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Proc. Natl. Acad. Sci. USAVol. 93, pp. 6197-6202, June
1996Developmental Biology
Cell-type and promoter-context dependent retinoic acid
receptor(RAR) redundancies for RAR832 and Hoxa-1 activation in F9
andP19 cells can be artefactually generated by gene knockouts
(genetic redundancy/specific synthetic retinoids/RAR mutants/EC
cell differentiation/RAR-retinoic X receptor heterodimers)
RESHMA TANEJA*, BIDYUT ROY*t, JEAN-Luc PLASSAT*, CHRIS F. ZuSIt,
JACEK OSTROWSKIS, PETER R. RECZEKS,AND PIERRE CHAMBON*§*Institut de
Genetique et de Biologie Moleculaire et Cellulaire, Centre National
de la Recherche Scientifique, Institut National de la Sante et de
la RechercheMedicale, Universit6 Louis Pasteur, College de France,
BP 163, 67404 Illkirch-Cedex, France; and Pharmaceutical Research
Institute, Bristol-Myers-Squibb, 100Forest Avenue, Buffalo, NY
14213-1091
Contributed by Pierre Chambon, February 28, 1996
ABSTRACT By using RAR type (a, 3B, or y)-specificsynthetic
retinoids and a pan-retinoic X receptor (RXR)-specific ligand, we
have investigated the contribution ofRARsand RXRs in the activation
of RA target genes and thedifferentiation ofembryonal carcinoma
cells. We demonstratecell-type- and promoter context-dependent
functional redun-dancies that differ between the three RAR types
for mediatingthe induction of RARf32 and Hoxa-1 in wild-type,
RARy-/-and RARa-/- F9 cells and in P19 cells. The extent
ofredundancy between RARs is further modulated by the syn-ergistic
activation of RXRs with a pan-RXR agonist. We alsodemonstrate that
the expression ofRARI32 is auto-inducible inRARy-/- but not in
wild-type F9 cells, indicating that thefunctional redundancies
observed between RARs in genedisruption studies can be
artefactually generated. Thus, eventhough all three RARs can
functionally substitute each otherfor inducing the expression of RA
target genes and celldifferentiation, one RAR can cell-specifically
override theactivity of the other RARs. Interestingly, only RARy
canmediate the retinoic acid-induced differentiation of wild-typeF9
cells, whereas the differentiation of P19 cells can bemediated by
either RARa or RARy.
The effects of retinoic acid (RA) and other retinoid
derivativesofvitaminA on embryonal carcinoma (EC) cell
differentiationappear to be mediated by retinoic acid receptors
(RARs) andretinoid X receptors (RXRs) (refs. 1-4 and references
there-in). RARs (a, 3, and y and their isoforms) and RXRs (a, 3,and
y and their isoforms) are members of the nuclear
receptorsuperfamily that act as ligand-activated transcriptional
regu-lators transducing the RA signal to control the expression
oftarget genes. RARs bind and are activated by all-trans RA(T-RA)
and 9-cis RA, whereas RXRs bind and are activatedby 9-cis RA only
(reviewed in refs. 5-11). RAR/RXR het-erodimers bind much more
efficiently than the respectivehomodimers to RA response elements
(RAREs) in vitro, andseveral lines of evidence support the notion
that these het-erodimers represent the main functional units
transducing theretinoid signal (reviewed in refs. 6-8, 11, and 12).
In vitrostudies with isolated receptors or cells transfected with
RARand RXR expression vectors have suggested that RXR may infact be
a silent partner when the heterodimer is bound to aDR5 response
element (13, 14), although several exceptions tothis "RXR silence"
have been reported (refs. 13-16 andreferences therein). Moreover,
we (3) and others (17), lookingat the effect of retinoids on
endogenous responsive genes incultured cells, have recently
reported that, at limiting ligand
The publication costs of this article were defrayed in part by
page chargepayment. This article must therefore be hereby marked
"advertisement" inaccordance with 18 U.S.C. §1734 solely to
indicate this fact.
concentrations, both RAR and RXR partners of RAR/RXRheterodimers
can be ligand-activated to induce synergisticallythe expression of
a variety of RA target genes.We have previously shown that
disruption ofRARy, but not
of RARa, in F9 EC cells is associated with
differentiationdefects and altered RA responsiveness of Hoxa-1, but
not ofRARP gene expression (1, 2). Here, by using RAR-
andRXR-specific synthetic retinoids, we have further dissected
thecontributions of each type of RAR and RXRs as ligand-dependent
transcription factors in RA-induced differentiationevents. We
demonstrate that, in wild-type (WT), RARy-/-and RARa-/- F9 cells,
and in P19 cells, the expression ofHoxa-1 and RARf32 isoform is
differentially regulated throughthe various RARs, in a cell-type-
and promoter context-dependent fashion. We show also that the
induction of bothgenes and EC cell differentiation can be
synergistically inducedby coactivation with a limiting
concentration of a RAR-specific retinoid together with a
pan-RXR-specific retinoid.Furthermore, only RARy can mediate F9
cell differentiation,whereas P19 cells can differentiate in
presence of either aRARa- or a RARy-specific retinoid. Most
interestingly, wereport that RAR32 expression is auto-inducible in
RARy-/-,but not inWT and RARa-/- F9 cells, which not only
confirmsthat RAR(32 is auto-inducible as previously observed in
P19cells (3), but it also demonstrates that gene knockouts
canartefactually create situations of redundancy of gene
function.
MATERIALS AND METHODS
Cell Culture. F9 WT, RARa-/-, and RARy-/- cells weregrown in
monolayer culture in Dulbecco's modified Eagle'smedium (DMEM)-F12
containing 10% fetal calf serum asdescribed (1) and P19 EC cells in
monolayer culture inDMEMcontaining 44 mM sodium bicarbonate and
7.5% fetal calfserum (18). Retinoids (T-RA, BMS753, BMS453,
BMS961,and BMS649) were dissolved in ethanol and added at
appro-priate concentrations for the indicated time periods.
Controlcells were treated with ethanol (vehicle) alone.RNA
Isolation and Semiquantitative Reverse Transcrip-
tase-PCR. Total RNA was isolated by the method of Chom-czynski
et al. (19). The conditions for RT-PCR have beendescribed (3). The
blots were analyzed on a PhosphorImager
Abbreviations: RA, retinoic acid; RAR, retinoic acid receptor;
RXR,retinoic X receptor; WT, wild type; RARE, retinoic acid
responseelement; EC, embryonal carcinoma; T-RA, all-trans RA; RT,
reversetranscriptase.tPresent address: Anthropometry and Human
Genetics Units, Bio-logical Science Division, Indian Statistical
Institute, 203-BarrackporeTrunk Road, Calcutta 700035, India.§To
whom reprint requests should be addressed.
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6198 Developmental Biology: Taneja et al.
analyzer, and fold inductions were quantitated relative
toethanol-treated cells which were given a value of 1.0.
RESULTS
Synthetic Retinoids Specific for RARa- and RARy-Mediated RARt82
Expression in WT, RARa-/-, and RARy-/-F9 Cells. The synthetic
retinoids Am80 (20) and CD666 (21)have been reported to be
selective for RARa and RARy in avery narrow range of 1 nM and 10
nM, respectively (3). Athigher concentrations, both ligands lose
their selectivity andact as pan-RAR agonists, which limits their
use to study thespecificity ofRARa and RARy on target gene
activation. Theselectivity of two novel synthetic retinoids
BMS188,753(BMS753) and BMS188,961 (BMS961), has been
establishedhere by using RARa-/- (2) and RARy-/- (1) mutant F9
cells.Endogenous RAR32 gene transcripts were determined
aftertreatment of WT, RARa-/-, and RARy-/- F9 cells withincreasing
concentrations of these synthetic retinoids, relativeto control
vehicle-treated (ethanol) cells (Fig. 1 and Table 1).Increasing
concentrations of BMS753 resulted in low levels ofRARP32
transcripts in WT cells, even at the highest concen-tration, while
100 nM BMS753 efficiently induced RARP32transcripts in RARy-/-
cells (Fig. 1, compare lanes 4 and 8;Table 1). In contrast, no
activation was seen in RARa-/- cellsup to 100 nM BMS753,
demonstrating that it is specific forRARa (even 1 t,M BMS753
resulted in very low RAR32expression in RARa-/- cells; data not
shown). BMS961induced RARf32 expression inWT and RARa-/- cells,
with nodetectable induction in the RARy-/- cells up to the
highestconcentration tested (1 tLM; data not shown), establishing
theRARy-specificity of this retinoid. Note that, at identical
WT RAR7/- RARa-/- WT RARy/- RARa-/-_ IH I I I Ln)
o.C o o O O O
an RARP2
-indicate,ad n d wh r t to34 t s (,36B4
1 2 3 4 5 6 7 8 9 10 1112 131415 161718 19 20 21 22
2324expression is not altered upon retinoid treatment.BMS753 (nM)
BMS961 (nM)
FIG. 1. Relative levels ofRAR2 transcripts in F9 WT, RARa-/-and
RARy-/- cells in presence of increasing concentrations of aRARa- or
a RARy-specific synthetic retinoid. Transcript amountswere
estimated by RT-PCR after treatment of cells for 24 h asindicated,
and normalized with respect to 36B4 transcripts (22),
whoseexpression is not altered upon retinoid treatment.
BMS961 concentrations, RAR32 expression was higher inRARa-/-
than in WT cells.
In WT cells, 100 nM BMS961 was as efficient as T-RA forRARP2
induction, whereas 100 nM BMS753 was markedly lessefficient, thus
indicating that the induction ofHRARt2expres-sion could be
primarily mediated through RAR- in WT cells.Note, however, that the
relative efficiency of BMS753 wasmuch higher in RARy-/- cells,
which not only is in agreementwith our previous observations that
RARa and RARy can befunctionally redundant (1, 2, 23), but also
suggests that thepresence of RARy prevents RARa from inducing
RAR32expression in WT cells.
Differential Induction of Hoxa-1and RAR/]2 ExpressionThrough
RAR. in F9 and P19 Cells. RA-induced Hoxa-1
Table 1. Relative levels of RARf32 and Hoxa-1 transcripts in WT,
RARa-/-, and RARy-/- F9 cells and in WTP19 cells
F9 cells
WT RARa-/- RARy-/- P19 cellsLigand RAR32 Hoxa-1 RAR32 Hoxa-1
RAR32 Hoxa-l RARf2 Hoxa-1
Ethanol, vehicle 1 1 1 1 1 1 1 1T-RA, 100 nM 20 9 22 11 19 3 25
80RARa-specificBMS753
1 nM 1 ND 1 ND 1 ND 1 110 nM 1 1 1 1 1 1 1 1100 nM 2 2 1 1 14 4
7 7
RAR3-specificBMS453, 0.5 /M 1 1 1 1 8 1 1 2
RARy-specificBMS961
1 nM 1 ND 2 ND 1 ND 1 110 nM 6 2 12 3 1 1 1 2100 nM 19 7 20 9 1
1 2 4
pan-RXR-specificBMS649, 1 pM 1 1 1 1 1 1 1 1
1 ,M BMS649 andBMS753
1 nM ND ND ND ND ND ND 2 910 nM 10 3 1 1 16 5 3 20100 nM ND ND
ND ND ND ND 30 42
BMS453, 0.5 ,iM 1 1 1 1 20 1 16 22BMS961
1 nM ND ND ND ND ND ND 3 810 nM 22 4 18 5 1 1 4 10100 nM ND ND
ND ND ND ND 24 30
The induction of RARf32 and Hoxa-1 transcripts in presence of
the synthetic retinoids (as indicated), expressed relative
toethanol (vehicle)-treated cells (taken as 1), is given after 24 h
of treatment. The numbers correspond to the average values(±20%)
from at least three independent experiments similar to that shown
in Fig. 1. ND, not determined.
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expression is markedly decreased in RARy-/- F9 cells,
whereasRAR/32 induction is unaffected by the loss of either RARy
orRARa (1, 2). To further investigate which receptor-type caninduce
the expression of Hoxa-1 and RAR32 in F9 cells, thetranscripts of
these endogenous genes were analyzed in WT,RARa-/-, and RARy-/-
cells treated with retinoids selective forRARa (BMS753), RAR3
(BMS453; ref. 24), and RARy(BMS961). RAR/32 and Hoxa-1 RNAs were
not efficiently in-duced by 100 nM BMS753 in WT F9 cells (Table 1),
whereasthese transcripts were induced in RARy-/- cells but not
inRARa-/- cells, as expected. Strikingly, the
RAR3-selectivesynthetic retinoid BMS453 did not activate the
expression ofeither gene in WT or RARa-/- cells, but specifically
andefficiently induced RAR32, but not Hoxa-1 transcripts inRARy-/-
cells. On the other hand, the RARy-selective retinoidBMS961
efficiently induced the expression of both genes in WTcells, in a
dose-dependent manner, and this RARy-selectiveinduction was
slightly higher in RARa-/- cells than in WT cells(Table 1).The
extent of activation of RARf32 and Hoxa-1 expression
was also investigated with the same retinoids in P19 cells.
Inthese cells, and in contrast toWT F9 cells, the
RARa-selectiveretinoid BMS753 was more efficient than the
RARy-selectiveretinoid BMS961. These data are in keeping with our
previousobservations showing that, in P19 cells and at selective
con-centrations, an RARa agonist (Am80) more efficiently in-duced
the expression of RA-responsive genes than a RARyagonist (CD666)
(3). As reported (3), BMS453 did not, or onlyvery weakly, induce
the expression of RAR32 and Hoxa-1 inP19 cells (Table 1).
Differential Synergism Between RAR-Selective SyntheticRetinoids
and a Pan-RXR-Specific Ligand for the Induction ofRARB82 and Hoxa-1
Transcripts in F9 and P19 Cells. Thesynergism between a
RAR-selective ligand and the pan-RXR-specific agonist BMS649
(SR11237; see ref. 3), which isinactive on its own (refs. 3 and 25;
see also Tables 1 and 2), wasinvestigated in WT F9 and P19 cells,
as well as in RAR mutantF9 cells. In WT F9 cells, at a limiting
concentration of theRAR-selective ligand (10 nM), the expression of
both RARJ2and Hoxa-l was synergistically induced by the
concomitantactivation of either RARa (BMS753)/RXR or
RARy(BMS961)/RXR (Table 1). In RARa-/- cells, only the acti-vation
of RARy/RXR effectively induced the expression ofRARP2 and Hoxa-1.
No induction of either gene was seen uponactivation of RAR/3
(BMS453)/RXR in either WT orRARa-/- cells, whereas activation
ofRARa (BMS753)/RXRin RARy-/- cells led to the synergistic
induction of both genes.Strikingly, activation of RARf (BMS453)/RXR
in the sameRARy-/- cells specifically and efficiently induced the
expres-sion of RAR32, but not of Hoxa-1.The expression of both
RARf2 and Hoxa-1 genes was
synergistically activated in P19 cells by the addition of
eitherone of the three RAR-selective retinoids together with
thepan-RXR-specific agonist (Table 1). Interestingly, a
combina-tion of the RARP-specific retinoid BMS453 and of the
pan-RXR-specific BMS649, which was completely ineffective
atinducing the expression of either RAR32 or Hoxa-1 in WT F9cells,
resulted in a strong synergistic induction of both genes inP19
cells.
Retinoid-Induced Differentiation of WT F9 Cells Is Spe-cifically
Mediated Through RARy, Whereas That of P19Cells Can Be Mediated
Through Either RARa or RARy.RA-induced differentiation is
dramatically delayed inRARy-/- cells but not in RARa-/- cells (1,
2). To deter-mine whether the selective activation of either RARa
orRARy could lead to morphological differentiation, F9 WTcells were
treated with either BMS753 or BMS961, respec-tively (Fig. 2 and
Table 2). WT F9 cells differentiated in thepresence of 100 nM
BMS961, whereas cells treated withBMS753 at the same concentration
remained undifferenti-
Table 2. Effect of synthetic retinoids on collagen type IV
(al)transcript induction and morphological differentiation of WT
andmutant F9 cells
Collagen type IV (al) transcript induction(A) and morphological
differentiation (B)
of F9 cells
WT RARa-/- RARy-/-Ligand A* Bt A* Bt A* Bt
Ethanol 1.0 None 1.0 None 1.0 NoneT-RA, 100 nM 16.0 ++ 17.0 ++
2.5 +/-BMS753, 10 nM 1.0 None 1.0 None 1.0 NoneBMS753, 100 nM 1.0
None 1.0 None 1.0 NoneBMS453, 0.5 /aM 1.0 None 1.0 None 1.5
+/-BMS961, 10 nM 1.0 None 1.0 None 1.0 NoneBMS961, 100 nM 10.0 ++
16.0 ++ 1.0 NoneBMS753, 10 nM 2.0 +/- 1.0 None 1.0 None+ BMS649,1
/aM
BMS753, 100 nM ND + ND None ND +/-+ BMS649,1 JLM
BMS453, 0.5 /LM 1.0 None 1.0 None 2.0 +/-+ BMS649,1 uLM
BMS961, 10 nM 4.0 + 10.0 ++ 1.0 None+ BMS649,1 iLMND, not
determined.
*Levels of collagen transcripts after a 96-hcontrol
ethanol-treated cells taken as 1.
treatment relative to
tSymbols are as follows: + +, more than 80-90% of the cells
exhibiteda differentiated phenotype; + and +/-, 50-70% and not more
than10%, respectively, of the cells appeared differentiated.
ated. At lower concentrations (1 and 10 nM), neitherBMS753 nor
BMS961 could trigger morphological differen-tiation. These results
were biochemically confirmed by de-termining the expression of a
differentiation-specificmarker, collagen type IV (al) (Table 2, and
data not shown),which was clearly upregulated only in cells treated
with 100nM BMS961, whereas no expression was seen in cells
treatedwith lower BMS961 concentrations or with BMS753 (Table2, and
data not shown).Most interestingly, BMS753, which could not induce
the
differentiation of F9 cells at 100 nM, did induce the
morpho-logical differentiation of P19 cells even at a concentration
of10 nM (Fig. 2), whereas BMS961 was inactive at the sameligand
concentration. However, at a higher concentration(lOOnM), the
RARy-specific BMS961 induced P19 cell dif-ferentiation as
efficiently as the RARa-specific BMS753 (Fig.2). In contrast,
neither F9WT cells nor P19 cells differentiatedin the presence of
the RAR,3-specific ligand BMS453 at anyconcentration (refs. 3 and
24; and see below).
Synergistic Differentiation of WT and Mutant F9 CellsTreated
with a RARa-, a RARp-, or a RARy-SpecificRetinoid Together with a
Pan-RXR-Specific Retinoid. Theability of the RAR a-, 3-, and
y-specific retinoids to inducedifferentiation of WT, RARa-/-, or
RARy-/- F9 cells wasalso tested over a 96-h period of treatment in
the absence andin the presence of the pan-RXR-specific-agonist
BMS649(Fig. 2 and Table 2). When the cells were treated with
eitherone of these retinoids alone, only the RARy-selectiveBMS961
at a high concentration (100 nM) could induce thedifferentiation of
WT and RARa-/- cells (Fig. 2 and Table2; data not shown). In
contrast, a suboptimal concentration(10 nM) of the RARy-selective
BMS961 together with thepan-RXR agonist BMS649 efficiently induced
the differen-tiation of WT and RARa-/- F9 cells, but not of P19
cells
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6200 Developmental Biology: Taneja et al.
F9 CELLSWT
BMS753 (100nM)
RARa--BMS961 (100nM).'.'.-;., '.;/.··/.
-,; 4ogt ~j ~,-.,C .1 '.·
RARa-/- RARy--BMS96+1 (lOnM) RARy-- RARy- BMS45 (0.5gLM)BMS649
(llM) BMS453 (0.51M) BMS91 1MOOnM BMS649 (1aM)1
P19 CELLSEthano T-RA (100nM)
B'11AiM'S 3...--'
BMS961(1OHM) BMS753 (1O0nM)
BMS753 (10nM)
FIG. 2. Morphological differentiation of P19 cells and ofWT,
RARa-/-, and RARy-/- F9 cells after a 96-h treatment with synthetic
retinoidsas indicated (see also Table 2).
(data not shown), whereas activation of both RARa (10 nMBMS753)
and RXR only poorly induced the differentiationof WT F9 cells (Fig.
2 and Table 2). However, a higherconcentration of BMS753 (100 nM)
together with the RXR-specific ligand more efficiently induced a
synergistic mor-phological differentiation of WT F9 cells (Fig. 2
and Table2). No or very little differentiation ofWT or mutant F9
cellscould be triggered by the RAR/3-selective synthetic
retinoidBMS453 alone or associated with the pan-RXR agonistBMS649
(Fig. 2 and Table 2). Similarly, P19 cells did notdifferentiate in
the presence of either the RAR3-specificBMS453 alone or together
with the pan-RXR agonist (data
not shown; see ref. 3). As expected, RARy-/- cells
wereinsensitive to differentiation in the presence of BMS961alone
or together with BMS649 (data not shown). However,in contrast to WT
and RARa-/- F9 cells and to P19 cells,a low degree of
differentiation of RARy-/- F9 cells was seenin presence of BMS453
and BMS649, but apparently not inthe presence of BMS753 (10 nM) and
BMS649 (Table 2; datanot shown).
In general, the extent of morphological differentiationbrought
about by the various retinoids was well correlated withthe relative
level of collagen type IV (al) transcripts presentin the treated
cells (Table 2).
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Proc. Natl. Acad. Sci. USA 93 (1996) 6201
DISCUSSIONWe have previously shown that low concentrations of
syntheticretinoids selective for either RARa, -j3 or -y do not or
veryinefficiently induce the expression of several RA target
genesnor trigger differentiation of F9 and P19 cells, while the
sameretinoids could synergize with the pan-RXR agonist BMS649,which
had no activity on its own (3). These results wereconsistent with
the proposal that RAR-RXR heterodimers arethe functional units
which mediate the induction of expressionof RA-responsive genes in
these EC cells and induce theirdifferentiation. Furthermore, the
magnitude of this synergismvaried depending on both the RAR (a, 13,
or y)/RXR com-bination and the promoter context of the responsive
genes (3).This previous study also suggested that there could be
asignificant degree of functional redundancy between the threeRAR
types. Functional redundancy between RARa, -3, and-y was also
suggested by studies in which inactivation ofRARy(1) resulted in a
reduced expression ofHoxa-1, whereas the lossof neither RARy nor
RARa had any effect on the expressionof RARfI2 (2). This latter
result indicated that all three RARscould be redundant for
mediating the induction of RARf32expression, and that the induction
of RAR32 could be auto-catalytic. We have further investigated here
the RAR/RXRsynergism and the redundancy between RARs for
RA-inducedHoxa-1 and RARP2 expression and morphological
differenti-ation of F9 and P19 EC cells. This investigation was
madepossible by the synthesis of two new synthetic retinoids,BMS753
and BMS961, which are shown here to be RARa- andRARy-specific
agonists, respectively, over a wide concentra-tion range.
Functional Redundancy Between RARs for the Induction ofRARf32
and Hoxa-1 Expression Is Cell-Type- and PromoterContext-Dependent
and Can Be Artefactually Generated bythe Knockout ofRARy in F9
Cells. The results summarized inTable 1 show that a specific
activation of RARy by a highconcentration of BMS961 can efficiently
induce the expressionof both RAR/32 and Hoxa-1 in WT F9 cells, but
not in P19 cells.In contrast, the specific activation of RARa by a
high con-centration of BMS753 results in a moderate induction of
bothgenes in P19 cells, but only in a weak activation in WT F9
cells.Although still apparent, these cell type-dependent
differentialfunctional redundancies between RARa and RARy, whichcan
be correlated with the predominance of RARy in F9 cellsand RARa in
P19 cells (23,26), become less clear cut when theRXRs are
concomitantly activated by the pan-RXR agonistBMS649. In both cell
types the expression of both genes isefficiently induced by the
association of the RXR-specificligand with either the RARa- or the
RARy-specific ligand ata concentration where they do not, or only
weakly, activate ontheir own. Note that, for efficient RARf2 and
Hoxa-1 induc-tion, higher concentrations of BMS753 and BMS961
arerequired in P19 cells than inWT F9 cells. In contrast,
BMS453,which specifically activates RARf3, while inhibiting RARa
andRAR3y (24), cannot induce on its own the expression of
eitherRARf2 or Hoxa-1 in WT F9 or P19 cells. Interestingly,
theassociation of BMS453 with the pan-RXR agonist BMS649,results in
an efficient induction of both genes in P19, but notin WT F9
cells.Taken collectively, these results clearly support the
conclu-
sion that the pattern of functional redundancy betweenRARa, -P,
and -y for the induction of RARf32 and Hoxa-1expression depends
both on the cell-type and on whetherRXRs are concomitantly
activated. Furthermore, the syner-gistic effects of RAR- and
RXR-specific ligands, which isparticularly striking in the case of
the RAR3-specific BMS453in P19 cells, strongly support our previous
suggestion thatRAR-RXR heterodimers represent the functional units
trans-ducing the inductive retinoid signal.
The observation that RAR32 and Hoxa-1 transcripts can
beefficiently induced by the RARa-specific ligand BMS753 inRARy-/-
but not in WT F9 cells clearly indicates that thepresence of RARy
prevents RARa to efficiently mediate theRAR,32 and Hoxa-1 induction
in WT F9 cells. Similarly, theinduction of RAR,t2 by the
RAR3-specific agonist BMS453 inRARy-/- but not in WT F9 cells
indicates that the RARj3-mediated induction of RAR,32 expression is
repressed by thepresence of RARy in WT F9 cells. The mechanism for
theserepressive effects is presently unknown. Interestingly, in
thesame RARy-/- F9 cells, the expression of the Hoxa-1 gene,whose
regulatory region contains a RARE identical to that ofRAR32 gene
(27, 28), is not induced by the RAR3-specificBMS453, in agreement
with our previous observations thatoverexpression of RARa in
RAR-y-/- cells is much moreefficient than that of RARf32 for
rescuing RA-induced expres-sion of Hoxa-1 to WT levels (23). Taken
together with theobservation that the induction of both RAR(32 and
Hoxa-1transcripts can be mediated by RAR3 in WT P19 cells,
thispromoter-context dependance of RAR/3 activity in F9
cellssuggests that these cells may lack a factor which synergizes
withRAR(3 on the Hoxa-1 promoter and is present in P19 cells.Note
that the present results, particularly the BMS453-inducedRAR,32
expression in RARy-/- cells in the absence of thepan-RXR agonist
BMS649, strongly support our previoussuggestion that RAR32
expression can be autocatalyticallycontrolled (3, 23).
Thus, several important conclusions can be drawn from ourpresent
results concerning the functional specificity and re-dundancy of
the three RAR types for the induction of RAtarget gene expression.
First, even in cases where all threeRARs possess the capability to
mediate the induction of anRA target gene, the actual involvement
of a given RAR (e.g.,RARt2) in this induction is dependent on the
cell type and onthe promoter context of the responsive gene, most
probablydue to the existence of cell- and promoter-specific
synergizingfactors. Second, the concomitant activation of RXRs
canmarkedly affect the capability of a given RAR type (e.g.,RAR/3
and RARy in P19 cells) to induce the expression of agiven gene,
most likely reflecting cell-specific synergistic ef-fects between
heterodimeric partners. Third, a given RAR(e.g., RARy in WT F9
cells, but not in P19 cells) cancell-specifically override the
other RARs for mediating theinduction of target genes, even though
these RARs are activein the absence of the "dominant" RAR. Thus,
the extensivefunctional redundancy between the three RARs, that
wasinferred from studies of single and double RAR mutations(see
ref. 12 for a review), may reflect, at least to some extent,the
nonphysiological situations created by the gene knockouts.In other
words, that RARs can functionally substitute for oneanother
following the knockout of one of them does not provethat they are
actually functionally redundant under normalWT conditions. In this
respect, RAR type-specific retinoids,such as those used in the
present study, should be useful indetermining which RAR type(s) may
specifically mediate agiven retinoid-induced event under normal WT
conditions,and to evaluate the possible synergizing effects of a
concom-itant activation of RXRs.The Cell Type-Specific Differential
Role of the RARs in
Morphological Differentiation of F9 and P19 EC Cells IsModulated
by the Synergistic Effect of a Pan-RXR Agonist.Our data (Fig. 2 and
Table 2) indicate that RARa on its own,specifically activated by a
low concentration of BMS753 (10nM), can trigger the morphological
differentiation of P19 cells,whereas it has no effect on WT or
mutant F9 cells even at ahigher concentration (100 nM). On the
other hand, RARy onits own, specifically activated by a high
concentration ofBMS961, can trigger the morphological
differentiation of bothF9 and P19 cells, whereas the RAR/-specific
BMS453 alonehas no effect on the differentiation of these EC cell
lines.
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Furthermore, BMS753-activated RARa together withBMS649-activated
RXRs synergistically trigger to some extentthe differentiation of
F9 cells, whereas the coactivation ofRAR/3 and RXRs cannot
efficiently trigger the morphologicaldifferentiation of either F9
or P19 cells. Moreover, a subop-timal concentration (10 nM) of the
RARy-specific BMS961together with the pan-RXR agonist (BMS649) can
synergis-tically induce the morphological differentiation of F9,
but notof P19, cells. Thus, this study clearly establishes that
themorphological differentiation of F9 and P19 EC cells
isdifferentially triggered by the three RAR types, and that
theireffects can be strongly modulated by a concomitant
activationof the RXRs.
It is important to realize that in all of the above cases, it
isunknown whether the observed phenotypic modifications trig-gered
by the various combinations of RAR- and pan-RXR-specific ligands
correspond to identical cell differentiationstates underlain by
similar cascades of retinoid-inducible mo-lecular events. However,
it is clear that specific RAR agonists,acting either alone or
synergistically with a pan-RXR-agonist,are more restricted than
T-RA in their effects on both RAtarget gene expression and
morphological differentiation ofthe two EC cells studied here. The
cellular and tissular effectsof the administration of synthetic
retinoids may therefore bemuch more selective than those resulting
from RA adminis-tration. This increased selectivity may extend the
therapeuticuse of retinoids by dissecting the beneficial effects
from thenumerous others generated by the administration of
retinoicacid which indiscriminately activates all RARs and
RXRs.
We thank J. L. Plassat and N. Chartoire for technical
assistance; thecell culture facility for providing cells; and C.
Werli, B. Boulay, andthe secretarial staff for help with
preparation of the manuscript. Wealso thank H. Gronemeyer and J. Y.
Chen for stimulating discussions,and J. Clifford and S. Ward for a
critical reading of the manuscript.This work was supported by funds
from the Centre National de laRecherche Scientifique, the Institut
National de la Sante et de laRecherche M6dicale, the College de
France, the Centre HospitalierUniversitaire Regional, the
Association pour la Recherche sur laCancer, the Fondation pour la
Recherche M6dicale, and the HumanFrontier Science Program. R.T. was
supported by fellowships from theCentre National de la Recherche
Scientifique and Fondation pour laRecherche M6dicale and Universit6
Louis Pasteur.
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