(Received for publication, June 5, 1995; and in revised form, October 11, 1995)
From the
Cellular responsiveness to retinoic acid and its metabolites is
conferred through two distinct families of receptors: the retinoic acid
receptors (RARs) and the retinoid X receptors (RXRs). Herein, we report
on the identification and characterization of several conformationally
restricted retinoids, which selectively bind and activate RX receptors.
Under the influence of retinoids, HL-60 myelocytic leukemia cells
differentiate into granulocytes. This effect is mediated by RAR,
as has been demonstrated through the use of a selective RAR
antagonist (Apfel, C., Bauer, F., Crettaz, M., Forni, L., Kamber, M.,
Kaufmann, F., LeMotte, P., Pirson, W., and Klaus, M.(1992) Proc.
Natl. Acad. Sci. U. S. A. 89, 7129-7133). Here, we show that
conformationally restricted RXR-specific retinoids, at doses that are per se inactive, are able to potentiate by up to one order of
magnitude the pro-differentiating effects of all-trans retinoic acid and an RAR
-selective synthetic retinoid. We
also present evidence that these RXR-selective ligands are able to bind
to a DNA RXR
RAR heterodimer complex. This finding demonstrates
that agonists for RARs and RXRs can synergistically promote HL-60
differentiation, which could be mediated through a heterodimer of these
receptors.
A subclass of nuclear hormone receptors have been described,
termed retinoid X receptors
(RXR)()(2, 3, 4) , which are
ligand-inducible transcription factors responsive to the 9-cis isomer of retinoic acid(5, 6) . A related
subclass of receptors, the retinoic acid receptors
(RAR)(7, 8, 9, 10, 11, 12) ,
also responds to 9-cis retinoic acid (9-cis RA), as
well as to the all-trans isomer of retinoic acid (t-RA). A
striking feature of RXRs is their ability to heterodimerize with
several members of the steroid receptor superfamily, including the RA
receptors, thyroid receptors, peroxisome proliferator-activated
receptors, vitamin D receptor, and chicken ovalbumin upstream
promoter-transcription factor (13, 14, 15, 16, 17, 18, 19, 20, 21) .
Growing evidence suggests that heterodimers are the active species for
binding to promoter response elements.
In view of the involvement of RXRs in multiple signaling pathways, RXR-selective ligands can possibly be expected to show medical usefulness through their modulation of the above mentioned and perhaps also other hormone-regulated processes. The pharmacological effects of RXR ligands, which are attributable solely to activity on RXRs, are difficult to assess using 9-cis RA as an activating ligand since 9-cis RA also functions as an RAR ligand and consequently elicits the full range of classical retinoid effects. Therefore, to gain deeper insight into RXR function as well as to evaluate their pharmacological usefulness, we have designed and characterized a number of RXR-selective compounds and report on some intriguing pharmacological properties. These compounds are sterically restricted analogues of 9-cis RA, which cannot isomerize. They exhibit high potency and high selectivity for RXRs versus RARs and belong to different structural classes than other RXR-selective compounds recently reported(22, 23, 24) . We have characterized these compounds with regard to their binding affinity to RXRs and RARs, to their transactivation potency of these receptors in transfected cells, and to their ability to induce DNA binding of RXR receptors to RXR response elements.
HL-60 is a human
myeloid leukemia cell line, which is exquisitely sensitive to
retinoid-induced differentiation(25) . The predominant
expression of RAR in these cells suggests that retinoids act
through this receptor(26, 27) . We have recently found
additional evidence supporting this assumption by the use of an
RAR
-selective antagonist, which was able to suppress
retinoid-induced differentiation (1) . Therefore, we have used
HL-60 as a model system to evaluate the role of RXR in
RAR
-mediated biological responses. We now show that these new
RXR-selective agonists synergistically enhance the differentiation of
HL-60 cells induced by all-trans RA or an RAR
-selective
retinoid.
An RAR/RXR reporter gene was constructed using a synthetic
oligonucleotide containing three copies of the RAR response element
from the RAR promoter (33) in front of the basal promoter
of TK and the luciferase coding region in the plasmid pGL2-basic
(Promega)
RARE-TK-luc. COS-1 cells were transiently transfected as
described above, and at the end of incubation the cells were assayed
for luciferase activity.
For transfection experiments in Drosophila cells (Schneider SL3), human RXR was expressed
using the Drosophila
-actin promoter(34) . Two
RXR reporter systems were constructed using synthetic oligonucleotides,
one containing three copies of the RXR response element from the rat
CRBP II gene (35) or the other containing three copies of the
RARE response element from the
RAR gene, each with the alcohol
dehydrogenase gene promoter (2) cloned in front of the
luciferase coding region in the plasmid pGL2-basic (Promega). The
transfection was performed by the calcium phosphate-DNA coprecipitation
method(32) , using 5 µg of total plasmid DNA (RXRE
reporter, RXR
expression vector = 1:10) per well of a
6-well dish in Dulbecco's modified Eagle's medium plus 15%
FCS. 18 h later, the cells were replated on 96-well plates, and
3-4 h later, various concentrations of retinoids were added to
duplicate wells. At the end of incubation (36-48 h), the cells
were assayed for luciferase activity.
The SeAP assays were performed
as described in detail previously(1) . For the luciferase assay
the medium was aspirated, and 40 µl of lysis buffer was added (25
mM Tris-phosphate, pH 7.8, 2 mM dithiothreitol, 2
mM CDTA, 10% glycerol, and 1% Triton X-100). After 15 min
incubation at room temperature, 20 µl were transferred into a
96-well plate, and the reaction was triggered by adding 100 µl of
luciferin buffer (270 µM CoA, 470 µMD-luciferin, 530 µM ATP, 20 mM tricine,
1.07 mM Mg(CO), 2.67 mM MgSO
,
0.1 mM EDTA, 33.3 mM dithiothreitol, adjusted pH
7.8).
The luminescent reaction products were measured in a 96-well
luminometer (Luminoscan, Flow). Transcription activation is expressed
as percent receptor activation, with 100% representing the activity
observed with RARs in the presence of 10M t-RA (3-8-fold induction) and 100% defining the RXR
activity observed in the presence of 10
M 9-cis RA respectively (CRBP II, 10-15-fold
induction:
RARE, 100-500-fold induction). Duplicate values
varied by 5-10%. Two to four dose-response experiments were
performed. The retinoid concentration giving half-maximal effect in
transactivation (EC
) was determined from the plots.
Reproducibility was found to be within a factor of 2.
To determine the activity of these compounds toward RXRs and RARs we
have measured their binding to the receptors in competition binding
assays. Fig. 1A shows competitive binding on RXR
using
H-9-cis RA as tracer. Ro 48-2250 and Ro
47-8652 compete nearly as well as unlabeled 9-cis RA for
binding to RXR
, whereas Ro 47-5944 is severalfold less potent. In
contrast, t-RA and Ro 40-6055 (Table 1) exhibit virtually no
binding to RXR
. On RAR
, the three synthetic compounds show
only marginally detectable binding in a competition assay with
H-t-RA as tracer, while t-RA and 9-cis RA exhibit
potent binding (Fig. 1B).
Figure 1:
Competition binding of t-RA, 9-cis RA, and synthetic retinoids to RXR (A) and RAR
(B). The radioligand was
H-9-cis RA for
RXR
and
H-t-RA for RAR
. Percent bound radioligand
is plotted against the log of the concentration of the retinoid, t-RA
(
), 9-cis RA (
), Ro 47-5944 (
), Ro 47-8652
(
), and Ro 48-2250 (
). Results of one representative
experiment are shown.
To test the ability of
these compounds to transactivate receptor-responsive promoters, we used Drosophila SL3 cells as a cellular system. It has been shown
in the literature that the RARE of the rat cytoplasmic retinol binding
protein gene (CRBP II) is only activated by RXR homodimers while the
RARE of the RAR gene is activated by RAR-RXR heterodimers as well
as by RXR homodimers(20) . Only after cotransfection of the
RXR
expression vector and different reporter constructs (CRBP
II-adh-luciferase or
RARE-adh-luciferase) does 9-cis RA
show a dose-dependent activation of the reporter gene (Fig. 2, A and B). All three compounds showed good activity,
being nearly as active as 9-cis RA on both response elements.
Using a palindromic thyroid hormone response element-luciferase
reporter gene, which is activated by either RAR
RXR heterodimers
or RXR homodimers (20) , we found the same results (data not
shown). For an RAR-selective retinoid (Ro 13-7410, TTNPB(38) ),
no activation was detectable on all three tested response elements
(data not shown).
Figure 2:
Transactivation dose-response curves for
RXR and RAR
-ER. A, activation of the
RARE-adh-luciferase reporter gene. B, activation of the
CRBP II-adh-luciferase reporter gene. Schneider SL-3 cells
cotransfected with the corresponding reporter gene and an RXR
expression plasmid were treated with various concentrations of
retinoids; 2 days later, they were assayed for luciferase activity. C, activation of the vit-TK-SeAP reporter gene. COS-1 cells
cotransfected with a vit-TK-SeAP reporter gene and the chimeric
RAR
-ER expression plasmid were treated with various concentrations
of retinoids; 2 days later, they were assayed for SeAP activity. t-RA,
; 9-cis RA,
; Ro 47-5944,
; Ro 47-8652,
; Ro 48-2250,
. Results of a representative experiment are
shown.
Transactivation measured using chimeric RAR-ERs in
COS cells showed that these three compounds are roughly three orders of
magnitude less active on RARs than t-RA or 9-cis RA. Fig. 2C shows as an example dose-response curves of
9-cis RA, t-RA, and the three compounds on RAR-ER
transactivation. Table 1summarizes activation and binding data
of these compounds to RAR
, -
, and -
and to RXR
. As
can be seen from these values, the three RXR-selective compounds show
approximately a 100-fold preference for RXR versus RAR in
transactivation.
Figure 3:
DNA-dependent ligand binding assay. Same
molar ratios of RAR and RXR
(A) or molar ration of
0.1/1 (B) were incubated with biotinylated oligonucleotides
containing the
RARE (DR5). Incubations were performed in the
presence of
H-t-RA (20 nM) (black bars),
H-9-cis RA (20 nM) (white bars),
or
H-9-cis RA (20 nM) plus Ro 13-7410
(1000 nM) (striped bars) with the addition of the
indicated unlabeled ligands (2000
nM).
In a second experiment, we
dropped the ratio of RAR to RXR from 1 to 0.1. Under these conditions,
the binding of H-t-RA fell to near background levels (data
not shown). This result suggests that under these conditions the RXR
homodimer and not the heterodimer is preferentially, if not
exclusively, bound to the DNA. This interpretation was supported by
binding studies using
H-9-cis RA with and without
Ro 13-7410 (Fig. 3B). As expected for an RXR homodimer
complex, we see nearly complete competition with RXR-selective ligands.
Figure 4:
Transactivation of RARRXR
heterodimer using the
RARE-TK-luc reporter gene. CV-1 cells were
cotransfected with a
RARE-TK-luc reporter gene and the empty
expression vector pSG5 or the indicated receptor expression plasmid for
RAR
and RXR
, treated with constant concentrations of
retinoids; 2 days later, they were assayed for SeAP activity. Lane
1, Me
SO; lane 2, 9-cis RA (100
nM); lane 3, Ro 13-7410 (100 nM); lane
4, Ro 48-2250 (100 nM); lane 5, Ro 47-5944 (100
nM); lane 6, Ro 48-2250 (100 nM) + Ro
13-4710 (10 nM); lane 7, Ro 47-5944 (100 nM)
+ Ro 13-4710 (10 nM). Results of one representative
experiment are shown.
Figure 5:
Ligand effects on the binding of RXR
to the RXR response elements (CRBP II and
RARE). In vitro translated RXR
(first column (=) contains no RXR
)
was preincubated with the indicated retinoids or Me
SO
alone(-) and then tested for DNA binding activity in the gel
retardation assay. A double-stranded oligonucleotide containing (A) the CRBP II sequence or (B) the
RARE
sequence was used as probe. Where indicated, a 100-fold molar excess of
unlabeled CRBP II or
RARE or estrogen response element (ERE) was added.
indicates the specific RXR homodimer
complex,
marks nonspecific band observed also with unprogrammed
reticulocyte lysate.
Figure 6:
Induction of differentiation of HL-60
cells by retinoids. OD is proportional to the number of
differentiated HL-60 cells. t-RA,
; RAR
-selective retinoid,
Ro 40-6055;
, Ro 47-5944;
, Ro 47-8652 (
); Ro
48-2250,
. Results of one representative experiment are
shown.
Figure 7:
Induction of differentiation of HL-60
cells by combination of retinoids. OD is proportional to
the number of differentiated HL-60 cells. Panels A, B, and C show dose-response curves with t-RA in
combination with a constant concentration, as indicated, of
RXR-selective retinoids; panels D, E, and F show dose-response curves with the RAR
-selective retinoid Ro
40-6055 in combination with a constant concentration, as indicated, of
RXR-selective retinoids. Results of one representative experiment are
shown.
In this report, we have presented three synthetic retinoids
that function as RXR-selective ligands. All three compounds contain the
polyene side chain in a 9-cis-like fixed configuration through
the introduction of a ring system. Since 9-cis RA is the
natural RXR ligand, these analogs were planned to mimic structural
features of 9-cis RA, and indeed they do show nearly equal
potency on RXR as compared to 9-cis RA. Gratifyingly,
even though 9-cis RA is also a ligand for the RARs, these
synthetic compounds exhibit virtually no activity on RARs in binding or
transactivation assays. This could be indicative of a distinctly
different retinoid binding pocket in RARs versus RXRs. It
could, on the one hand, imply that the binding pocket in RARs cannot
accept the additional size of a ring system at this position of the
side chain, although with the introduction of a three-membered ring in
Ro 48-2250, the size of the molecule is only minimally increased.
Alternatively, 9-cis RA may adopt different conformations in
binding to RARs and RXRs, and the RAR-required conformation may not be
accessible to these ring-constrained synthetic derivatives. As a third
possibility, mutual isomerization of the natural ligands t-RA and
9-cis RA under the assay conditions cannot be rigorously
excluded.
The activity of these RXR-selective compounds is evidenced
first by their binding affinity for RXR, which is nearly as high
as that of 9-cis RA (Fig. 1, Table 1). Second,
these compounds can induce the formation of RXR homodimers on RXR
response elements (Fig. 5). Third, these derivatives show potent
transactivation of RXR reporter genes (Fig. 2). Fourth, these
retinoids are able to potentiate the RAR
RXR heterodimer-mediated
transactivation by the RAR-selective retinoid 13-7410 (Fig. 4).
Taken together, these results suggest appropriate ligand-induced
conformational changes of the receptor, leading to RXR transcriptional
activity.
Furthermore, these selective ligands can be used for
exploring the functions of RXR. Since RXR is known to heterodimerize
with RAR, we have tested the influence of these compounds on a retinoic
acid-mediated process, HL-60 differentiation. Published reports suggest
that RAR is critical in mediating this
differentiation(1) . The RXR ligands per se exhibit
EC
concentrations higher than 100 nM in inducing
HL-60 differentiation (Fig. 6). This implies that liganded RXR
receptors are not sufficient to promote induction of HL-60 cells and
suggests that liganded RARs are necessary for this process. The weak
effect of the RXR-selective retinoids can be explained by their
residual RAR-activating activity (Fig. 2B). t-RA, a
potent ligand for RARs, induces HL-60 differentiation with an EC
of about 0.6 nM. When t-RA is added to cultures at a
concentration of 0.1 nM, essentially no differentiation is
induced. When cells are treated with 0.1 nM t-RA plus 100
nM of an RXR-selective ligand, substantial induction of
differentiation is obtained (Fig. 7, A-C). These
effects are somewhat more pronounced if a preferential RAR
ligand
(Ro 40-6055) rather than the unselective t-RA is used (Fig. 7, D-F).
From the results obtained in these combination
experiments, it is obvious that the effects of RXR-selective ligands on
HL-60 differentiation are more than additive and therefore cannot be
explained solely by their residual RAR-activating effects. This
indicates that RX receptors, in the presence of their ligand, are able
to enhance an RAR-mediated process. This could well be through the
activity of an RARRXR heterodimer in the presence of both
receptor ligands. In support of this hypothesis, we have presented
evidence (Fig. 3) that both ligands do indeed bind to a
heterodimer complex on a DNA response element (
RARE) and show
together enhanced transactivation of this element in transient
transfections of a reporter gene (Fig. 4). A recent study has
demonstrated by direct ligand binding experiments that within an
RXR
RAR heterodimer bound to a DR5 or DR1 response element, RXR is
excluded from binding its ligand(39) . These data appear to
contradict our conclusion that both ligands are able to bind such a
complex and would support alternative hypotheses, for example, that two
independent RAR and RXR pathways exist, and only the activation of both
pathways leads to a maximal response. There may be several reasons why
Kurokawa et al.(39) did not see binding of an RXR
ligand in the heterodimer. One reason may be that the synthetic
compound they used is unable to bind in the heterodimer, whereas the
new ligands we describe here are accessible to the heterodimer. In
addition, it is not clear exactly which response element they use, and
it is well possible that on one response element the RXR ligand binding
is blocked but not on another.
The fact that the addition of both an
RAR and RXR ligand is apparently able to activate better than either
alone has also been reported by two other groups. Durand et al.(40) could show that activation on an RARRXR
heterodimer response element (
RARE, DR5T-TK/CAT) in HeLa cells is
more than additive. They found a poor activation by an RXR-selective
ligand, a moderate activation by t-RA (not a very RAR-selective
retinoid) and a synergistic activation by both ligands, to the same
level as 9-cis RA alone. Lotan et al.(41) describe that a combination of suboptimal
concentrations of RAR-selective retinoids with RXR-selective retinoids
shows more than additive effects on the inhibition of cervical
carcinoma cell proliferation.
The fact that these RXR ligands can enhance a biological response mediated by a known heterodimer partner, RAR, may serve as an indication that they could also be useful for enhancing processes controlled by other receptor partners, such as thyroid receptors, vitamin D receptor, or peroxisome proliferator-activated receptors, as has also been suggested by combination experiments with 9-cis RA(18, 42) . With a view to clinical usefulness, the RXR-selective ligands could, by increasing the potency of partner receptor ligands, allow lower doses to be administered and thus, by reducing the side effects, provide superior treatment regimens for the various indications where such compounds are commonly used.