAR
,
RAR
(32, 34) , RXR
, RXR
, and very low
levels of RAR
. As shown in Fig. 1, all receptors are
constitutively expressed except for RAR
, which is induced by RA.
and RXR
mRNA are
constitutively expressed, in contrast to RAR
, which is strongly
induced by RA. Poly(A)
RNA (1 µg) from cells
treated with 10
M RA for the indicated time
periods was subjected to electrophoresis, blotted, and hybridized with
the indicated probes on the left. RNA size markers are
indicated at the right.
Regulation of Protein Complexes Binding to the
Next, we wished to determine the nature of the nuclear
proteins that mediate RA-regulation of RARRARE
gene transcription. For
this purpose, we examined the binding of proteins, present in nuclear
extracts obtained from cells undergoing the same treatment as described
above, to a labeled DNA probe containing the minimal
RARE (see Fig. 5B) in EMSA. The results are shown in Fig. 2. In untreated cells, a single retarded complex is
observed (Complex I), which essentially remains unchanged over the
first 24 h of RA treatment, although there appears to be more binding
at the 8 h time point. However, after 48 h of RA-treatment, Complex I
has completely disappeared. Interestingly, a new complex (Complex II)
with a higher mobility becomes apparent at the 24 h time point, and
after 48 h it has greatly increased in intensity and is the only
remaining complex. Binding of both these complexes is specific, as
illustrated by the DNA competition studies with the 24 h time point
sample (both Complexes I and II are present in this extract). Fig. 2shows that both complexes can be effectively competed by a
50-fold excess of cold
RARE but not by two unrelated
oligonucleotides representing the CCAAT-box transcription factor
binding site and the minimal promoter region of the Herpes simplex
virus thymidine kinase gene. Interestingly, DR1, which can bind
RAR
RXR and RXR
RXR dimers, also competes. These results
indicate that the
RARE could be occupied by a different set of
retinoid receptors in untreated cells (Complex I) than in RA-treated
cells (Complex II). In agreement with this, we find that addition of
9-cis-RA and RA slightly enhances the mobility of Complex I
and II, respectively, without significantly affecting the binding
efficiency (not shown). These possibilities are explored in the next
section.
RARE (-61/-29)
show differences in binding. As a source of Complex I, nuclear extract
from untreated cells (0 h) was used; for Complex II, the 48 h
RA-treated cells were used. Circles refer to protected guanine
residues (dimethyl sulfate, DMS); squares refer to
protected thymine residues (KMnO
). Opensymbols denote weak protection (intensity between 30 and
70% of corresponding band in free probe lane); solidsymbols denote strong protection (intensity less than 30%
of the corresponding band in the free probe lane). A,
autoradiograms of chemical modification interference experiments (see
``Materials and Methods''). Coding and noncoding strands are
indicated above the gel (see also below). F and B indicate free and bound probe, respectively. For orientation, the
upstream and downstream half-sites are indicated by an open,
and solidblackarrow, respectively,
alongside the gel. Protected nucleotides are indicated in relation to
the half-sites. B, summary of interference patterns shown
above.
RARE. Nuclear
extracts were prepared from cells treated with 10
M RA for the time points indicated above the
gel and analyzed by EMSA, using the
RARE as probe. Cold competitor
DNA, as indicated below the gel, was added in 50-fold excess
(see text). Arrows indicate the presence of Complexes I and
II. P, probe alone.
Immunological Characterization of Complexes I and II
Reveals the Presence of RXR
To establish the identity of complexes I
and II, we made use of a battery of RAR- and RXR-specific antibodies.
When used in the EMSA with in vitro translated receptors,
these antibodies can ``supershift'' retarded complexes if the
epitope is accessible and specifically change their mobility in the gel
to a position with lower mobility (data not shown). For simplicity, we
focused our attention on the 0 and 48 h time points, inasmuch as they
each contain just one complex (either I or II, respectively). For a
fair comparison of the proteins in Complexes I and II in this
experiment, we used 3 µg of the 0 h time point and 1.5 µg of
the 48 h time point nuclear extract, respectively, because these
amounts give equal levels of shifted probe in both complexes.
and RXR
, and of RXR
and
RAR
, Respectively
-specific antibody (RAR
).
The results are shown in Fig. 3A. Contrary to our
expectations, none of the RAR antibodies affected the mobility of
Complex I in the untreated extract, indicating that they either do not
contain RARs or that the epitope is inaccessible. In contrast, both RAR
antibodies completely supershifted Complex II at the 48 h timepoint,
whereas preimmune serum and a nonrelevant antibody had no effect. This
shows that Complex II contains at least RAR
.
RARE in the EMSA using antibody supershifts with
various specific RAR
RXR antisera. As a source of Complex I,
nuclear extract from untreated cells (0 h) was used; for Complex II,
the 48 h RA-treated cells were used, as indicated above the gels. The arrows with associated roman numerals point to Complex I and
II. Supershifted complexes are also indicated on the left by arrows, marked SS. Antibodies are added as indicated
below the gel; -, no antibody added; *, nonspecific,
serum-induced binding. A, detection of RARs. Antibodies used
were as follows:
Gal, anti-
-galactosidase; Pre, preimmune antisera; RAR
, specific
RAR
antibody; RARc, nonisoform-specific RAR antibody. B and C, detection of RXRs in Complexes I (B) and II (C). Antibodies used were as follows: RXR
(H), RXR
-specific antibody raised against
hinge-region epitope; RXR
(N), RXR
-specific antibody
raised against N-terminal epitope; RXR
and RXR
, antibodies raised against N-terminal epitopes in
RXR
and RXR
, respectively. P, probe alone.
Next, we used a
battery of RXR antibodies, consisting of two different RXR
antibodies (designated H and N, that recognize epitopes in the hinge
region, or in the N terminus, respectively), as well as RXR
and
RXR
antibodies (all raised against N-terminal epitopes). Fig. 3B shows the following results. Complex I can be
partially supershifted by both RXR
antibodies and slightly less
well by the RXR
antibody. Addition of more antibodies did not
increase the amount of shifted probe (data not shown). However, the
combination of both RXR
(type H) and RXR
antibodies
quantitatively supershifts the entire complex, whereas no shift is
observed with two different preimmune sera or the RXR
antibody.
(Note that all our non-IgG-purified antisera give rise to a
nonspecific, serum-dependent shifted band that migrates with a lower
mobility than the antibody-specific supershifted complex. It is
nonspecific because nonrelated antisera give the same result, they
cannot be competed with the immunizing peptide, and there is no
concomitant reduction in the intensity of the complexes of interest.)
This suggests that Complex I consists of, at least, RXR
and
RXR
.
antibody (RXR
H) but not at all by the
RXR
antibody raised against the N-terminal epitope (RXR
N).
Neither one of the other RXR antibodies, or preimmune sera, affected
the mobility of the complex. This not only suggests that Complex II
contains RXR
, but also that the N-terminal epitope of RXR
is
now inaccessible, unlike the epitope in Complex I. Again, addition of
more antisera did not change the amount of retarded probe, suggesting
that these effects are not due to limiting amounts of antibodies (not
shown), but may be due to different intra-dimer protein-protein
interactions between the RXR dimers and the RXR
RAR heterodimers.
However, we cannot exclude the possibility that there are also other
unknown nuclear protein(s) present in both complexes, although
antibodies against thyroid hormone receptors
and
, chick
ovalbumin upstream activator transcription factor, estrogen receptor,
glucocorticoid receptor, and even a monoclonal antibody against
RAR
did not affect the mobility of either complex (data not
shown). However, there is no support in the literature for higher order
RXR
RAR complexes on the
RARE. It is also possible that the
receptors could undergo posttranslational modification(s) upon
treatment with RA, which could cause epitope masking, or as yet
uncharacterized isoforms could be induced, but again, we unaware of any
studies giving credence to these hypotheses. Together, these data
indicate that, with respect to retinoid receptors, Complex I consists
mainly, if not completely, of RXR
and RXR
, whereas Complex II
at least contains RXR
RAR
heterodimers.
in Complex II, which should bind with higher affinity. At the
48 h time point, there may be enough RAR
protein produced that all
RXRs are titrated, such that Complex II completely replaces Complex I.
This interpretation is further strengthened by the results of the
Western blot shown in Fig. 4. Proteins from 10 µg of nuclear
extract of cells treated with RA for a period for up to 48 h were
separated by SDS-polyacrylamide gel electrophoresis, blotted onto
nitrocellulose membrane, and probed with a RAR
antiserum. A
specific band, migrating at about the expected mobility for RAR
,
is induced between 8 and 24 h of treatment. The kinetics of appearance
of the RAR
protein, therefore, match the observed DNA-binding
activity by RAR
in Complex II in the EMSA. Interestingly, as shown
before, the mRNA for RAR
can already be detected within 2 h of
treatment. Thus, according to our detection methods, translation of
RAR
mRNA lags at least 6 h behind transcription of the gene.
protein between 8 and 24 h of treatment
with RA. 10 µg of nuclear extract of cells treated for the
indicated time periods was electrophoresed through 10% SDS slabgel,
electroblotted onto nitrocellulose, and probed with RAR
antisera
(see ``Materials and Methods''). The arrow denotes
the position of the RAR
protein; the asterisk a
nonspecific band.
Chemical Modification Interference Assays of Complex I
and II Show Differential Protection of Bases in the
The RARE
RARE is an element with dyad symmetry, and
RARs
RXRs are known to bind as (homo/hetero)dimers. Our results
suggest that RXR
RXR
and RXR
RXR
homodimers, perhaps in combination with RXR
RXR
heterodimers, are the receptors that occupy the
RARE in untreated
cells. This is surprising, since this element is only very weakly bound
by RXRs in
vitro(21, 27, 28, 42, 43) ,
in contrast to RAR
RXR
heterodimers(17, 18, 20, 21, 22, 23) .
If this interpretation is true, it is possible that Complexes I and II
form different contact points with the bases in the
RARE
oligonucleotide. To test this hypothesis, we performed a chemical
modification interference assay of both complexes (time points 0 and 48
h) using dimethyl sulfate and KMnO
to identify protected G
and T residues, respectively. The results, shown in Fig. 5, show
that Complex I does not protect any T residues and only weakly protects
a few G residues at -41 in the coding strand and at -38,
-47, -49, -60, and -61 in the noncoding strand.
Except for the latter two bases, they appear to cluster around the two
half-sites in the probe (indicated by arrowsabove the sequence in Fig. 5B). In contrast, Complex II
gives a very clear and much stronger protection pattern of both G and T
residues. Particularly strong protection occurs at residues -41,
-52, and -53 in the coding strand and at -37,
-38, -48, and -49 in the noncoding strand. Weaker
protection is also observed between the two half-sites on this strand.
Thus, the contact sites for Complexes I and II are close to the two
established half-sites, but protection of bases is much stronger in
Complex II. These data are consistent with the hypothesis that there
are RXR dimers in Complex I that bind specifically, but with low
affinity (as reported for in vitro translated receptors) to
the
RARE, and that the RAR
RXR heterodimers in Complex II
bind with much higher affinity to this probe. In agreement with this,
the pattern of protection of this element by RAR
RXR
heterodimers in RA-induced P19 embryonic carcinoma (EC) cells is
essentially the same as that in Complex II (44) . Because it is
reasonable to assume that other types of RAR
RXR heterodimers
would also give strong protection, this argues against the possibility
that there are significant amounts of RARs in Complex I that escaped
our detection.
RAR
The above described results indicate that
RXR dimers mediate the retinoid-dependent induction of the RAR Gene Expression Is More Rapidly Induced by
9-cis-RA Than by RA
gene, instead of RAR
RXR heterodimers. This hypothesis can be
tested by comparing the effects of RA and 9-cis-RA on RAR
induction. RARs bind RA and 9-cis-RA with high affinity, but
RXRs bind 9-cis-RA with much higher affinity than
RA(45, 46) . If RAR
RXR heterodimers are
involved, it might be expected that the effects of RA and
9-cis-RA will be of comparable magnitude, because both ligands
activate RARs. If, on the other hand, RXR (homo)dimers are the
activating species, then it might be expected that 9-cis-RA
will generate a quicker response than RA. To test the hypothesis, in
this experiment the cells were grown in media that was stripped of
thyroid/steroid hormones to exaggerate the effects of both ligands. S91
cells still grow and differentiate in this media upon retinoid
treatment (data not shown). Total RNA was isolated from cells that were
untreated (0 h) or treated with RA or 9-cis-RA at the
indicated time points in Fig. 6. Fifteen µg of total RNA was
subjected to electrophoresis through an agarose gel, and analyzed by
Northern blotting (Fig. 6). The results show that, in cells
treated with RA, RAR
mRNA induction becomes apparent between 4 and
6 h, which is about 2 h slower than cells grown in regular serum. In
contrast, RAR
mRNA levels show a more rapid and potent increase
(between 2 and 4 h) after 9-cis-RA treatment. These results,
therefore, are supportive of our model of RXR-mediated RAR
induction by retinoids. The classic RA response (see Fig. 1) may
be due to metabolic conversion of RA to the 9-cis-RA
stereoisomer, which is how 9-cis-RA was
identified(45, 46) .
gene than RA. A, Total RNA (15 µg) from
cells, which were grown in thyroid/steroid hormone-depleted media and
treated with 10
M RA for the indicated time
periods, were subjected to electrophoresis, blotted, and hybridized
with the indicated probes on the left. B, quantitation of this
Northern blot by PhosphorImager scanning. The number of counts at time
point 0 (no RA treatment) for the RAR
probe was arbitrarily set at
1. All other values are normalized to this value. The cyclophilin probe
was used to normalize the data for loading/transfer
differences.
and RAR
and very small amounts, if any, of RAR
. Of
these RAR isoforms, only the latter is up-regulated by
retinoids(32, 34) , which we confirmed in this study.
We completed the inventory of retinoid receptors in these cells by
performing Northern blots with RXR-specific probes, and we showed that
S91 cells also constitutively express RXR
and RXR
, but no
RXR
.
appears to correlate with the induction of growth arrest. This
particular isoform has been suggested to have an important function in
neoplastic cells, i.e. its absence or aberrant expression has
been observed in many tumors and tumor cell lines and appears to
correlate with malignancy(3) . As a first step in understanding
the role of RAR
, we wished to determine which RARs and/or RXRs
mediate the retinoid-dependent activation of the RAR
2 promoter.
Although the RARE in this promoter has been well characterized (35, 36, 37) , the nature of the endogenous,
intracellular receptors that bind to this element has not been
established. We employed EMSA with the
RARE as probe incubated
with nuclear extracts of cells that were treated for various periods of
time with RA and analyzed the retarded complexes with a battery of RAR-
and RXR-specific antibodies. Our analysis shows a single retarded
complex (Complex I) in melanoma cells in their fully undifferentiated,
malignant state. This complex can be quantitatively supershifted with a
combination of antibodies against RXR
and RXR
, but it does
not react at all with two different RAR antibodies. This observation
can be most easily explained by assuming that this complex consists of
RXR
RXR
and RXR
RXR
homodimers or perhaps
in combination with RXR
RXR
heterodimers (this cannot
currently be determined). However, we cannot exclude the possibility
that other unknown nuclear protein(s) can form heterodimers with RXRs,
or could be part of the RXR dimer complex, although this has never been
reported to occur on the
RARE. In any case, our observations
suggest that binding to the
RARE, and, therefore, probably
transcriptional activation of the RAR
gene is largely mediated by
RXRs rather than RARs or RAR
RXR heterodimers.
RARE represents a DR5 element, which is
very poorly bound by RXR dimers in vitro but strongly bound by
RAR
RXR heterodimers, as illustrated in our chemical modification
interference assays. Since the RNAs for all three RAR isoforms
(including RAR
), and that of two RXRs can be detected by Northern
blot analysis, we wondered why we did not observe any RAR
RXR
heterodimers binding to the
RARE. The mechanism that we propose is
that in untreated S91 cells there is an excess of RXRs over endogenous
RARs such that RXR dimers are formed and are able to bind the
RARE
because there is no other competitor complex (the RAR
RXR
heterodimer) present. For instance, it was recently shown that in human
skin there is 5 times more total RXR than total RAR
protein(47) , and in S91 cells this difference could
conceivably be even higher. This explanation would be consistent with
published in vitro binding data, which show that RXR
homodimers can bind to the
RARE with low affinity; binding is only
enhanced if RARs are mixed in(21, 28, 42) .
This would also explain our observations in the EMSA in Fig. 2.
The RXR dimer complex (Complex I), which binds the
RARE in
untreated cells, remains virtually unchanged up to 24 h of RA
treatment, although the 8 h time point shows more intense binding. This
may be expected, as the RXR mRNAs are only weakly regulated by RA (Fig. 1). Meanwhile, RAR
mRNA is being transcribed and
translated, and RAR
protein begins to accumulate. Between 8 and 24
h of treatment with RA, RAR
RXR heterodimers become visible
as Complex II, and by 48 h of treatment, all endogenous RXRs are
titrated into the RAR
RXR heterodimer complex, leading to the
complete disappearance of Complex I. This would more satisfactorily
explain the abrupt change in complexes at the 48 h time point than
assuming that the RXRs are suddenly degraded or inactivated. This also
implies that Complex II not only consists of RAR
RXR
but
also contains RAR
RXR
heterodimers, since both RXRs were
identified in Complex II.
, is
inaccessible to our antibody in the heterodimer complex (see Fig. 3C), in contrast to that in the non-RAR containing
RXR complex (Fig. 3B). As our RXR
antibody
recognizes an N-terminal epitope and both types of RXRs can be expected
to form similar complexes with RARs, it probably explains why we did
not observe any supershifts with this antibody on Complex II but only
with Complex I. Again, we favor this explanation over that of assuming
that only RXR
somehow is no longer able to bind DNA between 24 and
48 h of treatment with RA. Interestingly, all RXR antibodies are able
to supershift quantitatively RAR
RXR heterodimers using
proteins that were obtained through the rabbit reticulocyte lysate
translation system, suggesting that either intracellular receptors are
different from their in vitro counterparts or contain other,
as yet unknown, nuclear partners. Also, the mobility of these in
vitro translated heterodimers is quite different from those of
Complex II (data not shown).
transcription is induced in
many cells, in particular EC cells. Is the same mechanism for
RXR-mediated activation that we propose here operative in those cells
as well? Minucci et al.(44) studied RA-induced
differentiation of P19 EC cells. In the absence of RA, very little
binding is observed to the
RARE in EMSA, with no protection of
residues using in vivo genomic footprinting techniques. In the
presence of RA, binding is strongly enhanced (with virtually identical
protection patterns as our Complex II), concomitant with induction of
RAR
gene expression. We propose that constitutively expressed RARs
(
and
) may be involved in the initial activation in a
heterodimer complex with RXR
. However, without the use of
antibodies, this has not yet been proven conclusively. It is also
interesting, that they too observe similar binding of two complexes to
the
RARE, of which only the lower complex (corresponding to our
Complex II) increases significantly in intensity after RA treatment.
Thus, RXRs might also play some role in activation of the RAR
gene
in P19 EC cells. The observation that, in RAC65 cells that are
RA-resistant P19 EC cells due to a truncation of the RAR
gene (48) or in F9 EC cells in which the RAR
gene has been
deleted(49) , RA-induced RAR
expression is virtually
unchanged is certainly not at odds with this interpretation. On the
other hand, Clifford et al.(32) , who used synthetic
retinoids like
4-[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)1E-propenylbenzoic
acid (TTNPB) and
[4-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl)2-naphtalenylcarbamoyl]benzoic
acid (Am80), which are specific for RARs, showed induction of the
RAR
gene in S91-C2 cells, although only treatment with TTNPB
reached the same induction level as treatment with RA. A possible
explanation for this apparent discrepancy may be that at the very high
doses that were used (1 µM), these retinoids are less
specific in their activities and also activate RXRs.
RXR heterodimers present in
untreated cells, which are below our detection levels and which may be
able to activate the RAR
gene. Also, it is even possible that
Complex I contains RAR
RXR heterodimers with inaccessible
epitopes, perhaps due to posttranslational modifications or the
association of (unknown) additional protein(s). However, it appears
that RXRs are the key mediators of retinoid-induced transcription of
the RAR
gene in S91 cells. Nonetheless, it would be interesting to
look at the kinetics of induction by TTNPB and Am80 compared with
9-cis-RA as we did between RA and 9-cis-RA in Fig. 6. Also, the availability of new RXR-specific retinoids
would be helpful to these studies(50) .
mRNA levels plateau after about 24 h of
treatment with RA (Fig. 1), whereas between 24 and 48 h of
treatment relatively large amounts of RAR
RXR heterodimers
begin to appear (Fig. 2), and the concentration of RAR
protein also continues to increase (Fig. 4). Thus, if
heterodimers completely replace RXR dimers by 48 h of treatment, one
could expect an increase in transcription between 24 and 48 h of
treatment, since the
RARE is much more potently activated by
RAR
RXR heterodimers than RXR dimers. A possible explanation for
this phenomenon may be that the cells have already differentiated to a
certain extent by 24 h of treatment, such that they no longer sustain
transcription of certain genes. A similar phenomenon was observed in
embryonic carcinoma cells. After RA-induced differentiation, an
adenovirus EIA
-like activity, which is required for
RAR
expression, disappears(51, 52, 53) .
For instance, we found that the DNA-binding activity of octamer binding
factor 1 (Oct1) disappears between 8 and 24 h of treatment with RA (not
shown). It is conceivable that Oct1, as well as other nuclear factors,
plays an important role in these processes, and may contribute to the
decline or transcriptional arrest of the RAR
gene after 24 h of RA
treatment.
RARE takes place after treatment with retinoids
in S91 cells. We believe that in the melanoma state (untreated cells),
RXRs alone occupy the
RARE, and are mainly responsible for
transcriptional activation of the RAR
gene. Between 24 and 48 h of
treatment, when the cells have essentially been converted to a benign,
melanocytic state, there appears to be a switch in receptor occupancy
so that the RXR dimer complex is replaced by a RAR
RXR
heterodimer. It will be interesting to see whether similar switches
also occur at other RAREs and whether our observations also apply to
other cell types.
(H)
antibody, and Dr. Benjamin Neel for the RAR antibodies.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.