Institut de Génétique et de Biologie Moléculaire et Cellulaire, Centre National de la Recherche Scientifique/Institut National de la Santé et de la Recherche Médicale/Université Louis Pasteur, Collège de France, 67404 Illkirch-Cedex, France
We have generated F9 murine embryonal
carcinoma cells in which either the retinoid X receptor
(RXR) and retinoic acid receptor (RAR)
genes or
the RXR
and RAR
genes are knocked out, and compared their phenotypes with those of wild-type (WT),
RXR
/
, RAR
/
, and RAR
/
cells. RXR
/
/
RAR
/
cells were resistant to retinoic acid treatment
for the induction of primitive and parietal endodermal
differentiation, as well as for antiproliferative and apoptotic responses, whereas they could differentiate into
visceral endodermlike cells, as previously observed for
RXR
/
cells. In contrast, RXR
/
/RAR
/
cells
were defective for all three types of differentiation, as
well as antiproliferative and apoptotic responses, indicating that RXR
and RAR
represent an essential receptor pair for these responses. Taken together with results obtained by treatment of WT and mutant F9 cells
with RAR isotype- and panRXR-selective retinoids,
our observations support the conclusion that RXR/
RAR heterodimers are the functional units mediating
the retinoid signal in vivo. Our results also indicate that
the various heterodimers can exert both specific and redundant functions in differentiation, proliferation, and
apoptosis. We also show that the functional redundancy
exhibited between RXR isotypes and between RAR
isotypes in cellular processes can be artifactually generated by gene knockouts. The present approach for multiple gene targeting should allow inactivation of any set
of genes in a given cell.
RETINOIDS exert their pleiotropic effects on vertebrate development, cellular differentiation, proliferation, and homeostasis through two classes of
ligand-dependent transactivators: the retinoic acid receptors (RARs)1, and the retinoid X receptors (RXRs) (for
reviews see De Luca, 1991 The F9 murine embryonal carcinoma (EC) cell line expresses all types of RARs and RXRs (Zelent et al., 1989 To further investigate the roles of RXRs and RARs in
differentiation, proliferation, and apoptosis, we have now
generated F9 cells lacking either RXR Cell Culture
F9 cells were cultured and induced to differentiate into primitive, parietal,
and visceral endodermlike cells as previously described (Boylan et al.,
1993 Targeting of the RAR The RAR Electroporation, selection of neomycin-resistant clones, Cre-mediated
excision of the resistance genes, and Southern blot analysis were performed as previously described (Metzger et al., 1995 Western Blotting and Electrophoretic Mobility Shift
Assays (EMSA)
Isolation of whole cell extracts from F9 cells and transfected COS-1 cells,
Western blot analysis, and electrophoretic mobility shift assays were performed as previously described (Rochette-Egly et al., 1991 Reverse Transcription (RT)-PCR
RNA preparation, RT-PCR, and Southern blotting were performed as
previously described (Bouillet et al., 1995 Analysis of Cellular Growth
Cells were plated in triplicate 3-cm culture wells (5 x 102 cells per well),
and cell counting experiments were performed as previously described
(Clifford et al., 1996 Analysis of Apoptosis
Apoptosis was analyzed by both Hoechst staining of nuclei and FACS®
analysis, as previously described (Clifford et al., 1996 Targeted Disruption of the RAR The RXR
Southern blot analysis using a 3 To establish F9 cells in which both RXR Function of RXRs and RARs in the Retinoid-induced
Differentiation of F9 Cells into Primitive and Parietal
Endodermlike Cells
WT F9 cells differentiate into primitive and parietal endodermlike cells, when grown in monolayer culture in the
presence of tRA alone and tRA in combination with dibutyryl cAMP (bt2cAMP), respectively (Strickland, 1981
The extent of differentiation of WT and mutant F9 cells
was further investigated biochemically by determining the
mRNA levels of collagen type IV Table IV.
Summary of the Involvement of the Various RARs and RXRs in the Transduction of the Retinoid Signal in F9 Cells, as
Deduced from the Present and Previous Studies of RAR and RXR Mutant Cells and the Use of Receptor-specific Retinoids
; Blomhoff, 1994
; Chambon,
1994
, 1996
; Kastner et al., 1995
; Mangelsdorf and Evans,
1995
). RARs are activated by all-trans retinoic acid (tRA)
and by 9-cis retinoic acid (9C-RA), whereas RXRs are activated by 9C-RA only. The various RAR (RAR
,
, and
) and RXR (RXR
,
, and
) isotypes are encoded by
different genes, and their isoforms, which differ in their
NH2-terminal regions, are generated by differential promoter usage and alternative splicing. The multiple RAR
and RXR isotypes and isoforms are conserved in vertebrate evolution, and display distinct spatiotemporal expression patterns in developing embryos and adult tissues,
suggesting that each receptor performs some unique functions (for reviews see Leid et al., 1992a
; Chambon, 1994
;
Kastner et al., 1994
). RXR/RAR heterodimers bind much
more efficiently to retinoic acid response elements (RAREs)
than their respective homodimers in vitro (for reviews see
Leid et al., 1992a
; Chambon, 1994
, 1996
; Giguère, 1994
;
Glass, 1994
; Kastner et al., 1994
; Mangelsdorf et al., 1994
; Gronemeyer and Laudet, 1995
; Keaveney and Stunnenberg, 1995
; Mangelsdorf and Evans, 1995
), and several lines
of evidence support the idea that these heterodimers represent the functional units transducing the retinoid signal in
vivo (Kastner et al., 1995
; Chambon, 1996
).
;
Martin et al., 1990
; Wan et al., 1994
), and upon retinoic
acid treatment, it differentiates into cells resembling three
distinct extraembryonic endoderm (primitive, parietal,
and visceral), depending on the culture conditions (for reviews see Strickland, 1981
; Hogan et al., 1983
; Gudas et al.,
1994
). Retinoid-induced differentiation is accompanied by
an apoptotic response and a dramatic decrease in the rate
of proliferation (Sleigh, 1992
; Atencia et al., 1994
; Clifford
et al., 1996
). Thus, F9 cells provide an attractive system for
the analysis of retinoid signaling in vivo.
and RAR
, or
RXR
and RAR
, and then compared their phenotypes
with those of wild-type (WT), RXR
/
(Clifford et al.,
1996
), RAR
/
(Boylan et al., 1995
), and RAR
/
(Boylan et al., 1993
; Taneja et al., 1995
) F9 cells. Multiple gene targeting in a given cell has been achieved by using a
Cre/loxP system (Sauer and Henderson, 1990
; Metzger et
al., 1995
), which allows removal of the antibiotic resistance
gene from a targeted locus, and therefore subsequent mutagenesis of the second allele of a given gene with the
same targeting construct, as well as the targeting of additional genes. We demonstrate that tRA-treated RXR
/
/
RAR
/
cells differentiate poorly into primitive and parietal endodermlike cells and are impaired in both antiproliferative and apoptotic responses, whereas they fully differentiate into visceral endoderm (VE)-like cells, as previously
observed for RXR
/
cells (Clifford et al., 1996
). In contrast, RXR
/
/RAR
/
cells are defective for all three
types of endodermal differentiation, as well as for the antiproliferative and apoptotic responses, indicating that the
absence of both RXR
and RAR
cannot be functionally compensated by the other retinoid receptors in these cells.
Taken together with results obtained by treatment of
WT and mutant F9 cells with panRXR- and RAR isotype-
selective retinoids, our findings support the conclusion
that RXR/RAR heterodimers are the functional units mediating the retinoid signal in vivo. Furthermore our results indicate that RXR/RAR heterodimers can exert both specific and redundant functions in differentiation, proliferation,
and apoptosis. We also show that functional redundancy
between RXR isotypes and between RAR isotypes can be
artifactually generated by gene knockouts.
Materials and Methods
; Clifford et al., 1996
). The retinoids (tRA, Am80, BMS753, BMS453,
BMS961, and BMS649) were dissolved in ethanol.
or RAR
Genes in RXR
-null
F9 Cells.
targeting vector, pRAR
(LNL), was previously described
(Metzger et al., 1995
). The RAR
targeting vector, pRAR
(LPL), was derived from pD
6.5A (a gift from D. Lohnes, IGBMC, CNRS/INSERM/ ULP, Illkirch, France), which contains a 6-kb genomic fragment including
exons 5 and 8. A unique SmaI site, followed by stop codons in all three
reading frames, was introduced into pD
6.5A at the KpnI site located in
exon 8 of RAR
by inserting the oligonucleotides 5
-CCCCGGGTAGGTAGATAGCGTAC-3
and 5
-GCTATCTACCTACCCGGGGGTAC-3
, yielding the pRAR
T4 construct. An XhoI-BamHI fragment containing the phosphoglycerate kinase (PGK) promoter-driven, puromycin- resistance (puro) gene, flanked by loxP sites, was isolated from pHRLpuro1,
and blunt ended with T4 DNA polymerase, followed by ligation into the
SmaI site of pRAR
T4. pHRLpuro1 was constructed from VS-1, a plasmid containing a loxP site-flanked PGKpuroA+ cassette, by mutating the
SalI site. The PGKpuroA+ cassette was obtained by ligating the PGK promoter (a 500-bp EcoRI-PstI fragment isolated from pKJ-1 [Adra et al.,
1987
]) to the coding sequence of the puro gene (a 600-bp HindIII-ClaI
fragment isolated from pLXPB [Imler et al., 1996
]), and by inserting the
SV-40 polyadenylation signal (a 160-bp BglII-XbaI fragment isolated from
pSG5 [Green et al., 1988
]) using synthetic oligonucleotides. This cloning resulted in the loss of the PstI, HindIII, and ClaI restriction sites, and the
introduction of HindIII and EcoRI sites at 5
and 3
ends of the PGK promoter, respectively. KpnI, ApaI, XhoI, and BglII restriction sites, and
BamHI and SacI sites are located at 5
and 3
ends of the loxP-flanked
cassette, respectively.
; Clifford et al., 1996
).
Puromycin selection (500 ng/ml) was carried out for 10 d.
; Gaub et al.,
1992
; Boylan et al., 1993
).
; Roy et al., 1995
). The PCR
primers and end-labeled oligonucleotide probes for collagen IV
1, laminin B1,
-fetoprotein (AFP), and 36B4, were described previously (Clifford et al., 1996
). Transcript levels were quantified using a BAS 2000 bio-imaging analyzer (Fuji Ltd., Tokyo, Japan), and were normalized to the
corresponding 36B4 signals.
). [3H]Thymidine incorporation assays were performed essentially as described (Clifford et al., 1996
), with the following
modifications. Cells were cultured for 4 d in six replicate 3-cm wells, in the presence or absence of 1 µM tRA, and three of the six wells were treated
with 8 µCi/well [3H]methylthymidine (20.0 Ci/mmol; Dupont-NEN, Boston, MA) for 2 h before harvesting. The cell cycle profile of WT and mutant cells was determined as previously described (Clifford et al., 1996
).
).
Results
or RAR
Genes in
RXR
Null F9 Cells
-null F9 cell line, C2RXR
(L)/
(L), which constitutively expresses a ligand-dependent chimeric Cre-
recombinase (Cre-ER; Metzger et al., 1995
), was electroporated with the targeting constructs pRAR
(LNL) (Fig.
1 A) or pRAR
(LPL) (Fig. 2 A) to generate F9 cells disrupted in either the RXR
and RAR
genes or the RXR
and RAR
genes. These targeting constructs contain
translation stop codons and loxP site-flanked neomycin- or puromycin-resistance genes in exon 9 of the RAR
gene or exon 8 of the RAR
gene, respectively (Materials
and Methods; Figs. 1 A and 2 A). Since these exons encode
the B region, which is common to all isoforms of a given
RAR isotype (Kastner et al., 1990
; Leroy et al., 1991
;
Chambon, 1994
), the expression of RAR
and RAR
proteins is suppressed by these mutations. Homologous recombination (HR) and Cre-mediated excision of the resistance genes were verified by Southern blotting (Figs. 1 B
and 2 B).
Fig. 1.
Disruption of both alleles of the RAR gene by HR in
a RXR
(L)/
(L) cell line. (A) Schematic diagram of the pRAR
(LNL)
targeting construct, the WT RAR
locus, and the recombined locus after integration (HR[I]) and after Cre-mediated excision
(HR[E]). Dark boxes indicate exons. The exons 4-8 encoding the
NH2-terminal part of minor isoforms (RAR
3-7) (Leroy et al.,
1991
) are not represented. Restriction enzyme sites and the location of probes are indicated. The neo and a1 probes have been
previously described (Metzger et al., 1995
). The numbers in the
lower part of diagram are in kb. K, KpnI; L, loxP recombination
site; S, SalI; ST, two translation stop codons; Xb, XbaI; Xh, XhoI.
(B) Southern blot analysis indicating the targeting of the RAR
gene in a RXR
(L)/
(L) cell line. The genotypes of different cell
lines (e.g., 9) and their subclones (9a, etc.) are indicated at the top
of each lane, and correspond to all three panels. (C) Western blot
analysis indicating the absence of RAR
protein in RXR
(L)/
(L)/
RAR
(L)/
(LNL) cell lines. Lanes 1 and 2 contain 2 µg of whole
cell extracts from COS cells transfected with either the pSG5
(Green et al., 1988
) or mRAR
ø expression construct (Zelent et
al., 1989
), and lanes 3-6 contain 60 µg of whole cell extracts from
WT and mutant F9 cells, as indicated. RAR
protein was detected using the rabbit polyclonal antibody RP
(F), followed by
chemiluminescence detection. Mol wt is shown in kD.
[View Larger Version of this Image (30K GIF file)]
Fig. 2.
Disruption of both alleles of the RAR gene by HR in
a RXR
(L)/
(L) cell line. (A) Schematic diagram of the pRAR
(LPL)
targeting construct, the WT RAR
locus, and the recombined locus after integration (HR[I]) and after Cre-mediated excision
(HR[E]). Dark boxes indicate exons. The exons 6 and 7 encoding
the NH2-terminal part of minor isoforms (RAR
4 and 6; Kastner
et al., 1990
) are not represented. Restriction enzyme sites and the
location of probes are indicated. The puro probe corresponds to a
0.7-kb EcoRI-XbaI fragment derived from pHRLpuro1. The r1
probe corresponds to a 1.5-kb BamHI-EcoRI fragment derived
from the RAR
genomic clone
G1mRAR
(Lohnes et al.,
1993
). The r2 probe corresponds to a 1.6-kb EcoRI-PstI fragment
derived from pRAR
(LPL). The numbers in the lower part of the
diagram are in kb. B, BamHI; Bg, BglII; E, EcoRI; H, HindIII; L,
loxP recombination site; S, SalI; ST, three translation stop codons
inserted in all reading frames. Asterisk indicates that these sites
are not present in the WT gene, and dashed line represents vector
sequence. (B) Southern blot analysis indicating the disruption of
the RAR
gene in a RXR
(L)/
(L) cell line. The genotypes of
different cell lines (e.g., 25) and their subclones (25a, etc.) are indicated at the top of each lane and correspond to all four panels.
(C) EMSA indicating the absence of RAR
protein in RXR
(L)/
(L)/
RAR
(L)/
(LNL) cells. A radiolabeled oligonucleotide corresponding to the Hoxa-1/RAR
RARE was incubated with 20 µg of
whole cell extracts from WT cells (lanes 1 and 4), RXR
(L)/
(L)/
RAR
(L)/
(LNL) cells (lanes 2 and 5) and RAR
/
cells (lanes 3 and 6), or with 2 µg of whole cell extracts from COS cells transfected with either the pSG5 (lanes 7 and 9; Green et al., 1988
) or
mRAR
ø expression construct (lanes 8 and 10; Zelent et al.,
1989
), together with 0.5 µg of whole cell extracts from COS cells
transfected with mRXR
ø expression construct (Leid et al.,
1992b
). The arrows indicate the shifted complex formed in the
presence of mouse monoclonal antibodies Ab2
(F) and Ab10
(A2).
[View Larger Version of this Image (36K GIF file)]
probe (a1) located outside
of the pRAR
(LNL) targeting construct (Fig. 1 A), indicated
that, after electroporation, 5 out of 96 C2RXR
(L)/
(L)
neomycin-resistant clones had one targeted RAR
allele
(Fig. 1, A, HR[I]; and B, compare lanes 3 and 4 with lanes 1 and 2; data not shown). One RXR
(L)/
(L)/RAR
+/
(LNL)
cell line (clone nine) was treated with estradiol (E2) to excise the loxP-flanked cassette (
[LNL] and
[L]) designate the targeted allele before and after Cre-mediated excision, respectively). Southern blot analysis using a1 and
"neo" probes revealed that excision of the resistance gene
occurred in two out of six subclones treated with E2 (Fig.
1, A, HR[E]; and B, compare lane 5 with lanes 3 and 4; see
also Metzger et al., 1995
). The second allele of the RAR
gene was targeted in the RXR
(L)/
(L)/RAR
+/
(L) cell
line (Fig. 1 B, clone 9a) using the same targeting construct and strategy. 2 of 96 neomycin-resistant clones were positive for the desired recombination event, resulting in
RXR
(L)/
(L)/RAR
(L)/
(LNL) cell lines (Fig. 1 B, clones
9a-10 and 9a-26; compare lanes 6 and 7 with lanes 1-5). No
wild-type RAR
transcripts were detected in RXR
(L)/
(L)/
RAR
(L)/
(LNL) cells by semi-quantitative RT-PCRs (data
not shown). Similarly, no RAR
protein could be detected
in RXR
(L)/
(L)/RAR
(L)/
(LNL) cells (called hereafter
RXR
/
/RAR
/
) by Western blotting using the polyclonal antibody RP
(F) (Gaub et al., 1992
), directed
against the F region of the RAR
protein (Fig. 1 C, compare lanes 3 and 4 with lanes 2, 5, and 6).
and RAR
genes are inactivated, C2RXR
(L)/
(L) cells were electroporated with the pRAR
(LPL) targeting construct (Fig. 2
A). Southern blot analysis using the r1 probe, located 5
to
the pRAR
(LPL) sequence (Fig. 2 A), revealed that 3 out of
96 puromycin-resistant clones had one targeted RAR
allele (Fig. 2, A, HR[I]; and B, compare lanes 3 and 4 with
lanes 1 and 2; and data not shown). One RXR
(L)/
(L)/
RAR
+/
(LPL) cell line (clone 25) was transiently transfected
with a Cre recombinase expression construct (pPGK-Cre)
(Clifford et al., 1996
), since for unknown reasons the loxP-flanked, puromycin-resistance gene was not excised by
treatment of the cells with E2 (data not shown;
[LPL]
and
[L] designate the targeted allele before and after Cre-mediated excision, respectively). The pattern obtained by Southern blot analysis, using r1, r2, and puro
probes, clearly indicated that 2 out of 96 subclones had lost
the puromycin-resistance cassette (Fig. 2, A, HR[E]; and
B, compare lane 5 with lane 4; and data not shown). The
second allele of the RAR
gene was inactivated in one
RXR
(L)/
(L)/RAR
+/
(L) cell line (Fig. 2 B, clone 25a)
using the same targeting construct and strategy, yielding a
RXR
(L)/
(L)/RAR
(L)/
(LPL) cell line (Fig. 2 B, clone
25a-3, compare lane 6 with lanes 1-5). No wild-type RAR
RNA was detected in RXR
(L)/
(L)/RAR
(L)/
(LPL) cells
(data not shown). The absence of RAR
protein was verified by EMSA using the monoclonal antibodies Ab2
(F)
and Ab10
(A2) (Rochette-Egly et al., 1991
), directed against
the F and A2 regions of the RAR
protein, respectively.
No antibody-shifted complex was observed in RXR
(L)/
(L)/
RAR
(L)/
(LPL) cells (called hereafter RXR
/
/RAR
/
)
(Fig. 2 C, compare lane 5 with lanes 4, 6, and 10). Note
that the RXR
loci, which contain loxP sites, were not rearranged during excision of the resistance genes at the
RAR
or RAR
loci (data not shown). Note also that the
knockout of a given receptor(s) did not result in major
variations of those remaining (Chiba et al., 1997
).
;
Hogan et al., 1983
). Previous studies have shown that
these two types of differentiation are severely impaired in
RAR
/
and RXR
/
cells (Boylan et al., 1993
; Clifford
et al., 1996
). The differentiation patterns of RXR
/
/
RAR
/
and RXR
/
/RAR
/
cells were compared
with those of WT and single knockout cell lines. After 4 d
of treatment, <10% of RXR
/
/RAR
/
cells became
flatter and irregular in shape, or rounded with long cell
processes, which are the morphological characteristics of
primitive or parietal endodermal differentiation of WT F9
cells, respectively. The same results were obtained with
9a-10 and 9a-26 RXR
/
/RAR
/
cell lines (Fig. 3 A,
compare g-i with a-f, and data not shown). No morphological differentiation at all was observed in RXR
/
/
RAR
/
cells after 4 d of treatment, and <0.1% of the
cells exhibited a differentiated morphology after 6 d of
treatment (Fig. 3 A, j-l, and data not shown). This undifferentiated phenotype persisted after 10 d of treatment.
Fig. 3.
RXR/
/RAR
/
F9 cells do not differentiate
into primitive and parietal endodermlike cells. (A) WT (a-
c), RXR
/
(d-f), RXR
/
/
RAR
/
(g-i) and RXR
/
/
RAR
/
(j-l) cells were
treated with control vehicle
(a, d, g, and j), 1 µM tRA
alone (b, e, h, and k) or 1 µM
tRA and 250 µM bt2cAMP (c, f, i, and l) for 4 d. Cells
were photographed under a
phase-contrast microscope
at x125 magnification. (B)
Total RNA from WT and
mutant cells, treated with
control vehicle or 1 µM tRA
for 48 h, was analyzed by RT-PCR analysis for collagen
type IV
1, laminin B1, and
36B4. (C) RT-PCR analysis
was performed as in B, for
three separate experiments.
The levels of RNA transcripts were expressed relative to the amount present in
tRA-treated WT cells, which was taken as 100. The white
and black bars correspond to
transcript levels expressed in
vehicle- and tRA-treated cells,
respectively. Bar, 100 µm.
[View Larger Version of this Image (103K GIF file)]
1 and laminin B1, two
markers of endodermal differentiation (Fig. 3, B and C).
After 48 h of 1 µM tRA treatment, the induction of collagen type IV
1 and laminin B1 was reduced in RAR
/
cells (10-fold and 6-fold lower levels, respectively) and in RXR
/
cells (3-fold and 6-fold lower levels, respectively) when compared to WT cells, whereas these inductions were not altered in RAR
/
cells (Boylan et al.,
1993
, 1995
; Clifford et al., 1996
; Taneja et al., 1996
). The
induction of both transcripts was also impaired in RXR
/
/
RAR
/
cells (fivefold and sevenfold lower levels, respectively), whereas it was fully abrogated in RXR
/
/
RAR
/
cells. Thus, RA-induced primitive and parietal
endodermal differentiation of WT F9 cells appears to be
mainly mediated by the RXR
/RAR
pair, whereas it
cannot be mediated by combinations of RXR(
+
) with
either RAR
or RAR
(see Table IV).
Function of RXRs and RARs in the Retinoid-induced Differentiation of F9 Cells into VE-like Cells
When F9 cells are grown in suspension as aggregates, low
levels of tRA induce a VE phenotype in the outermost
layer of cells, which display an irregular surface (Strickland, 1981; Hogan et al., 1983
; see also Fig. 4 A, WT, a and
b, brackets). We have previously shown that, in contrast to
primitive and parietal endodermal differentiation, VE differentiation can be induced by tRA in RXR
/
F9 cells
(Clifford et al., 1996
). Similarly, after 10 d of treatment, >80% of the outer layer of RAR
/
and RXR
/
/
RAR
/
cell aggregates differentiated into VE-like cells,
which were indistinguishable from those of WT and
RXR
/
cells (Fig. 4 A, compare f with panels b and d;
and Table I). In contrast, <10% of the outer layer of
RAR
/
cells exhibited VE conversion after 10 d of
treatment, whereas full VE differentiation was eventually
achieved after 14 d of treatment (Fig. 4 A, compare h with
g and b; and Table I). In RXR
/
/RAR
/
cells, the surface of the aggregates was as smooth after 10 d of treatment as in untreated controls (Fig. 4 A, compare j with a
and i), and <10% of the aggregates displayed only a spotty
VE conversion after 12 or 18 d of treatment (Table I). To
exclude the possibility that this very poor differentiation
of the RXR
/
/RAR
/
cells could be due to some
clonal variation, rather than the presence of the RXR
-null mutation in the RAR
-null background, we expressed
the RXR
cDNA in RXR
/
/RAR
/
cells. As expected, cells expressing the RXR
cDNA exhibited a phenotype identical to that of RAR
/
cells, i.e., the RA-
induced VE differentiation was restored at late time (14 d)
of RA treatment (data not shown).
Table I. Effect of Various Retinoids on Morphological Differentiation of WT and Mutant F9 Cells into Visceral Endoderm (VE)-like Cells |
We also analyzed the mRNA levels of collagen type
IV1, laminin B1 and AFP in WT and mutant F9 cells
(Fig. 4 B). After 10 d of aggregate culture in the presence
of 50 nM tRA, the three markers were similarly induced in
WT, RXR
/
, RAR
/
, and RXR
/
/RAR
/
cells.
In RAR
/
cells, the induction of laminin B1 was similar
to that of WT cells, whereas the induction of collagen IV
1
was slightly reduced (twofold lower than in WT cells). In
contrast, the induction of AFP, a specific marker of VE
differentiation, was hardly detectable in RAR
/
cells after
10 d of RA treatment (Fig. 4 B). There was no induction of
either collagen IV
1, laminin B1 or AFP in RXR
/
/
RAR
/
cells, in agreement with their lack of morphological differentiation into VE. Thus, RXR
and RAR
play an essential role in VE differentiation of WT F9 cells.
To further investigate the functions of RARs and RXRs
in VE differentiation, WT and mutant F9 cells were treated
for 10-18 d with tRA or synthetic retinoid agonist selective
for RAR (BMS188,753 [BMS753]; Taneja et al., 1996
),
RAR
(BMS189,453 [BMS453]; Chen et al., 1995
), RAR
(BMS188,961 [BMS961]; Taneja et al., 1996
) and all three
RXRs (panRXR, BMS188,649 [BMS649]; also known as SR11237; Lehmann et al., 1992
; see Roy et al., 1995
) (Table I). In WT cells, VE differentiation was triggered by 100 nM of the RAR
agonist as effectively as by 50 nM tRA,
and it was synergistically induced by a combination of 10 nM RAR
and 1 µM panRXR agonists (Table I). In contrast, VE differentiation of RAR
/
cells was triggered
by 10 nM RAR
agonist as efficiently as by 50 nM tRA,
and it was even synergistically induced by 1 nM of the
RAR
agonist in combination with 1 µM panRXR agonist, indicating that RAR
partially hinders the RAR
function in WT cells. The RAR
agonist alone, or together with the panRXR agonist, was more efficient in
RXR
/
and RXR
/
/RAR
/
cells than in WT cells, but
weaker than in RAR
/
cells, demonstrating that RXR
prevents an efficient synergism between RXR(
+
) and
RAR
(Table I; see Table IV). As expected, no VE differentiation was observed in RAR
/
and RXR
/
/
RAR
/
cells treated with the RAR
/panRXR agonist
combination.
The RAR agonist, BMS753, on its own did not trigger
VE differentiation of WT and RXR
/
cells, whereas the
combination of 100 nM RAR
and 1 µM panRXR agonists was much less efficient than the RAR
/panRXR agonist combination. As expected, no VE differentiation was
seen in RAR
/
and RXR
/
/RAR
/
cells upon
treatment with the RAR
/panRXR agonist combination. In contrast, RAR
/
cells weakly differentiated into VE-like cells upon treatment with 100 nM RAR
agonist
alone, and this differentiation was markedly enhanced by
addition of 1 µM panRXR agonist. This synergistic stimulation was almost abrogated in RXR
/
/RAR
/
cells.
A combination of RAR (BMS453) and panRXR agonists did not trigger VE differentiation in WT, RXR
/
,
RAR
/
, and RXR
/
/RAR
/
cells. Interestingly, this
combination synergistically induced VE differentiation of
RAR
/
cells, and this effect was almost absent in
RXR
/
/RAR
/
cells, as in the case of the RAR
/panRXR agonist combination (Table I). Thus, RAR
strongly
prevents RAR
and RAR
to synergize with RXR
, and
mutation of RAR
artefactually generates functional redundancy between RARs for VE differentiation of F9
cells (see Table IV).
Function of RXRs and RARs in the Retinoid-induced
Antiproliferative Response of F9 Cells and
Retinoid-induced Proliferation of RXR/RAR
Null
F9 Cells
The effect of tRA on proliferation of WT, RXR/
,
RXR
/
/RAR
/
, and RXR
/
/RAR
/
F9 cells was
investigated (Fig. 5 A). After 6 d of 1 µM tRA treatment, the inhibition of growth as determined by cell counting,
was lower for RXR
/
than for WT cells (58 and 79% inhibition relative to untreated control cells, respectively)
(Clifford et al., 1996
). The antiproliferative effect of tRA
was decreased to the same extent for RXR
/
/RAR
/
cells (54% inhibition) and RXR
/
cells. On the other
hand, tRA did not reduce, but slightly increased the number of RXR
/
/RAR
/
cells. The rate of DNA synthesis was also compared for WT and mutant cell lines by
measuring [3H]thymidine incorporation during the anti-proliferative response to tRA (Fig. 5 B). After 4 d of 1 µM
tRA treatment, [3H]thymidine incorporation was reduced
by 54% in WT cells relative to vehicle-treated control cells,
and only by 20% in RXR
/
and RXR
/
/RAR
/
cells. In contrast, there was no inhibition of [3H]thymidine
incorporation in RXR
/
/RAR
/
cells.
FACS® analysis has previously shown that tRA treatment of WT F9 cells results in an accumulation of cells in
the G0 and G1 phases of the cell cycle, and that this accumulation was decreased in RXR/
cells (Clifford et al.,
1996
) (Fig. 5 C). The cell cycle profile of untreated
RXR
/
/RAR
/
and RXR
/
/RAR
/
cells was the
same as that of WT and RXR
/
cells. After 5 d of 1 µM
tRA treatment, the proportion of cells in the G0 and G1
phases, was 71% for WT cells, whereas it was lower for
RXR
/
and RXR
/
/RAR
/
cells (38 and 40%, respectively; Fig. 5 C, compare b, d, and f). Interestingly, the
cell cycle profile of RXR
/
/RAR
/
cells was not significantly affected by tRA treatment, and was almost identical to that of untreated WT cells (Fig. 5 C, compare h
with g and a).
To further dissect the roles of RARs and RXRs in the
control of proliferation, WT and mutant F9 cells were
treated for 6 d with tRA or receptor-selective retinoids,
and cell numbers were counted (Table II). In WT cells,
100 nM RAR agonist efficiently reduced the proliferation, and 10 nM of the same agonist, which had no effect
on its own, synergized with 1 µM panRXR agonist. The effect
of these retinoids on proliferation was reduced, while not
abolished, in RXR
/
and RXR
/
/RAR
/
cells, indicating that RXR
can be partially replaced by RXR(
+
) for synergizing with RAR
(see Table IV). Interestingly,
the RAR
/panRXR agonist combination was more efficient in RAR
/
cells than in WT cells, revealing that
RAR
partially hinders the antiproliferative effect of
RAR
in WT cells (Table II; see Table IV). As expected,
this combination did not inhibit the proliferation of
RAR
/
and RXR
/
/RAR
/
cells. The combination
of 100 nM RAR
and 1 µM panRXR agonists, which reduced the proliferation of WT cells less efficiently than the
RAR
/panRXR agonist combination, was more efficient
in RAR
/
cells than in WT cells (Table II), indicating
that RAR
partially hinders the antiproliferative effect of
RAR
in WT cells (see Table IV). The RAR
/panRXR
agonist combination had no effect on the proliferation of
RXR
/
cells, showing that RAR
can only synergize
with RXR
to inhibit proliferation (see Table IV).
Table II. Effect of Various Retinoids on Proliferation of WT and Mutant F9 Cells |
Surprisingly, a treatment with 10 and 100 nM RAR agonist increased the number of RXR
/
/RAR
/
cells,
indicating that RAR
can mediate a proliferative effect in
the absence of both RXR
and RAR
. As expected, the
RAR
/panRXR agonist combination did not affect proliferation of RAR
/
and RXR
/
/RAR
/
cells. Neither
the RAR
agonist alone nor in combination with the panRXR agonist affected the proliferation of WT, RXR
/
,
RAR
/
, and RXR
/
/RAR
/
cells, whereas this combination synergistically reduced the proliferation of RAR
/
cells. Note that 500 nM RAR
agonist alone increased the
cell number of RXR
/
/RAR
/
cells, and that this effect was enhanced by addition of 1 µM panRXR agonist.
Thus, the presence of RAR
not only hinders the antiproliferative effect of the RXR
/RAR
pair, but also the
proliferation-promoting effects of the combinations of
RXR(
+
) with RAR
, showing again that knockouts
generate artifactual effects not observed under WT conditions, as already seen above in the case of F9 cell differentiation (see Table IV).
Function of RXRs and RARs for the Retinoid-induced Apoptotic Response of F9 Cells
Since retinoids can induce apoptosis, which also contributes to the decrease in cell number in retinoid-treated F9
cells (Atencia et al., 1994), we determined the extent of
the tRA-induced apoptotic response of WT and mutant F9
cells by FACS® analysis. Sub-2N-size, DNA-containing
particles corresponding to "apoptotic bodies" appeared in
WT cells after 5 d of tRA treatment, whereas they were
not detected in tRA-treated RXR
/
(see also Clifford et
al., 1996
), RXR
/
/RAR
/
, and RXR
/
/RAR
/
cells (Fig. 5 C, compare d, f, and h with b [arrow]). Apoptosis was confirmed by staining with the DNA-binding fluorochrome Hoechst 33258. Apoptotic particles and condensed chromatin were similarly observed in tRA-treated
WT and RAR
/
cells (Fig. 6, a, b, e, and f; Table III). In
contrast, tRA-induced apoptosis was reduced in RAR
/
cells, rarely seen in RXR
/
(Clifford et al., 1996
) and
RXR
/
/RAR
/
cells, and abolished in RXR
/
/
RAR
/
cells (Fig. 6, c, d, and g-l; Table III). Note that a
background level of apoptosis occurred at high cell density
even in the absence of tRA, as previously mentioned (Clifford et al., 1996
).
Table III. Effect of Various Retinoids on Apoptosis of WT and Mutant F9 Cells |
To further investigate the role played by the different
RAR and RXR isotypes in apoptosis, WT and mutant F9
cells were treated for 6 d with receptor-selective retinoids,
and stained with Hoechst dye (Table III). In WT cells, 100 nM RAR agonist triggered apoptosis, and the addition of
1 µM panRXR agonist resulted in a synergistic effect. The
effect of these retinoids was markedly reduced in RXR
/
and RXR
/
/RAR
/
cells, indicating that RXR
can
only poorly be replaced by RXR(
+
) for this response
(Table IV). In contrast, the RAR
/panRXR agonist combination was more efficient in RAR
/
cells than in WT
cells, indicating that RAR
partially prevents the apoptotic response mediated by RAR
in WT cells (Table IV). As expected, this combination had no effect on the apoptosis of RAR
/
and RXR
/
/RAR
/
cells. Neither
the RAR
/panRXR nor the RAR
/panRXR agonist
combination induced the apoptosis of WT, RXR
/
,
RAR
/
, RXR
/
/RAR
/
, and RXR
/
/RAR
/
cells, whereas they weakly triggered apoptosis of RAR
/
cells (Table III). 100 nM Am80, which acts as panRAR agonist at this concentration (Hashimoto et al., 1990
), was as
efficient as 100 nM RAR
agonist for WT, RXR
/
,
RAR
/
, and RXR
/
/RAR
/
cells. The panRAR/
panRXR combination was more effective than either the
RAR
/panRXR or the RAR
/panRXR combination in
RAR
/
cells, whereas these retinoids had no effects on
the apoptosis of RXR
/
/RAR
/
cells (Table III).
Thus, RAR
fully prevents the weak apoptotic response
that can be mediated by the RXR
/RAR
and RXR
/
RAR
pairs, and mutation of RAR
artifactually generates some functional redundancy (Table IV).
In vitro studies using either cell-free systems or cultured
cells cotransfected with vectors overexpressing the various
retinoid receptors and cognate recombinant reporter
genes, have suggested that RXR/RAR heterodimers could
be the functional units transducing the retinoid signal in
vivo. These studies have also indicated that the various
RXR/RAR heterodimers, resulting from the combination of either one of three RXRs (,
, or
) with either one of
the three RARs (
,
, or
), could be differentially involved in the numerous physiological events that are controlled by retinoids in vivo (Chambon, 1994
, 1996
). The results of RAR and RXR gene knockout studies in the
mouse have supported these suggestions, but their interpretation remains equivocal, in particular because cell-
autonomous and non-cell-autonomous effects cannot be
distinguished in the intact animal (Kastner et al., 1995
,
1997
).
The aim of the present study was to determine the actual role of the various RXR/RAR combinations as retinoid transducers in a well-established, cell-autonomous system, namely that provided by the retinoid-responsive F9
EC cells. To this end, differentiation into primitive, parietal, and visceral endoderms, as well as antiproliferative and apoptotic responses have been studied in RXR and
RAR single or compound mutant F9 EC cells cultured in
the presence of either tRA or panRXR- and/or RAR isotype-selective synthetic retinoids. Our present results are
summarized in Table IV with relevant data from previous
reports (Roy et al., 1995; Clifford et al., 1996
; Taneja et al.,
1996
), and lead to several important conclusions, which
are in keeping with those recently drawn from a study of the expression of RA-responsive genes in the same mutant
F9 EC cells (Chiba et al., 1997
).
Taken together, our genetic data and those obtained
with selective retinoids in WT or mutated cells establish
that RXR/RAR pairs are always involved in the transduction of the retinoid signal, irrespective of the nature of the
retinoid-induced event examined (differentiation, antiproliferative, or apoptotic effects). This is obvious from both
the comparison of single and double mutants, and the
combined use of the panRXR ligand with suboptimal concentrations of either one of the RAR isotype-specific, synthetic retinoids. Thus, since the panRXR-specific agonist
is never active on its own, all cellular events induced by retinoids in F9 EC cells appear to be mediated by RXR/
RAR heterodimers. Note that the "subordination" of
RXRs to RARs (i.e., that a RXR cannot be transcriptionally activated unless its heterodimer RAR partner is
liganded), which has been repeatedly observed in different cell systems (Roy et al., 1995; Chen et al., 1996
; Horn et al., 1996
; Taneja et al., 1996
), as well as in some in vitro studies (Durand et al., 1994
; Apfel et al., 1995
; Forman et al.,
1995
), may be important to prevent the promiscuous activation of the retinoid and other signaling pathways (e.g.,
those of thyroid hormones and vitamin D3) by RXR
ligands (Mangelsdorf and Evans, 1995
; Chambon, 1996
).
The dispensability of the RXR ligand that can be observed
in some instances when a saturating amount of a RAR-
selective ligand (notably in the case of RAR
) is used, has
been previously noted (Roy et al., 1995
). This dispensability most probably reflects the fact that the RAR activation
functions of RXR/RAR heterodimers alone are sufficient
to trigger the expression of the genes involved in the cellular event considered, whereas the synergistic effect of the
activation functions of the RXR heterodimeric partner becomes indispensable at lower concentrations of the RAR
ligand (Clifford, J., unpublished results), which are probably closer to physiological retinoic acid concentrations.
The second important conclusion of the present study is
that the various RXR/RAR heterodimers that can be
formed in F9 EC cells exhibit some functional specificity.
Indeed, each of the cellular events that are RA-induced in
F9 cells appears to preferentially involve a specific RXR/
RAR isotype combination (or set of combinations) (Table
IV). It appears that in all cases the RA signal is transduced by RXR/RAR
heterodimers in WT F9 cells. However,
both the RXR
/RAR
and RXR
/RAR
heterodimers
can mediate the RA-induced inhibition of WT F9 cell proliferation. Thus, in WT F9 cells, depending on the cellular
event considered, different RXR/RAR isotype heterodimers possess both specific functions and redundant functions
shared with other heterodimers. Interestingly, additional
redundant functions, not seen in WT cells, are revealed
when either RXR
or RAR
are not expressed. The presence of RAR
often hinders or blocks the activity of RAR
and RAR
, where the presence of RXR
can hinder the
activity of RXR(
+
) (Table IV). In several instances, the retinoid-induced cellular events mediated by RXR
/RAR
in WT cells can be mediated by RXR(
+
)/RAR
heterodimers in the absence of RXR
, and by RXR
/RAR(
and/or
) heterodimers in the absence of RAR
(Table
IV). Again, these redundancies vary according to the cellular event examined, further supporting the conclusion that the different RXR/RAR isotype heterodimers possess
some functional specificity.
The third conclusion is that gene knockouts generate artifactual conditions unmasking potential functional redundancies, which actually do not occur in the WT situation
(Table IV). For instance, in the case of visceral endoderm
differentiation, RXR(+
)/RAR
heterodimers can efficiently substitute for RXR
/RAR
heterodimers in the
absence of RXR
; in addition, either RXR
/RAR
or
RXR
/RAR
heterodimers can efficiently substitute for
RXR
/RAR
heterodimers in the absence of RAR
, even
though RXR
/RAR
heterodimers essentially mediate
this differentiation in WT F9 EC cells. How the presence of RXR
/RAR
heterodimers prevents potentially functionally redundant heterodimers from transducing the RA
signal is unknown, but it could be related to their differential affinities for the RAREs of the target genes involved
in the cellular processes induced by RA in F9 cells. In any
event, it is clear that the functional redundancies that are
revealed by gene knockout cannot be taken as evidence
for a lack of functional specificity of the knockout gene
product under WT physiological conditions. It is not unlikely that many of the functional redundancies that have
been so far revealed by mouse gene knockouts are similarly artifactually generated.
Interestingly, our present study also reveals that different RXR/RAR heterodimers can have opposite effects on
cell proliferation of F9 cells. RXR/RAR
or RAR
heterodimers are involved in the transduction of the antiproliferative effect of RA, but in the absence of RXR
and
RAR
, both RXR(
+
)/RAR
and RAR
heterodimers can mediate a proliferative effect of RA (Tables II and
IV). Note that induction of proliferation of certain cell
types by retinoids has been previously reported (Amos
and Lotan, 1990
; Koshimizu et al., 1995
). Our present observations on retinoid-induced cell antiproliferative and
proliferative effects, strengthen the conclusion that different RXR/RAR heterodimers can exert specific functions. These observations also suggest that the actual set of retinoid receptors present in a given cell may have a profound
influence on the effects generated by a retinoid treatment.
It is interesting to note that morphological differentiation of WT F9 EC cells can be efficiently triggered by a
combination of panRXR/RAR-specific (BMS961) agonists, but not by a combination of panRXR/RAR
-specific (BMS753) agonists, nor by a combination of a panRXR/RAR
-specific (BMS453) agonists. In contrast, P19 EC cell differentiation can be triggered by either a panRXR/RAR
or a panRXR/RAR
agonist combination,
but not by a panRXR/RAR
agonist combination (Taneja
et al., 1996
), whereas the differentiation of the NB4 acute
promyelocytic leukemia cells, and HL60 myeloblastic leukemia cells can be triggered by a combination of a panRXR/RAR
or a panRXR/RAR
agonists, but not by a
panRXR/RAR
agonist combination (Chen et al., 1996
).
Similarly, the apoptosis of NB4 cells can be induced by a
panRXR/RAR
agonist combination (Chen et al., 1996
),
which on the other hand is inefficient in the case of F9 cells
(Table III). Thus, different RAR isotype-specific agonists acting synergistically with a panRXR agonist are not only
more restricted than tRA in their effects on various cellular events in a given cell type (e.g., differentiation and apoptosis), but also affect differentially these events in a cell-specific manner. These cell type-specific effects of synthetic
retinoids may extend their potential for therapeutical use.
Finally, to our best knowledge, this study is the first report of multiple gene targeting (two alleles of two genes) in a mammalian cell-autonomous system. Similar approaches will allow the inactivation of any set of genes in a given cell, which will undoubtedly and particularly useful to elucidate the molecular mechanisms underlying complex biological events.
Table IV. Summary of the Involvement of the Various RARs and RXRs in the Transduction of the Retinoid Signal in F9 Cells, as Deduced from the Present and Previous Studies of RAR and RXR Mutant Cells and the Use of Receptor-specific Retinoids |
Received for publication 29 April 1997 and in revised form 25 August 1997.
Address all correspondence to P. Chambon, Institut de Génétique et de Biologie Moléculaire et Cellulaire, Centre National de la Recherche Scientifique/Institut National de la Santé et de la Recherche Médicale/Université Louis Pasteur, Collège de France, BP 163, 67404 Illkirch-Cedex, France. Tel.: (33) 388-65-32-13. Fax: (33) 388-65-32-03. E-mail: IGBMC@ IGBMC.U-STRASBG.FRWe are grateful to J-M. Bornert and P. Unger for technical assistance; to P.R. Reczek (Bristol-Meyers-Squibb, Pharmaceutical Research Institute, Buffalo, NY) for the gift of synthetic retinoids; K. Shudo (University of Tokyo, Tokyo, Japan) for Am80; to J.L. Imler (Transgène, Société Anonyme, Strasbourg, France) for pLXPB; to J. Brocard, P. Kastner, D. Lohnes, and R. Taneja for various oligonucleotides and plasmids; to C. Ebel, and C. Waltzinger for help with FACS® analysis; and to M.P. Gaub, and C. Rochette-Egly for various antibodies and helpful discussions. We thank H. Gronemeyer, P. Kastner, and all members of the retinoid group for helpful discussions. We also thank the cell culture facility for providing cells, I. Colas, F. Ruffenach, and E. Troech for oligonucleotide synthesis, the secretarial staff for typing, and R. Bucher, S. Metz, C. Werlé, B. Boulay, and J.M. Lafontaine for preparing the figures.
This work was supported by funds from the Centre National de la Recherche Scientifique, the Institut National de la Santé et de la Recherche Médicale, the Collège de France, the Centre Hospitalier Universitaire Régional, the Association pour la Recherche sur le Cancer, the Fondation pour la Recherche Médicale, the Human Frontier Science Program, and Bristol-Myers-Squibb. J. Clifford was supported by a fellowship from the Association pour la Recherche sur le Cancer and the US National Institutes of Health (grant No. IF32GM15857), and H. Chiba by a fellowship from the Centre National de la Recherche Scientifique.
AFP, -fetoprotein;
E2, estradiol;
EC, embryonal carcinoma;
EMSA, electrophoretic mobility shift assays;
HR, homologous recombination;
PGK, phosphoglycerate kinase;
RAR, retinoic acid receptor;
RXR, retinoid X receptor;
RARE, retinoic acid response element;
RT, reverse transcription;
tRA, all-trans retinoic acid;
VE, visceral endoderm;
WT, wild-type.
1. | Adra, C.N., P.H. Boer, and M.W. McBurney. 1987. Cloning and expression of the mouse pgk-1 gene and the nucleotide sequence of its promoter. Gene (Amst.). 60: 65-74 |
2. | Amos, B., and R. Lotan. 1990. Retinoid-sensitive cells and cell lines. Methods Enzymol. 190: 217-225 |
3. |
Apfel, C.,
M. Kamber,
M. Klaus,
P. Mohr,
S. Keider, and
P.K. LeMotte.
1995.
Enhancement of HL-60 differentiation by a new class of retinoids with selective activity on retinoid X receptor.
J. Biol. Chem.
270:
30765-30772
|
4. | Atencia, R., M. Garcia-Sanz, F. Unda, and J. Arechaga. 1994. Apoptosis during retinoic acid-induced differentiation of F9 embryonal carcinoma cells. Exp. Cell Res. 214: 663-667 |
5. | Blomhoff, R. 1994. Overview of vitamin A metabolism and function. In Vitamin A Health and Disease. R. Blomhoff, editor. Marcel Dekker, New York/ Basel/Hong Kong. 1-35. |
6. | Bouillet, P., M. Oulad-Abdelghani, S. Vicaire, J-M. Garnier, B. Schuhbaur, P. Dollé, and P. Chambon. 1995. Efficient cloning of cDNAs of retinoic acid-responsive genes in P19 embryonal carcinoma cells and characterization of a novel mouse gene, Stra1 (Mouse LERK-2/Eplg2). Dev. Biol. 170: 420-433 |
7. |
Boylan, J.,
D. Lohnes,
R. Taneja,
P. Chambon, and
L.J. Gudas.
1993.
Loss of
retinoic acid receptor ![]() |
8. |
Boylan, J.,
T. Lufkin,
C.C. Achkar,
R. Taneja,
P. Chambon, and
L.J. Gudas.
1995.
Targeted disruption of retinoic acid receptor ![]() ![]() ![]() |
9. | Chambon, P.. 1994. The retinoid signaling pathway: molecular and genetic analyses. Semin. Cell Biol. 5: 115-125 |
10. |
Chambon, P..
1996.
A decade of molecular biology of retinoic acid receptors.
FASEB (Fed. Am. Soc. Exp. Biol.) J.
10:
940-954
|
11. | Chen, J-Y., S. Penco, J. Ostrowski, P. Balaguer, M. Pons, J.E. Starrett, P.R. Reczek, P. Chambon, and H. Gronemeyer. 1995. RAR-specific agonist/antagonists which dissociate transactivation and AP1 transrepression inhibit anchorage-independent cell proliferation. EMBO (Eur. Mol. Biol. Organ.) J. 14: 1187-1197 [Abstract]. |
12. | Chen, J-Y., J. Clifford, C. Zusi, J. Starrett, D. Tortolani, J. Ostrowski, P.R. Reczek, P. Chambon, and H. Gronemeyer. 1996. Two distinct actions of retinoid-receptor ligands. Nature (Lond.). 382: 819-822 |
13. | Chiba, H., J. Clifford, D. Metzger, and P. Chambon. 1997. Distinct retinoid X receptor heterodimers are differentially involved in the control of expression of retinoid target genes in F9 embryonal carcinoma cells. Mol. Cell. Biol. 17: 3013-3020 [Abstract]. |
14. |
Clifford, J.,
H. Chiba,
D. Sobieszczuk,
D. Metzger, and
P. Chambon.
1996.
RXR![]() |
15. |
De Luca, L.M..
1991.
Retinoids and their receptors in differentiation, embryogenesis, and neoplasia.
FASEB (Fed. Am. Soc. Exp. Biol.) J.
5:
2924-2933
|
16. | Durand, B., M. Saunders, C. Gaudon, B. Roy, R. Losson, and P. Chambon. 1994. Activation function 2 (AF-2) of retinoic acid receptor and 9-cis retinoic acid receptor: presence of a conserved autonomous constitutive activating domain and influence of the nature of the response element on AF-2 activity. EMBO (Eur. Mol. Biol. Organ.) J. 13: 5370-5382 [Abstract]. |
17. | Forman, B.M., K. Umesono, J. Chen, and R.M. Evans. 1995. Unique response pathways are established by allosteric interactions among nuclear hormone receptors. Cell. 81: 541-550 |
18. |
Gaub, M-P.,
C. Rochette-Egly,
Y. Lutz,
S. Ali,
H. Matthes,
I. Scheuer, and
P. Chambon.
1992.
Immunodetection of multiple species of retinoic acid receptor ![]() |
19. | Giguère, V.. 1994. Retinoic acid receptors and cellular retinoid binding proteins: complex interplay in retinoid signaling. Endocr. Rev. 15: 61-79 |
20. | Glass, C.K.. 1994. Differential recognition of target genes by nuclear receptor monomers, dimers, and heterodimers. Endocr. Rev. 15: 391-407 |
21. | Green, S., I. Issemann, and E. Scheer. 1988. A versatile in vivo and in vitro eukaryotic expression vector for protein engineering. Nucl. Acids Res. 16: 369 |
22. | Gronemeyer, H., and V. Laudet. 1995. Transcription factors 3: nuclear receptors. Protein Profile. 2: 1173-1308 |
23. | Gudas, L.J., M.B. Sporn, and A.B. Roberts. 1994. Cellular biology and biochemistry of the retinoids. In The Retinoids: Biology, Chemistry and Medicine. M.B. Sporn, A.B. Roberts, and D.S. Goodman, editors. Raven Press, New York. 443-520. |
24. | Hashimoto, Y., H. Kagechika, and K. Shudo. 1990. Expression of retinoic acid receptor genes and the ligand-binding selectivity of retinoic acid receptors (RARs). Biochem. Biophys. Res. Commun. 166: 1300-1307 |
25. | Hogan, B.L.M., D. Barlow, and R. Tilly. 1983. F9 teratocarcinoma cells as a model for the differentiation of parietal and visceral endoderm in the mouse embryo. Cancer Surv. 2: 115-140 . |
26. |
Horn, V.,
S. Minucci,
V.V. Ogryzko,
E.D. Adamson,
B.H. Howard,
A.A. Levin, and
K. Ozato.
1996.
RAR and RXR selective ligands cooperatively induce
apoptosis and neuronal differentiation in P19 embryonal carcinoma cells.
FASEB (Fed. Am. Soc. Exp. Biol.) J.
10:
1071-1077
|
27. | Imler, J.L., C. Chartier, D. Dreyer, A. Dieterle, M. Sainte, Maie, T. Faure, A. Pavirani, and M. Methali. 1996. Novel complementation cell lines derived from human lung carcinoma A549 cells support the growth of E1-deleted adenovirus vectors. Gene Ther. 3: 75-84 |
28. |
Kastner, P.,
A. Krust,
C. Mendelsohn,
J.M. Garnier,
A. Zelent,
P. Leroy,
A. Staub, and
P. Chambon.
1990.
Murine isoforms of retinoic acid receptor ![]() |
29. | Kastner, P., M. Leid, and P. Chambon. 1994. The role of nuclear retinoic acid receptors in the regulation of gene expression. In Vitamin A in Health and Disease. R. Blomhoff, editor. Marcel Dekker, New York. 189-238. |
30. | Kastner, P., M. Mark, and P. Chambon. 1995. Nonsteroid nuclear receptors: what are genetic studies telling us about their role in real life? Cell. 83: 859-869 |
31. |
Kastner, P.,
M. Mark,
N. Ghyselinck,
W. Krezel,
V. Dupé,
J.M. Grondona, and
P. Chambon.
1997.
Genetic evidence that the retinoid signal is transduced by
heterodimeric RXR/RAR functional unit during mouse development.
Development (Camb.).
124:
313-326
|
32. | Keaveney, M., and H.G. Stunnenberg. 1995. Retinoic acid receptors. In Inducible Gene Expression. vol. 2. P.A. Bauerle, editor. Birkhäeuser, Boston. 187-242. |
33. | Koshimizu, U., M. Watanabe, and N. Nakatsuji. 1995. Retinoic acid is potent growth activator of mouse primordial germ cells in vitro. Dev. Biol. 168: 683-685 |
34. | Lehmann, J.M., L. Jong, A. Fanjul, J.F. Cameron, X.P. Lu, P. Haefner, M.I. Dawson, and M. Pfahl. 1992. Retinoids selective for retinoid X receptor response pathways. Science (Wash. DC). 258: 1944-1946 |
35. | Leid, M., P. Kastner, and P. Chambon. 1992a. Multiplicity generates diversity in the retinoic acid signaling pathways. Trends Biochem. Sci. 17: 427-433 |
36. | Leid, M., P. Kastner, R. Lyons, H. Nakshatri, M. Saunders, T. Zacharewski, J-Y. Chen, A. Staub, J-M. Garnier, S. Mader, et al . 1992b. Purification, cloning, and RXR identity of the HeLa cell factor with which RAR or TR heterodimerizes to bind target sequences efficiently. Cell. 68: 377-395 |
37. |
Leroy, P.,
A. Krust,
A. Zelent,
C. Mendelsohn,
J-M. Garnier,
P. Kastner,
A. Dierich, and
P. Chambon.
1991.
Multiple isoforms of the mouse retinoic acid
receptor ![]() |
38. |
Lohnes, D.,
P. Kastner,
A. Dierich,
M. Mark,
M. LeMeur, and
P. Chambon.
1993.
Function of retinoic acid receptor ![]() |
39. | Mangelsdorf, D.J., and R.M. Evans. 1995. The RXR heterodimers and orphan receptors. Cell. 83: 841-850 |
40. | Mangelsdorf, D.J., K. Umesono, and R.M. Evans. 1994. The retinoid receptors. In The Retinoids: Biology, Chemistry and Medicine. M.B. Sporn, A.B. Roberts, and D.S. Goodman, editors. Raven Press, New York. 319-349. |
41. | Martin, C.A., L.M. Ziegler, and J.L. Napoli. 1990. Retinoic acid, dibutyryl-cAMP, and differentiation affect the expression of retinoic acid receptors in F9 cells. Proc. Natl. Acad. Sci. USA. 87: 4804-4808 [Abstract]. |
42. | Metzger, D., J. Clifford, H. Chiba, and P. Chambon. 1995. Conditional site-specific recombination in mammalian cells using a ligand-dependent chimeric Cre-recombinase. Proc. Natl. Acad. Sci. USA. 92: 6991-6995 [Abstract]. |
43. |
Rochette-Egly, C.,
Y. Lutz,
M. Saunders,
I. Scheuer,
M-P. Gaub, and
P. Chambon.
1991.
Retinoic acid receptor ![]() |
44. |
Roy, B.,
R. Taneja, and
P. Chambon.
1995.
Synergistic activation of expression
of retinoic acid (RA)-responsive genes and induction of embryonal carcinoma cell differentiation by an RA receptor ![]() ![]() ![]() ![]() |
45. | Sauer, B., and N. Henderson. 1990. Targeted insertion of exogenous DNA into the eukaryotic genome by the Cre recombinase. New Biol. 2: 441-449 |
46. | Sleigh, M.J.. 1992. Differentiation and proliferation in mouse embryonal carcinoma cells. Bioessays. 14: 769-775 |
47. | Strickland, S.. 1981. Mouse teratocarcinoma cells: prospects for the study of embryogenesis and neoplasia. Cell. 24: 277-278 |
48. |
Taneja, R.,
P. Bouillet,
J.F. Boylan,
M-P. Gaub,
B. Roy,
L.J. Gudas, and
P. Chambon.
1995.
Reexpression of retinoic acid receptor (RAR)![]() ![]() ![]() ![]() |
49. |
Taneja, R.,
B. Roy,
J-L. Plassat,
C.F. Zusi,
J. Ostrowski,
P.R. Reczek, and
P. Chambon.
1996.
Cell-type and promoter-context dependent RAR redundancies for RAR![]() |
50. | Wan, Y-J., L. Wang, and T-C. Wu. 1994. The expression of retinoid X receptor genes is regulated by all-trans and 9-cis-retinoic acid in F9 teratocarcinoma cells. Exp. Cell Res. 210: 56-61 |
51. |
Zelent, A.,
A. Krust,
M. Petkovich,
P. Kastner, and
P. Chambon.
1989.
Cloning
of murine ![]() ![]() ![]() |