(Received for publication, March 16, 1995; and in revised form, May 25, 1995)
From the
Two retinoic acid (RA) receptors, retinoic acid receptors (RARs)
and retinoid X receptors (RXRs), have been identified.
All-trans-RA and its 9-cis-isomer are ligands for
RARs, but only 9-cis-RA binds RXRs with high affinity.
Activation-induced T cell hybridoma death is mediated via the
engagement of Fas by activation-up-regulated Fas ligand, and RA
prevents this type of apoptosis by inhibiting the induction of Fas
ligand expression. To investigate the mechanism of RA action, T
hybridoma cells were transfected with cDNA encoding RXR
Activation-induced apoptosis of immature thymocytes is an
important means of eliminating potentially autoreactive T
cells(1, 2, 3, 4) . Much of our
understanding of the molecules and pathways involved in this phenomenon
comes from studies with murine T cell hybridomas, which are made by
fusing normal peripheral T cells with a thymoma cell line. Activation
of T cell hybridomas via the antigen-specific T cell receptor rapidly
induces a G Retinoic acid (RA) ( In this report the
nature of the RA receptors involved in preventing activation-induced
apoptosis is explored in two ways. First, T cell hybridomas that
overexpress wild type or a dominant-negative form of RXR
Figure 1:
Western blot analysis of RXR
Figure 2:
Transactivation of an RA responsive
element-driven reporter. A, 10
Luciferase reporter constructs were also used to
evaluate the effect of RXR
Figure 3:
Anti-CD3 stimulation induction of IL-2,
growth inhibition, and cell death. CMVV, RXR
Figure 4:
Inhibition of activation-induced apoptosis
by 9-cis RA in RXR
Figure 5:
Prevention of activation-induced apoptosis
of T cell hybridomas by natural and synthetic retinoids.
[
Figure 6:
Effects of RAR- and RXR-selective
retinoids on activation-induced apoptosis.
[
Figure 7:
Regulation of FasL expression by
retinoids. CMVV, RXR
Figure 8:
Occupancy of RXRs and RARs efficiently
prevents activation-induced FasL induction. 2B4.11 cells were activated
with plastic-coated 2C11 (1 µg/well) in the presence of varying
concentrations of the indicated retinoids.
[
When occupied, steroid hormone receptors bind to specific DNA
responsive elements as homodimers and regulate gene expression. The
responsive elements of these receptors are symmetric, consisting of
palindromic repeats of core recognition motifs
(CRMs)(23, 47) . The situation is more complicated for
RA and its receptors(24, 47) . RA responsive elements
usually contain asymmetric direct repeats of CRMs separated by one,
two, or five base pairs (DR-1, DR-2, and DR-5). In vitro studies have shown that RARs and RXRs preferentially bind to all
of these responsive elements as RAR Fas is a
transmembrane protein whose ligation by ligand or antibodies leads to
apoptosis(52, 53) . Fas-mediated apoptosis is not
inhibited or delayed by protein and RNA synthesis
inhibitors(54) . lpr and gld mice have
defects in Fas and FasL, respectively(55, 56) , and
both develop lymphoproliferation and autoimmunity(57) .
Peripheral T cells from these mice did not undergo activation-driven
apoptosis(58, 59, 60) . We have recently
shown that 2B4.11 cells, like thymocytes and many transformed cell
lines, constitutively expresses Fas on their surface(18) . As
is the case for other T cell hybridomas (16, 17) ,
activation of 2B4.11 cells results in rapid of FasL, which in turn
engages Fas and leads to cell death. 9-cis-RA prevents
activation-induced FasL mRNA up-regulation and, therefore, apoptosis.
Here we show that the ability of different retinoids to inhibit FasL
expression correlates with their activity in preventing apoptosis, and
a dominant-negative RXR
or
dominant-negative RXR
. Cells that overexpressed RXR
were more
sensitive to 9-cis-RA rescue from activation-induced death
than cells transfected with vector alone. In contrast, cells expressing
the dominant-negative RXR
could not be rescued from death with
9-cis-RA. In wild type cells, an RAR-selective synthetic
retinoid had little effect on activation-induced apoptosis, while an
RXR-selective agonist prevented apoptosis but only at concentrations
about
10-fold greater than that required for 9-cis-RA.
Simultaneous addition of the RAR- and RXR-selective retinoids
completely prevented activation-induced apoptosis at concentrations
where either alone had relatively little protective effect. The same
hierarchy of efficacy was found for activation-induced Fas ligand
expression. These data demonstrate that binding of both RARs and RXRs
is required for efficient inhibition of activation-induced Fas ligand
up-regulation and T cell apoptosis by retinoic acid.
/S cell cycle block(5) , followed in
4-6 h by apoptotic death(6, 7) .
Activation-induced death can be blocked at different levels by a number
of independently acting agents. Immunosuppressive drugs like
cyclosporin A and FK 506(8, 9, 10) prevent
activation-induced apoptosis, presumably by inhibiting calcineurin
activity and thus interfering with T cell receptor-mediated
signaling(11, 12) . Unexpectedly, glucocorticoids,
which are themselves potent inducers of apoptosis in T cell hybridomas
and immature thymocytes, prevent the ability of T cell receptor
ligation or occupancy to induce apoptosis (mutual
antagonism)(13, 14, 15) . The mechanism of
action of these inhibitors of apoptosis has recently been clarified by
the finding that the interaction between Fas (also known as APO-1 or
CD95) and the activation-up-regulated ligand for Fas (FasL) signals the
cells to undergo apoptosis(16, 17, 18) . Both
CsA (19) and glucocorticoids (18) prevent
activation-induced up-regulation of FasL.
)also blocks activation-induced apoptosis of T cell
hybridomas and thymocytes, although unlike glucocorticoids RA itself
does not cause apoptosis(20, 21) . Moreover, retinoic
acid inhibits the ex vivo apoptosis of peripheral blood
lymphocytes from patients infected with human immunodeficiency
virus(22) . We have recently found that the ability of RA to
prevent activation-induced apoptosis is due to its inhibition of
activation-induced up-regulation of FasL(18) . Two RA receptor
types, RARs and RXRs, have been identified by gene cloning. Both of
them belong to the nuclear receptor superfamily and are
ligand-regulated transcription
factors(23, 24, 25) . RARs and RXRs exist as
at least three different subtypes, designated
,
, and
,
and at this time no clear functional differences between these subtypes
have been established. Although formation of RXR
RXR homodimers in
the presence of 9-cis-RA or RXR-selective retinoids has been
reported (26, 27) , the RA receptors bind
preferentially to responsive elements as RAR
RXR heterodimers and
thereby transactivate gene expression (28, 29, 30, 31, 32, 33) .
RXRs can also form heterodimers with other nuclear receptors, such as
those for vitamin D
and thyroid hormone, and transactivate
via the corresponding responsive
elements(34, 35, 36, 37) .
Furthermore, while both all-trans-RA and its stereoisomer
9-cis-RA are ligands for RARs, only 9-cis-RA binds
RXRs with high affinity(38, 39) . Since
9-cis-RA was found to be approximately 10-fold more potent
than all-trans-RA in preventing activation-induced apoptosis
of T cell hybridoma and thymocytes, we concluded that RXRs play a
critical role in this process(21) .
were
studied. Second, synthetic retinoids that are selective RAR and/or RXR
agonists were used. The results demonstrate that RXRs are required for
RA inhibition of Fas ligand expression and T cell apoptosis. However,
an RXR-selective retinoid is only a moderately good inhibitor. Ligands
that bind to both RXRs and RARs, or the combination of an RAR-selective
ligand plus an RXR-selective ligand, block activation-induced
up-regulation of FasL and cell death most efficiently. Therefore, both
RXRs and RARs are involved in prevention of activation-induced T cell
apoptosis by retinoids.
Cells and Reagents
2B4.11 is a murine T cell
hybridoma specific for peptide 81-104 of pigeon cytochrome c. The mouse leukemia cell lines L1210 and L1210-Fas, which
express very low and high levels of Fas on the cell surface,
respectively, were kindly provided by Dr. P. Golstein (Centre
d'Immunologie de Marseille-Luminy, Marseille, France). Cells were
cultured and assayed in RPMI 1640 (Biofluids Inc., Rockville, MD)
supplemented with 10% heat-inactivated fetal calf serum, 4 mM glutamine, 100 units/ml penicillin, 150 µg/ml gentamicin, and
5 10
M 2-mercaptoethanol.
145-2C11 (2C11) is a hamster anti-mouse CD3-
monoclonal
antibody and was purified from culture supernatant by affinity
chromatography over a Protein A-Sepharose column(40) .
All-trans-RA was purchased from Sigma. 9-cis-RA and
synthetic retinoids TTNPB (41) and LGD1069 (27) were
produced by Ligand Pharmaceuticals (La Jolla, CA). Retinoid 351 was
obtained from Roche (Nutley, NJ).
Plasmid Constructs and Stable
Transfection
Expression vectors containing mouse RXR and
its DNA-binding domain deletion mutant (DBD
) were
constructed by cloning the corresponding cDNA into the EcoRI
site of plasmid pCXN2, which has a cytomegalovirus enhancer and
-actin promoter to drive the expression of inserted
genes(31, 42) . The pRARE-Luc reporter construct was
kindly provided by Dr. Elwood Linney (Duke University, Durham, NC). The
pCRBP II-Luc was constructed by inserting two copies of the RA
responsive elements from rat cellular retinol-binding protein type II
gene upstream of the tk promoter and luciferase gene in
plasmid pW1(31, 43) . To obtain stable transfectants,
2B4.11 cells were transfected with pCXN2, pCXN2-RXR
, or
pCXN2-DBD
by electroporation, seeded into 96-well
tissue culture wells, and selected with geneticin (Life Technologies,
Inc.).
Immunoblotting
Nuclear extracts were prepared as
described (44) , separated on 10% SDS-polyacrylamide gel,
transferred to nitrocellulose, and immunoblotted with the anti-RXR
monoclonal antibody MOK13.17(37) .
DNA Fragmentation Assay
DNA fragmentation of
2B4.11 cells or transfectants was quantitated by using the principle
that high molecular weight DNA adheres to fiberglass filters while
fragmented DNA passes through(21) . Briefly, cells were
incubated with [H]thymidine (5 µCi/ml) for 3
h. After thorough washing, 5
10
cells were
distributed in triplicate into flat bottomed 96-well microtiter plates
and treated with the indicated reagents. After 16 h of culture at 37
°C, the cells were hypotonically lysed and harvested onto
fiberglass filters, and the activity on the filters was determined by
scintillation counting. The results are expressed as specific DNA
fragmentation: ((M - E)/M)
100%, where M is the cpm for cells cultured in medium and E is
the cpm for cells cultured under experimental conditions. For
experiments in which different cells were compared, the data are
expressed as percentage of maximum specific DNA fragmentation, where
activation-induced specific DNA fragmentation in medium alone was
designated as 100%. For the measurement of functional Fas ligand
expression, L1210 or L1210-Fas cells were labeled with
[
H]thymidine and cultured with effector T cell
hybridomas that had been stimulated with anti-CD3 for 4 h in the
presence or absence of retinoids. The cells were harvested after
incubation for another 4 h.
Transient Transfection and Luciferase Assay
T cell
hybridomas were transfected with pRARE-Luc or pCRBP II and
pCXN2-RXR by electroporation. Transfection efficiency was
determined by cotransfection with pCMV-
-galactosidase, and its
activity was measured with the Tropix substrate and amplifiers (Tropix,
Bedford, MA). Transfected cells were cultured for 6-8 h and
harvested. Luciferase activity was determined as relative fluorescence
units with Promega (Madison, WI) luciferase assay substrates and a
Monolight 2010 (Analytical Luminescence Laboratory, San Diego, CA).
Functional Assays
Anti-CD3-induced growth
inhibition of T cell hybridoma was measured by determining
[H]thymidine incorporation into DNA as
described(21) . IL-2 production by anti-CD3-stimulated T cell
hybridomas was quantitated with IL-2-dependent CTLL-2
cells(45) . Cell viability was assessed by trypan blue
exclusion and light microscopy.
Stable Transfection of RXR
The observation that
9-cis-RA is more potent than all-trans-RA in
preventing activation-induced apoptosis of T cell hybridomas implicated
the RXR in this phenomenon. To test this possibility, 2B4.11 T
hybridoma cells were transfected with pCXN2-RXR and a Dominant-negative
RXR
into 2B4.11 Cells
or
pCXN2-DBD
in order to overexpress wild type RXR or
express a dominant-negative form of RXR. The latter was created by
deleting the DNA-binding domain of RXR
. The resulting molecule is
able to heterodimerize with wild type RAR, but the dimer fails to bind
to RA responsive elements efficiently(31) . 2B4.11 T hybridoma
cells were also transfected with the empty expression vector (pCXN2) as
a control. G418-resistant clones were analyzed by immunoblotting for
expression of the transfected genes. Nuclear extracts from 2B4.11 cells
transfected with vector alone (CMVV) had low levels of RXR
in the
nuclear extract, whereas a G418-resistant clone that had been
transfected with pCXN2-RXR
(RXR
WT) expressed high levels of
RXR
(Fig. 1). Two G418-resistant cells that had been
transfected with pCXN2-DBD
(RXR
DN1 and DN2)
expressed high levels of the dominant-negative RXR
. As expected,
due to the internal deletion, the molecular mass of the
dominant-negative RXR
was 39.5 kDa compared with 45 kDa for the
wild type molecule.
WT
and dominant-negative RXR
in transfected 2B4.11 cells. Nuclear
extracts were made from 5
10
2B4.11 cells
transfected with vector alone (CMVV), RXR
WT, or dominant-negative
RXR
(RXR
DN1 and DN2). After separation by SDS-PAGE, proteins
were transferred to membranes, blotted with an anti-RXR
monoclonal
antibody, and developed with
I-labeled goat anti-mouse
antibody.
Transactivation of Reporter Genes in Wild Type and
Dominant-negative RXR
The RA responsive
element for cellular retinol-binding protein type II responds to RA
when cells are co-transfected with RXRs but not with RARs(43) .
We attempted to study the effect of the RXR Transfected Cells
dominant-negative
molecule by transfecting CMVV cells with the pCRBP II-Luc reporter
construct. As for many cell lines(31, 43) , no
increase of luciferase activity was observed in response to
9-cis-RA, presumably because of insufficient levels of RXR
expression, but in RXR
-overexpressing RXR
WT cells luciferase
activity was induced about 2-3-fold (data not shown). To boost
the signal so that the effect of the dominant-negative RXR
could
be evaluated, CMVV, RXR
DN1, and RXR
DN2 cells were
cotransfected with pCRBP II-Luc and the RXR
-expression construct
pCXN2-RXR
. One of four representative experiments is shown in Fig. 2A. Luciferase activity was highly induced by
9-cis-RA in CMVV cells. In contrast, induction of luciferase
was poor in RXR
DN1 and DN2 cells, requiring 10-30-fold more
9-cis-RA to achieve levels of activity comparable with that in
CMVV cells.
CMVV, RXR
DN1,
and RXR
DN2 cells were cotransfected with pCRBP II-Luc (10 µg)
and pCXN2-RXR
(10 µg). Two h later the concentrations of
9-cis-RA were added, the cells were incubated for another 6 h,
and luciferase activity was measured. B, 10
CMVV
and RXR
WT cells were transfected with 10 µg of pRARE-Luc and
incubated with the indicated concentrations of all-trans-RA
for 6 h, and luciferase activity was measured. The transfection
efficiency in different cells was similar as determined by measuring
the activity of co-transfected
pCMV-
-galactosidase.
overexpression. To increase the
sensitivity of the assay for quantitative analysis, use was made of
pRARE-Luc, which contains RARE from RAR
and is
transactivated by RAR
RXR heterodimers(31) . When
pRARE-Luc was transfected into CMVV cells, all-trans-RA was
able to induce increases in luciferase activity in a dose-response
manner (Fig. 2B). Strikingly, cells that overexpressed
RXR
were much more sensitive, requiring 10-100-fold less
all-trans-RA than CMVV cells to achieve the same response, a
result that was observed in three independent experiments. Thus, RXRs
are limiting in 2B4.11 cells; overexpression makes the cells more
responsive to RA, and expression of a dominant-negative form of
RXR
makes the cells relatively refractory to RA.
The Ability of 9-cis-RA To Inhibit Apoptosis Is Enhanced
by RXR
To determine if overexpression of
RXR Overexpression and Blocked by Expression of a
Dominant-negative RXR
or expression of a dominant-negative RXR
affects
biological responses to T cell activation, the transfected cell lines
were stimulated with immobilized anti-CD3 antibodies. All of the cells
responded, as assessed by IL-2 production, inhibition of cell growth,
and induction of cell death (Fig. 3). The
RXR
-overexpressing cells reproducibly produced the lowest amount
of IL-2, and the RXR
dominant negative-expressing cells the
highest. Although it is possible that this is due to clonal
variability, it is consistent with the previous observation that
retinoic acid causes an approximately 2-fold decrease in IL-2
production by 2B4.11 cells(21) . None of the cells produced
IL-2 in the absence of anti-CD3 stimulation (data not shown). The
susceptibility of the cells to activation-induced apoptosis allowed us
to determine whether manipulation of RXR function alters the response
of the cells to 9-cis-RA. One representative experiment of
five is shown in Fig. 4. As previously shown with untransfected
2B4.11 cells, 9-cis-RA inhibited the DNA fragmentation of CMVV
cells in a dose-dependent manner, with half-maximal inhibition
occurring at a concentration of
10
M.
Cells that overexpress wild type RXR
were
10-fold more
sensitive to 9-cis-RA, with a half-maximal response at
10
M. In contrast, cells expressing
the dominant-negative RXR
(RXR
DN1 and DN2) were resistant to
9-cis-RA, responding poorly if at all even at concentrations
of 10
M. Therefore, the ability of
9-cis-RA to prevent activation-induced T cell death is
enhanced by increasing RXR levels and is prevented by a
dominant-negative RXR.
WT, RXR
DN1, and
RXR
DN2 cells were stimulated with the indicated amount of
immobilized anti-CD3 antibody for 18 h. After an aliquot of supernatant
was taken for IL-2 measurement (A), the cells were pulsed with
[
H]thymidine and cultured for another 4 h before
harvesting to measure growth (B). The amount of
[
H]thymidine incorporated by each of the cell
lines ranged from 60,000 to 84,500 cpm/5
10
cells.
To measure cell death, cells cultured with anti-CD3 (1 µg/well)
were stained with trypan blue dye, and dead cells were enumerated by
light microscopy (C). The standard deviations of duplicate
cultures are shown.
overexpressing and dominant-negative
RXR
-expressing cells. [
H]thymidine labeled
CMVV, RXR
WT, RXR
DN1, and RXR
DN2 cells were stimulated
with plastic-coated anti-CD3 antibody (1 µg/well) in the presence
of the indicated concentrations of 9-cis-RA. After 18 h the
cells were harvested and the percent specific DNA fragmentation
determined. ▴, CMVV;
, RXR
WT;
, RXR
DN1;
, RXR
DN2.
Inhibition of Activation-induced Apoptosis by RAR- and
RXR-selective Retinoids
The effects of overexpressing wild type
RXR and a dominant-negative RXR
demonstrate the involvement
of RXRs in the prevention of apoptosis by RA. However, RXRs have been
shown to work as heterodimeric partners of RARs and several other
nuclear receptors. To further explore the mechanism of RA action, we
made use of synthetic retinoids that selectively bind RARs (TTNPB),
RXRs (LGD1069), or both (retinoid 351). 2B4.11 cells were induced to
undergo apoptosis by stimulation with plastic-adherent anti-CD3 in the
presence of varying amounts of RA or synthetic retinoids (Fig. 5). As shown previously, all-trans-RA and
9-cis-RA inhibited activation-induced apoptosis with an
approximately 10-fold difference in potency. The RAR-selective retinoid
TTNPB was much less potent than all-trans-RA, having no effect
on activation-induced apoptosis except at a very high concentration (10
µM). The synthetic retinoid 351, which is similar to
9-cis-RA in its binding specificity, was identical to
9-cis-RA in its effects on apoptosis. Interestingly, the
RXR-selective ligand LGD1069 was no more potent than
all-trans-RA and in fact had little effect on apoptosis except
at a very high concentration (10 µM), suggesting that
ligand-bound RXRs (in RXR homodimers or RAR
RXR heterodimers) were
not a very effective mediator for inhibition of activation-induced
apoptosis. Instead, the most effective retinoids (9-cis-and
retinoid 351) were those that bind to both RARs and RXRs with high
affinity. To directly determine whether occupancy of both RARs and RXRs
is required for maximum potency, a combination of RAR- and
RXR-selective retinoids was tested (Fig. 6). While retinoids
TTNPB (RAR-selective) and LGD1069 (RXR-selective) each caused
suboptimal decreases in DNA fragmentation of anti-CD3-stimulated 2B4.11
cells, the combination of TTNPB (10
M) plus
LGD1069 (10
M) almost completely blocked
DNA fragmentation. Therefore, the most effective inhibition of
activation-induced death requires that both RARs and RXRs bind and are
activated by their ligands.
H]thymidine-labeled 2B4.11 cells were stimulated
with plastic-coated 2C11 (1 µg/well) in 96-well microtiter plates
in the presence of the indicated concentrations of retinoids. After
overnight culture, specific DNA fragmentation was determined.
,
all-trans-RA;
, 9-cis-RA;
, LGD1069;
, TTNPB; ▪, ritinoid 351.
H]thymidine-labeled 2B4.11 cells were stimulated
with immobilized anti-CD3 antibody (1 µg/well) in the presence of
the RAR-selective retinoid TTNPB, the RXR-selective retinoid LGD1069,
or both for 18 h, and specific DNA fragmentation was measured.
, medium; ▪, LGD1069 (10
M).
Regulation of Fas Ligand by Retinoids
Since
activation-induced T cell hybridoma apoptosis is due to up-regulation
of FasL and its subsequent interaction with Fas, we asked how the
different receptor-selective ligands affect the regulated expression of
FasL. L1210 cells express very little Fas and are resistant to
FasL-induced death, whereas L1210-Fas cells express uniformly high
levels of Fas and are very sensitive to FasL-induced
death(18, 46) . These matched cells have been used as
targets to measure the level of FasL expressed by
``effector'' cells. Activation of 2B4.11 cells for 4 h
induces FasL mRNA, and co-culture of these cells for an additional 4 h
with L1210-Fas, but not L1210, cells results in DNA
fragmentation(18) . Activation of CMVV, RXR DN1, and
RXR
DN2 cells induced FasL, as measured by the ability of these
cells to kill L1210-Fas cells (Fig. 7). Death of L1210-Fas cells
induced by activated 2B4.11 and various transfectants was inhibited by
soluble extracellular Fas (Fas:Fc) but not by soluble extracellular
tumor necrosis factor receptor (TNFRp60:Fc) ( (18) and data not
shown). Just as for activation-induced T cell hybridoma apoptosis,
retinoid 351 inhibited FasL expression by CMVV cells but had little or
no effect on FasL expression in cells expressing the dominant-negative
RXR
receptor. In contrast to retinoids, dexamethasone prevented
FasL up-regulation in CMVV, RXR
DN1, and RXR
DN2 cells
equally well (data not shown). To determine whether RARs and RXRs
participate in inhibiting the up-regulation of FasL, 2B4.11 cells were
activated in the presence of an RXR-selective retinoid, an
RAR-selective retinoid, or both, and kill of L1210-Fas target cells was
measured. The RAR-selective retinoid TTNPB had little effect on
activation-induced apoptosis, while the RXR-selective retinoid LGD1069
partially inhibited Fas-mediated killing at high concentrations (Fig. 8). When added together, LGD1069 and TTNPB were better at
inhibiting up-regulation of FasL than either one alone, and it was
possible to achieve complete inhibition of functional FasL expression
on 2B4.11 cells. These data demonstrate that retinoids prevent
activation-induced apoptosis by inhibiting up-regulation of FasL, and
both RARs and RXRs are involved in this process.
DN1, and RXR
DN2 cells were activated
with plastic-coated 2C11 (1 µg/well) in the presence of varying
concentrations of retinoid 351.
[
H]thymidine-labeled L1210 or L1210-Fas cells
were added 4 h later, the cells were co-cultured for another 4 h before
harvesting, and DNA fragmentation was measured. Specific DNA
fragmentation of L1210-Fas cells in the absence of retinoid 351 was as
follows: CMVV, 40.8%; RXR
DN1, 26.1%; RXR
DN2, 48.3%. DNA
fragmentation of L1210 cells was <5% in all
cases.
H]thymidine-labeled L1210-Fas cells were added 4
h later, and the cells were co-cultured for another 4 h before
harvesting and measurement of DNA fragmentation.
, LGD1069;
, TTNPB; ▪, LGD1069 + TTNPB (10
M).
RXR heterodimers without
requiring ligand. Further studies found that transactivation of DR-5
and DR-2 responsive elements by RAR
RXR heterodimers in response
to RA requires RAR binding to the 3` CRM (48, 49, 50) . The importance of orientation
in nuclear receptor action is also exemplified by the vitamin
D
-thyroid hormone receptor heterodimer (VDR
TR), which
binds to responsive elements as 5`-TR
VDR-3` or 5`-VDR
TR-3`
but only transactivates when it binds the ligand for the downstream
receptor(51) . There is also some evidence that RXR homodimers
might mediate gene transactivation. It has been reported that RXRs can
bind to DR-1 responsive elements as homodimers in the presence of
9-cis-RA or RXR-selective retinoids(26, 27) .
Furthermore, using RAR- and RXR-selective ligands and chimeric RA
receptors, it was shown that although RAR
RXR heterodimers have a
higher affinity for a DR-1 element than RXR homodimers, they cannot
transactivate because the RAR binds to the 5` instead of the 3`
CRM(27) . Considering that RXR
RAR heterodimers are formed
preferentially, these results may explain why co-transfection of RXRs
but not RARs is required in many cells, including 2B4.11 cells, to
transactivate reporter constructs driven by DR-1 CRMs in response to
9-cis-RA. Another RA responsive element found in the promoter
region of cellular retinoic acid binding protein type II consists of
two CRMs separated by one base pair (DR-1). Using mutated RARs or RXRs
that cannot themselves transactivate but that can form heterodimers
with wild type partners, it was shown that these heterodimers bind to
this DR-1 element and transactivate in the presence of the ligand for
the wild type partner. Therefore, one partner of an RAR
RXR
heterodimer is capable of transactivating when bound by ligand.
Interestingly, 9-cis-RA is more potent than
all-trans-RA in inducing cellular retinoic acid binding
protein type II mRNA, which was attributed to the ability of
9-cis-RA to bind to both RARs and RXRs(28) . Our
finding that an RXR-selective retinoid was only a moderately good
inhibitor of apoptosis suggests that RXR
RXR homodimers or
RAR
RXR heterodimers bound with an RXR-selective ligand are not
the optimal mediator of the anti-apoptotic effect of retinoids.
Instead, both RXRs and RARs are involved in this process, since
efficient inhibition of activation-induced cell death requires the
presence of a ligand for each type of receptor.
disrupts the ability of retinoids to
suppress Fas ligand up-regulation and cell death. These data provide
further evidence that retinoids inhibit activation-induced apoptosis by
inhibiting FasL expression and raise questions about the mechanism of
RAR and RXR action. Compared with the understanding of gene
transactivation, limited progress has been made in understanding
negative regulation of gene expression by RARs/RXRs and other nuclear
receptors(61) . Studies on the glucocorticoid receptor have
shown that it can repress the function of transcription factor AP-1
through protein-protein
interactions(62, 63, 64) . Similarly,
regulation of the rat stromelysin gene by RA was found to be mediated
through the AP-1 site of its promoter region(65) . RA inhibits
collagenase promoter-driven gene transcription (66) because RA
receptors antagonize the required transactivation by
AP-1(67, 68, 69) ; the mechanism of this
antagonism is not well understood. Since AP-1 is also known to be
induced in T cells by activation(70) , it is possible that RA
receptors modulate FasL expression by antagonizing the function of
AP-1. It is also possible that there are RA responsive elements in the
regulatory region of the FasL gene, and that the effect of RA on FasL
is a direct effect on gene transcription. The gene for FasL has
recently been cloned(71, 72) , and sequencing of the
regulatory flanking sequences of the FasL gene should allow these
hypotheses to be tested.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.