COUP-TFI (Chicken Ovalbumin Upstream Promoter-Transcription Factor I) Regulates Cell Migration and Axogenesis in Differentiating P19 Embryonal Carcinoma Cells
Françoise Adam,
Tony Sourisseau,
Raphaël Métivier,
Yann Le Page,
Christine Desbois,
Denis Michel and
Gilles Salbert
Equipe Information et Programmation Cellulaire (F.A., T.S., Y.L.P.,
C.D., D.M., G.S.) Equipe dEndocrinologie Moléculaire de la
Reproduction (R.M.) UPRES-A CNRS 6026 Université de
Rennes I, Campus de Beaulieu 35042 Rennes Cedex, France
 |
ABSTRACT
|
---|
The developmental expression patterns of the
nuclear orphan receptors COUP-TFs (chicken ovalbumin upstream
promoter-transcription factors) have been correlated to neurogenesis in
several animal species. Nevertheless, the role of COUP-TFs in
neurogenesis remains unknown. We have studied the functional
involvement of COUP-TFI in retinoic acid (RA)-induced neuronal
differentiation of P19 embryonal carcinoma cells through two
complementary approaches: 1) deregulated expression of COUP-TFI, and 2)
inactivation of endogenous COUP-TFs by means of a dominant-
negative COUP-TFI mutant. Low levels of wild-type (wt)COUP-TFI
transgene expression did not inhibit neural cell fate and primarily
enhanced neuron outgrowth from RA-treated P19 aggregates. In contrast,
high COUP-TFI expression impeded the neuronal differentiation of P19
cells induced with RA, resulting in cell cultures lacking neurons. This
morphological effect was correlated to an elevated level of E-cadherin
mRNA. The dominant-negative COUP-TFI mutant induced cell packing after
RA treatment and inhibited neurite extension and neuron outgrowth from
aggregates. A RGD peptide interference assay indicated that endogenous
COUP-TFs could favor migration of neurons through an integrin-dependent
mechanism. Accordingly, vitronectin mRNA levels were shown to be
up-regulated by COUP-TFI by RT-PCR analysis, and COUP-TFI stimulated
the mouse vitronectin promoter activity in transient transfection
assays. Taken together, these data indicate that COUP-TFI is not simply
a global repressor of retinoid functions, but shows a high selectivity
for regulating genes involved in cellular adhesion and migration
processes that are particularly important for neuronal differentiation.
 |
INTRODUCTION
|
---|
Pattern formation and organogenesis in metazoan are orchestrated
by transcriptional regulators, cell adhesion molecules, and signal
transduction systems that have been tightly maintained along evolution.
The nuclear receptor superfamily (for review, see Ref. 1) includes very
ancient members such as retinoid X receptor (RXR) (2), FTZ-F1 (3), and
chicken ovalbumin upstream promoter-transcription factor (COUP-TF) (4, 5), which are important developmental regulators in vertebrates and
arthropods (6, 7, 8, 9, 10, 11, 12, 13, 14, 15) and have been reported in cnidarians (16). The
COUP-TFI orphan receptor was originally characterized as a
transcriptional activator of the chicken ovalbumin gene (17). Since
then, COUP-TFI and other closely related receptors, such as COUP-TFII
(18), have been extensively studied, both in terms of biochemical
properties (i.e. DNA binding, dimerization, transcriptional
activation, and repression) and tissue distribution, with a particular
emphasis on developmental processes (for review, see Ref. 19). In all
the species that were examined for the presence of COUP-TF during
development (from sea urchin to mouse), expression of this orphan
receptor was clearly associated with neurogenesis (20, 21, 22, 23, 24, 25).
Nonetheless, the precise role of COUP-TFI in the regulation of neuronal
growth and differentiation is still unknown. Recent studies suggest
that COUP-TFI is involved in the regulation of cell-cell contacts (26)
and modulates axonal growth (14, 27). Indeed, COUP-TFI gene disruption
in mouse results in decreased arborization of spinal nerves (14), in
abnormal morphogenesis of the ninth cranial nerve and ganglion (14),
and in defects in the guidance of axons emanating from thalamic neurons
that normally project to cortical layer IV (27). Disruption of the
mouse COUP-TFII gene induced early lethality (before day 10
postcoitum), thus making it difficult to analyze the role of COUP-TFII
in neurogenesis (28). Even if a certain degree of redundancy between
COUP-TFI and COUP-TFII is likely to exist, as in the case of retinoid
receptors (29), these knock-out experiments suggest that each mouse
COUP-TF gene ensures nonredundant functions.
Several studies have pointed out the possibility that COUP-TF genes
could be part of retinoid signaling pathways both in vivo
and in cell culture systems (30, 31, 32, 33). Notably, up-regulation of
COUP-TFI and COUP-TFII genes occurs during the differentiation programs
triggered by retinoic acid (RA) in mouse teratocarcinoma cells such as
P19 embryonal carcinoma (EC) cells (32). Pluripotent P19 EC cells can
be induced to differentiate into all three germ layer derivatives
(i.e. ectoderm, endoderm, and mesoderm) when appropriate
inducers and culture conditions are used (34, 35, 36). When P19 cells are
grown as aggregates, RA induces a neuroectodermal-like differentiation
pathway that generates neurons, glial, and fibroblast-like cells (34, 37). Therefore, P19 cells have been widely used to screen for genes
involved in neuronal differentiation (38, 39, 40). We report here the
consequences of supraphysiological doses of COUP-TFI, and of functional
inactivation of endogenous COUP-TFs, on the RA-induced differentiation
of P19 cells. Morphological studies show that a too-early (or a
too-high) expression of wild-type (wt)COUP-TFI impedes neural
differentiation. Remarkably, inhibition of endogenous COUP-TFs by
expression of a dominant-negative COUP-TFI mutant resulted in a
strengthening of cell-cell contacts, decreased axonal growth, and
slower migration of neurons. These data suggest a major function of
COUP-TFI during development in controlling cell adhesion.
 |
RESULTS
|
---|
C141
S COUP-TFI Acts as a
Dominant-Negative Mutant Inhibiting wtCOUP-TFI Binding to DNA
Mutation C141
S, by replacing the fourth
cysteine residue of the second zinc finger by a serine, is likely to
disrupt zinc coordination, and thus to disorganize the overall folding
of the DNA-binding domain (DBD). In accordance with this hypothesis,
this COUP-TF mutant does not bind DNA (41). Since the major
dimerization interface of nuclear receptors is contained in the
ligand-binding domain, we postulated that the mutant COUP-TFI
should still be able to dimerize with the wtCOUP-TFI. To test this
hypothesis, we used a yeast two-hybrid system (Fig. 1A
) and a
glutathione-S-transferase (GST)-pull-down assay (Fig. 1B
).
These two complementary approaches generated similar results and showed
that heterodimers of wt and mutant COUP-TFI can be formed. Indeed, in
the yeast system, ß-galactosidase activity was detected when the
Gal4DBD/
COUP-TFI construct was used in conjunction with either
Gal4AD/COUP-TFI or Gal4AD/ mutCOUP-TFI (Fig. 1A
). These data were
confirmed in pull-down assays showing that a
35S-labeled mutCOUP-TFI was retained on a
GST-wtCOUP-TF I matrix (Fig. 1B
). As the mutant COUP-TFI was defective
in DNA binding, it seemed reasonable to assume that the
wtCOUP-TFI/mutCOUP-TFI heterodimer would bind less stably to DNA than a
wtCOUP-TFI homodimer. This was verified by electromobility shift assays
(EMSA) in which in vitro translated proteins were incubated
with the high-affinity COUP-TF-binding site DR-1 (42) as a probe (Fig. 1C
). In vitro transcription/translation reactions were run
with a fixed amount of pcDNA/wtCOUP-TFI vector (100 ng - 1X, Fig. 1C
)
and increasing amounts of pcDNA/mutCOUP-TFI (from 0 to 900 ng), the
total amount of vector DNA being adjusted to 1 µg with the empty
pcDNA expression vector. Whereas a strong binding of wtCOUP-TFI
homodimers was seen on the DR-1 probe, no retarded complex was observed
in the presence of mutCOUP-TFI (Fig. 1C
). Cotranslation of wtCOUP-TFI
with increasing amounts of mutCOUP-TFI resulted in a progressive
decrease in the amount of retarded complex, suggesting that mutCOUP-TFI
could be used as a dominant-negative mutant to inhibit wtCOUP-TFI
activity.

View larger version (23K):
[in this window]
[in a new window]
|
Figure 1. Mutant C141 S COUP-TFI Dimerizes with
wtCOUP-TFI and Inhibits Its DNA Binding Activity
A, mutCOUP-TFI interacts with wtCOUP-TFI in a yeast two-hybrid
system. Y190 yeast cells were transformed with the different vectors as
shown. The resulting ß-galactosidase activities were assayed and are
shown as Miller units. Each bar represent the mean ±
SEM of four values obtained in two independent experiments.
B, mutCOUP-TFI is retained on a GST-wtCOUP-TFI matrix. 4
µl of 35S-labeled in vitro
translated mutCOUP-TFI were allowed to interact either with GST alone
(lane 2) or a GST- COUP-TFI fusion protein (lane 3) immobilized on
glutathione-agarose beads. After extensive washing, the retained
fractions were analyzed by SDS-PAGE. For comparison, one fifth of the
volume of 35S-labeled mutCOUP-TFI used for
interaction with the fusion protein was loaded directly on the gel
(input, lane 1). The position of the mutCOUP-TFI protein is indicated
by an arrow. C, mutCOUP-TFI inhibits wtCOUP-TFI DNA binding
in EMSA. In vitro cotranslated wtCOUP-TFI and mutCOUP-TFI
were incubated with a 32P-labeled DR-1 probe
before electrophoresis. wtCOUP-TFI homodimers generated a retarded
complex indicated by an arrow. The ratio between wt and
mutCOUP-TFI expression vector used for in vitro
transcription/translation is shown (1X corresponds to 100 ng of
vector). Total amount of vector DNA was systematically adjusted to 1
µg with the empty expression vector. D, COUP-TFI C141 S inhibits
wtCOUP-TFs in cotransfection assays. CHO cells were cotransfected with
the ApoA1-tk-CAT reporter gene and different combinations of
pECE-RAR , pECE-RXR , pcDNA-wtCOUP-TFI, and pcDNA-mutCOUP-TFI
expression vectors. Results are shown as the mean ±
SEM (n = 3) of the relative CAT activities.
E, Western blot analysis of COUP-TFII expression in yeast, CHO, Hela,
and HepG2 cells. An arrow indicates the position of
COUP-TFII.
|
|
To determine whether the mutated COUP-TFI protein could alter
wtCOUP-TFI-mediated transcriptional regulation, we cotransfected
Chinese hamster ovary (CHO) cells with a retinoid-responsive reporter
gene (ApoA1-tk-CAT) with or without retinoid receptor expression
vectors in various combinations with wt and mutant COUP-TFI plasmids.
This reporter gene contains a DR-2 element overlapping a DR-1 element
and is known to be activated by retinoid receptors, an activation that
can be counteracted by COUP-TF probably through binding to the DR-1
site (45). Surprisingly, when we cotransfected the reporter gene with
increasing amounts of mutCOUP-TFI vector alone, chloramphenicol acetyl
transferase (CAT) activities markedly increased (Fig. 1D
). This first
observation suggested that CHO cells express endogenous COUP-TFs that
are likely to repress the basal activity of the reporter gene and that
the mutated COUP-TFI can block this repression. In this respect, it
seemed logical to counteract the positive effect of mutCOUP-TFI by
cotransfecting the wtCOUP-TFI vector (Fig. 1D
). Retinoid receptors were
able to activate the transcription of the reporter gene in the presence
of 10-6 M RA, and this activation
was even higher when mutCOUP-TFI was expressed (Fig. 1D
). These data
imply first that endogenous COUP-TFs are able to partially repress
retinoid receptors, and second that mutCOUP-TFI does not negatively
interfere with retinoid receptor-mediated transcriptional activation.
This provides evidence for the specificity of mutCOUP-TFI action.
Finally, mutCOUP-TFI expression was able to abolish the retinoid
receptor inhibition mediated by exogenous wtCOUP-TFI (Fig. 1D
). These
transfection experiments thus allow the interpretation that mutCOUP-TFI
specifically interferes with wtCOUP-TF functions, and thus may not
alter other nuclear receptor signaling pathways. Since these
transfection data suggested that CHO cells did express endogenous
COUP-TF genes, we ran a Western blot analysis and found that, indeed,
CHO cells express detectable levels of COUP-TFII as shown in Fig. 1E
.
This was true also for Hela cells, whereas HepG2 cells did not express
COUP-TFII (Fig. 1E
). Analysis of COUP-TFI expression showed that HepG2
cells but not CHO and Hela cells were positive for
COUP-TFI in Western blot (data not shown). Thus, in view of the
transfection data, the mutant COUP-TFI is likely to be able to
interfere with both COUP-TFI and COUP-TFII functions since CHO cells
express only COUP-TFII (note that a similar transfection experiment as
the one shown in Fig. 1D
was run in Hela cells and gave identical
results). Even if in vitro data are suggestive of a direct
inhibitory effect of mutCOUP-TFI on its wild-type counterpart, one
cannot exclude that mutCOUP-TFI modulates wtCOUP-TFI or other nuclear
receptors by titrating corepressors or coactivators. However,
transfection data suggest that mutCOUP-TFI does not titrate the
coactivators required for the transcriptional response to
retinoids.
Stable COUP-TFI Expression in Transfected P19 Cells
The use of the cytomegalovirus (CMV) promoter to drive expression
of either wild-type or mutated COUP-TFI in P19 cells allowed us to
assess the effects of low vs. high levels of transgene
expression. Indeed, the CMV promoter can be activated by cAMP in the
teratocarinoma cell line PCC7 (43) as well as in P19 cells (Fig. 2A
) [note that the CMV promoter is also
activated about 3- to 4-fold above basal activity by RA in P19 cells
(data not shown)]. High levels of COUP-TFI mRNA were detected in cells
transfected with wt or mutant COUP-TFI vectors and treated with 1
mM cAMP for 24 h (Fig. 2A
), whereas low but
detectable levels were seen in the absence of cAMP (Fig. 2A
). No
COUP-TFI expression was detected in control cells treated or not with
cAMP, in agreement with the fact that undifferentiated P19 cells do not
express COUP-TFI (32). The presence of COUP-TFI in the transfected
cells was checked at the protein level by electromobility-shift assay
(Fig. 2
, B and C). For these experiments, we used the DR-1 binding-site
for COUP-TF and protein extracts from aggregated cells that had been
treated either with cAMP for 48 h (Fig. 2
, B and C) or with
different combinations of cAMP and RA for 48 h as indicated (Fig. 2E
). Extracts from P19 control cells (pcDNA) treated with cAMP
generated a single complex with the DR-1 probe (noted by an
asterisk, Fig. 2B
) that might be due to the interaction of
EAR2 (a COUP-TF-related orphan receptor) with the DR-1 element
according to Jonk et al. (32). When we used extracts from
wtCOUP-TFI-transfected cells that had been treated with cAMP, a slower
migrating complex appeared (arrow, Fig. 2B
) that reflected
interaction of the exogenous COUP-TFI with the DR-1 probe, as shown by
the antibody supershift (Fig. 2B
). The specificity of DNA recognition
by the overexpressed wtCOUP-TFI was assessed by competition
experiments in EMSA (Fig. 2C
) using the DR-1 element as a probe. The
various competitors could be ranked as follows from high affinity to
low affinity: DR-1, DR-7, DR-5, ERE, AP-1, and GRE, with the two last
showing very low to no affinity for COUP-TF. These data are in
accordance with the known affinities of COUP-TF for different binding
sites (21, 41, 44). Treatment of P19 control cell aggregates with
10-6 M RA resulted in the
appearance of a major complex mainly corresponding to COUP-TFI/DNA
interaction as shown by the supershift obtained in the presence of
COUP-TFI antibodies (Fig. 2D
). The lack of supershift in the presence
of COUP-TFII antibodies suggests that the induction of the COUP-TFII
gene might be delayed compared with the induction of the COUP-TFI gene.
In another experiment we compared the levels of DR-1 binding activity
in the different cell lines (Fig. 2E
). Cells containing the wtCOUP-TFI
transgene expressed low levels of this orphan receptor in the absence
of drugs, but high levels of wt COUP-TFI were observed in the presence
of 1 mM 8-Br-cAMP (arrow, Fig. 2E
).
The amount of complex generated from transgene expression was even
higher than the one formed by the endogenous COUP-TFs after
induction by 10-6 M RA.
Finally, when cells were treated with both RA and cAMP, the amount of
COUP-TF/DNA complex was slightly higher than when cells were treated
with cAMP alone. Interestingly, the presence of high amounts of
COUP-TFI inhibited the putative EAR2/DNA interaction (Fig. 2E
). Similar
results have been described by Jonk et al. (32). In cells
expressing the mutated form of COUP-TFI, cAMP did not induce the
formation of a retarded complex (Fig. 2E
) in accordance with the
fact that COUP-TF C141
S does not bind DNA
(41). Formation of the endogenous COUP-TF/DNA complex was inhibited by
expression of the mutated COUP-TF (Fig. 2E
) in the presence of RA and
completely abolished in the presence of RA and cAMP, indicating that
the mutated COUP-TF works as a dominant-negative mutant, sequestering
endogenous COUP-TFs.

View larger version (43K):
[in this window]
[in a new window]
|
Figure 2. Stable Expression of COUP-TFI in Transfected P19 EC
Cells
A, RT-PCR analysis of transgene expression. Control cells (pcDNA),
wild-type (wt), or mutant (mut) COUP-TF expressing cells were grown for
24 h as a monolayer in the presence or absence of cAMP as
indicated before RNA extraction and RT-PCR analysis of COUP-TFI and the
invariant PO gene expression. Results show the ethidium bromide
staining of a 2% agarose gel. B and C, EMSAs of control (pcDNA) and
wtCOUP-TFI cells treated as aggregates for 48 h with cAMP. An
asterisk indicates the putative Ear2/DNA complex (see
text), an arrow shows the position of the COUP-TFI/DNA
complex, and an open circle shows the position of the
supershifted complex in the presence of COUP-TFI antibodies (Ab). The
oligonucleotides indicated in panel C were added as competitors at
a 20-fold molar excess. D, DR-1 binding activity in P19 cell aggregates
treated with 10-6 M RA for
48 h. E, DR-1 binding activities in the different cell lines
cultured as aggregates for 48 h and treated as indicated with
all-trans-retinoic acid (RA) and/or cAMP. Symbols
are the same as in Fig. 1B . Note that the mutant COUP-TFI inhibits
endogenous COUP-TFs binding to the probe.
|
|
RA-Induced Neural Differentiation Is Severely Affected in
wtCOUP-TFI- Overexpressing Cells
Morphological characteristics of undifferentiated P19 cells were
not modified by the expression of either wt or mutated COUP-TFI
transgenes (Fig. 3
). Indeed, they
remained small cells with a high nucleus to cytoplasm ratio.
Furthermore, there was no difference in doubling time (
13 h) between
the three cell lines (data not shown). We next asked whether COUP-TFI,
which is known to inhibit retinoid pathways in cell culture systems
(43, 44, 45), could modulate neural differentiation of P19 cells. Ten
passages after the initial transfection, cells were aggregated in the
presence of 1 mM cAMP to stimulate the expression of the
CMV-COUP-TFI transgenes, or 1 µM all-trans-RA,
or both. After 3 days, the aggregates were dissociated, and individual
cells were plated onto cell culture dishes (Fig. 3
). After dissociation
of the aggregates, the replated cells were observed during the next
48 h of differentiation (Fig. 3
). After 24 h, the pcDNA cell
culture was already constituted of two different populations:
phase-dark cells growing attached to the plastic, and phase-bright
cells growing on the top of the first layer of cells and extending
neurites. This was observed for both treatments (RA or RA+cAMP). During
the following hours, the underlying cells became confluent in
RA-treated cells and the neurite network increased (Fig. 3
). In
RA+cAMP-treated pcDNA cells the underlying monolayer never became
confluent but neurite extension still went on. wtCOUP-TFI-expressing
cells behaved like pcDNA cells when treated with RA only. Conversely,
RA+cAMP- treated wtCOUP-TFI cells did not differentiate into
neuron-like cells, and only the phase-dark cells attached to the
plastic were observed (Fig. 3
). Thus, an early overexpression of
wtCOUP-TFI clearly inhibited neuronal differentiation but did not seem
to block other differentiation pathways. Similar results were obtained
when we first treated the wtCOUP-TFI cells with cAMP for 2 days
before adding RA (data not shown), indicating that high levels of
COUP-TFI before RA addition only block the neuronal differentiation
pathway, and thus that COUP-TFI is not a global inhibitor of retinoid
functions. When the mutant COUP-TFI cells were treated with RA alone,
cells remained closely associated in compact clusters during 48 h,
and a reduced number of phase-dark fibroblast-like cells and astrocytes
developed when compared with control cells. No neurite outgrowth could
be detected during the first 24 h, suggesting a delay in neural
differentiation. After 2 days the mutCOUP-TFI culture was
morphologically similar to the pcDNA cell culture with the exception
that the plastic-attached cells did not yet form a confluent monolayer
(Fig. 3
). In the presence of both RA and cAMP, most cells (
98%) did
not attach to the plastic after dissociation of the aggregates and died
within 24 h, rendering impossible the phenotypic analysis of these
cells. Such a cell death after treatment with RA and cAMP was also
observed, but to a much lower extent (
50% survival), for control
cells. These results suggest either that high levels of mutated
COUP-TFI are cytotoxic or cytostatic, or that endogenous COUP-TFs are
required for cell survival during differentiation. This last
possibility would be consistent with the observation that
wtCOUP-TFI-expressing cells did not undergo cell death when treated
with RA and cAMP (Fig. 3
). Finally, cells that had been treated with
cAMP alone did not differentiate as previously observed (46) and did
not show high levels of cell death (Fig. 3
).

View larger version (105K):
[in this window]
[in a new window]
|
Figure 3. Overexpression of mutCOUP-TFI Induces Cell Death in
Differentiating Cells
Cellular aggregates were treated with RA, cAMP, or both, for 3 days and
dissociated before plating onto tissue culture-grade plastic and
photographed 24 or 48 h later. Photographs show details of the
different cultures and are representative of the content of the entire
plates. Neurons are seen as phase bright cells growing on the top of
phase dark cells (magnification, x345). For comparison, the top
panels show micrographs of undifferentiated cells (pcDNA,
wtCOUP-TFI, and mutCOUP-TFI from left to right). Note
that cells treated with cAMP alone do not differentiate (bottom
panels).
|
|
Induction of the POU III gene Brn-2 by RA has been previously shown to
be a crucial step for neural differentiation of P19 cells (47, 48).
Semiquantitative RT-PCR analysis of Brn-2 expression in our different
cell lines did not reveal any change in the RA-induced Brn-2 mRNA
levels (Fig. 4
). These data did not
correlate well with the morphological analysis of wtCOUP-TFI cells
after RA induction, as almost no neurons were seen after cotreatment
with RA and cAMP (see Fig. 3
). This apparent discrepancy suggests that
high levels of COUP-TFI inhibit P19 cell neuronal differentiation
through a blockade at a differentiation state in which the Brn-2 gene
is already expressed. Schmidt et al. (49) suggested the
existence of an intermediate epithelial precursor, in the
neuroectodermal differentiation pathway of P19 cells, expressing high
levels of E-cadherin. We thus examined E-cadherin mRNA levels by RT-PCR
and found that, indeed, RA was able to dramatically suppress E-cadherin
expression in control cells as well as in cells expressing mutCOUP-TFI
and, to a lesser extent, in wtCOUP-TFI cells (Fig. 4
). Conversely, in
the presence of cAMP, RA was not able to inhibit E-cadherin expression
in wtCOUP-TFI cells.

View larger version (49K):
[in this window]
[in a new window]
|
Figure 4. RT-PCR Analysis of the Expression of Selected
Retinoid-Responsive Genes
RT-PCR analysis was run with RNA extracted from aggregates of pcDNA,
wt, and mutant COUP-TFI cells treated as indicated for 48 h.
Bmp-4, Bone morphogenetic protein-4; E-cad, E-cadherin.
|
|
Inhibition of Endogenous COUP-TFs Results in Impaired
Axogenesis
Since excessive cell death occurred in mutCOUP-TFI-expressing
cells treated simultaneously with RA and cAMP, we could not evaluate
the effects of a high expression of mutCOUP-TFI on neural
differentiation. However, low levels of mutCOUP-TFI were sufficient to
lead to a delayed neurite extension (see Fig. 3
), indicating a putative
role of endogenous COUP-TFs in the control of axonal growth. To test
this hypothesis further, we treated aggregated cells with RA alone for
3 days and then plated the aggregates without dissociation in
tissue-culture grade plastic dishes in the presence or not of 1
mM cAMP. Cells were maintained in culture for 5 days before
antineurofilament immunohistochemistry (Fig. 5
). As shown in Fig. 5
, extensive
neuritic outgrowth was seen for control cells and wtCOUP-TFI cells
in the presence or not of cAMP. Neurite extension was observed also for
mutCOUP-TFI cells cultured in the absence of cAMP. Strikingly, neurite
bundles as well as individual neurites were completely absent after
enhanced expression of mutCOUP-TFI with 1 mM cAMP, despite
the presence of numerous neurofilament-positive cell bodies (Fig. 5
).
Similar results were obtained when cells were plated from dissociated
aggregates. Interestingly, as observed in Fig. 5
, removal of cAMP from
the medium after 3 days allowed neurite extension, showing that
inhibition of axogenesis by the dominant-negative COUP-TF can be
reversed.

View larger version (90K):
[in this window]
[in a new window]
|
Figure 5. P19 Cell Endogenous COUP-TFs Control Axogenesis
Aggregated cells were plated after a 3-day RA treatment and further
cultured for 5 days in the presence or not of 1 mM
cAMP before anti-NF200 immunohistochemistry (magnification, x400).
Note the absence of neurites in mutCOUP-TFI cells treated with cAMP,
despite the presence of neurofilament (NF200)-positive cell bodies
(dark staining). Photographs show details of the
different cultures and are representative of the content of the entire
plates.
|
|
COUP-TFI Promotes Neuronal Migration
During the course of the preceding experiment, we noticed a clear
difference in the spreading of aggregates from the stable cell lines
(neurons tend to stay closely associated in mutCOUP-TFI cells). We next
decided to analyze in more detail the behavior of the cells after
plating undissociated aggregates previously treated for 3 days
with RA (Fig. 6
). After 2 days, the
aggregates had completely disappeared from wtCOUP-TFI cell cultures,
whereas most of them were still observable in mutCOUP-TFI cell cultures
(compare "wtCOUP-TFI-no peptide" with "mutCOUP-TFI-no peptide",
Fig. 6A
). Cells were then fixed and examined for the presence of
neurons by antineurofilament immunohistochemistry (Fig. 6A
). The
results revealed that neurons had migrated on the underlying
monolayer in the case of wtCOUP-TFI cells but not of mutCOUP-TFI cells
(Fig. 6A
), despite an equivalent spreading of the monolayer on the
plastic surface. Quantification of this phenomenon revealed that 100%
of the aggregates were totally spread, or with extensive neuron
outgrowth, in wtCOUP-TFI cells, against 44% for control cells and 18%
for mutCOUP-TFI cells (Fig. 6B
).

View larger version (54K):
[in this window]
[in a new window]
|
Figure 6. COUP-TFI Promotes Neuronal Migration
A, Aggregates of cells treated for 3 days with 1 µM
RA were plated onto tissue culture-grade plastic and further cultured
for 24 h before the RGDS and SDGRG peptides were added at the
indicated concentrations. After an additional day in culture, cells
were fixed and stained for the presence of NF200 by
immunohistochemistry (magnification, x220). Neurons and neurites were
intensely stained and either spread on the top of astrocytes
[wtCOUP-TFI (no peptide)], or packed together [mutCOUP-TFI (no
peptide), or wtCOUP-TFI RGDS 0.1 mg/ml]. B, The percentage of
aggregates presenting either migrating neurons out of the core of the
aggregates or a completely spread organization was determined and is
shown as the percentage of aggregates with neuron outgrowth in function
of the cell lines, in the absence of peptide (control) or in the
presence of SDGRG and RGDS peptides at 0.1 mg/ml. Results are shown as
the mean ± SEM for 150 aggregates of each cell
line scored in three independent experiments.
|
|
We then used a peptide interference assay to determine to which extent
the behavior of P19-derived neurons was dependent on extracellular
matrix (ECM)/receptor interactions. A number of ECM proteins
(e.g. fibronectin and vitronectin) share an RGD motif
involved in the recognition by different integrin
ß heterodimers
(50). Integrins play major roles in cell motility and migration (50, 51) and could thus be involved in P19 neuron migration. At high dosage
(1 mg/ml), the RGDS peptide totally inhibited cell spreading and
migration on the plastic surface whereas the control peptide SDGRG did
not. This was observed for all three cell lines (see Fig. 6A
for
wtCOUP-TFI cells). More interestingly, at low dosage (0.1 mg/ml),
astrocyte and fibroblast spreading and migration were not inhibited by
the RGDS peptide, but neuron migration on the surface of these cells
was clearly counteracted (Fig. 6
, A and B). In the resulting cultures,
neuron cell bodies stayed closely associated, as in the case of the
mutCOUP-TFI cells untreated with RGDS peptide. Nonetheless, these
grouped neurons still extended neurites that did not seem to project on
the underlying cells (data not shown). These experiments suggest that
the endogenous COUP-TFI promotes neuronal migration through a mechanism
involving integrin receptors. This function of COUP-TFI seems to be
unrelated to the control of axogenesis since neurite growth was
maintained in the RGDS-treated cells.
COUP-TFI Modulates ECM Synthesis
To test for differential adhesion/migration of our cell lines on
purified substrates, we next plated 3 days-RA-treated aggregates onto
plastic dishes coated with poly D-lysine, laminin, or
fibronectin, in neurobasal medium complemented with N2 instead of serum
to avoid possible interference between the purified substrates and
serum-contained molecules. The morphology of the cells was observed
during the following days and cells were photographed 3 days after
plating (Fig. 7
). The time lapse
necessary for cell attachment was identical (few minutes) between
cell lines on all substrates. The cultures became different thereafter,
showing almost no migration out of control and mutCOUP-TFI aggregates
attached to poly D-lysine and only limited migration on
both laminin and fibronectin (Fig. 7
). wtCOUP-TFI cells showed a much
higher rate of migration on all three substrates (Fig. 7
). Unlike
laminin and fibronectin, which are natural substrates for cell
attachment, migration, and neurite outgrowth (52, 53, 54), poly
D-lysine did not allow cell migration except for wtCOUP-TFI
cells (Fig. 7
). These data strongly suggested that wtCOUP-TFI cells had
a modified synthesis of ECM molecules, making them autonomous for
migration and spreading. We tested this last hypothesis by RT-PCR
analysis of components of the ECM known to interact with integrins
through their RGD sequence (50): fibronectin and vitronectin. Results
showed that the expression of fibronectin was not affected by wt or
mutant COUP-TFI (Fig. 8A
). Most notably,
the vitronectin gene seemed to be directly regulated by wtCOUP-TFI
since mRNA levels were dramatically increased in wtCOUP-TFI cells
treated with cAMP alone, RA, and RA + cAMP. Increased vitronectin mRNA
levels were detected both in aggregate and monolayer cell cultures,
indicating that COUP-TF could modulate the vitronectin gene
independently of the cell-cell interactions occurring in aggregates.
These results strongly support the idea that COUP-TFI may regulate
neurogenesis through the modulation of vitronectin levels, an ECM
molecule involved in neurite extension, motoneuron differentiation, and
possibly glial-guided neuronal migration (see Discussion and
Refs. 55, 56, 57). Alignment of the human and mouse vitronectin promoter
sequences showed the presence of two conserved nuclear receptor
half-binding sites (Fig. 8B
). Furthermore, prediction of transcription
factor binding-sites (Transfac database) revealed the presence of
recognition sequences for Sp1 in the mouse promoter and HNF-3 in both
promoters, two proteins that have been shown to cooperate with COUP-TFI
(58, 59). To determine whether COUP-TFI could directly regulate the
vitronectin promoter, we used a -528/+47 fragment of the mouse
vitronectin gene coupled to a luciferase reporter gene (60). This
plasmid was cotransfected in P19 cells with increasing amounts of
either wtCOUP-TFI or mutCOUP-TFI expression vectors (Fig. 8C
). Basal
expression of the reporter gene was high in P19 cells since the
luciferase activity was about 100-fold above background. This activity
was further enhanced after transfection of the wtCOUP-TFI expression
vector, whereas mutCOUP-TFI was not able to stimulate transcription
from the vitronectin promoter. These results implicate a direct
role of COUP-TFI in the control of the vitronectin gene and put
in a concrete form the role of COUP-TFI in the regulation of ECM
synthesis.

View larger version (127K):
[in this window]
[in a new window]
|
Figure 7. wtCOUP-TFI Enhances Migration on Different Cell
Substratum
Undissociated aggregates of cells treated for 3 days with 1
µM RA were plated onto plastic coated with poly
D-lysine, laminin, or fibronectin as indicated and
photographed 3 days after plating (phase contrast). The black
arrowheads indicate the position of the initially plated
aggregates that are still visible in pcDNA and mutCOUP-TFI cells. Note
the higher spreading of wtCOUP-TFI cells on all three substrates
(magnification, x180). This magnification did not allow the
visualization of neurites in wtCOUP-TFI cells, but examination at a
higher magnification showed the presence of numerous neurons extending
neurites whatever the substrate was.
|
|

View larger version (41K):
[in this window]
[in a new window]
|
Figure 8. COUP-TFI Regulates the Synthesis of Vitronectin
A, RT-PCR analysis was run with RNA extracted from aggregates (agg.) or
monolayer cell cultures (mon.) of pcDNA, wt, and mutant COUP-TFI cells
treated as indicated for 48 h. B, Alignment of the human and mouse
proximal promoter regions of the vitronectin gene (black
dots indicate identical nucleotides between mouse and human,
and dashes represent gaps that were introduced to
maximize sequence homology). The conserved nuclear receptor (NR)
half-binding sites are boxed as well as putative binding
sites for other transcription factors. C, The mouse
vitronectin promoter is activated by COUP-TFI in transient transfection
assays. P19 cells were cotransfected with a mouse vitronectin promoter
fragment (-528/+47) linked to a luciferase coding sequence and the
indicated amounts of wt or mutCOUP-TFI expression vectors. Results are
shown as the mean ± SEM (n = 3) of the
relative luciferase activities (raw luciferase activities divided by
ß-galactosidase activities).
|
|
 |
DISCUSSION
|
---|
The data presented here provide the first insights into the
molecular mechanisms underlying the involvement of COUP-TFI in
neurogenesis and suggest a major function for COUP-TFI in the
regulation of cell-cell and cell-matrix interactions.
Previous studies suggested that COUP-TF might be a global repressor of
the functions of retinoids and other signaling molecules. The
remarkable DNA-binding flexibility of COUP-TF orphan receptors had led
to the observation that they can repress several signaling pathways in
cell culture systems, mainly through competition for liganded-nuclear
receptor binding sites (44, 45, 61), and especially for RA response
elements (44, 45, 62). Additional evidence for a potential negative
role of COUP-TFs on retinoid signaling came from experiments showing
that COUP-TF I can form heterodimers with RXR on DNA, thus sequestering
this promiscuous partner for RARs (63). The existence of a regulatory
loop was then suggested by data demonstrating that RA acid itself
induces (or represses in some instance) the expression of COUP-TF genes
in different systems such as P19 cells, Xenopus, zebrafish
and mouse embryos (30, 31, 32, 33). The relevance of the P19 cell as a model
system for early differentiation is widely admitted (for review, see
Ref. 64). These cells behave very much like ES cells when treated with
RA (65, 66), and their use allowed the characterization of genes that
show spatially and temporally restricted expression patterns during
mouse development (38, 40, 66, 67). Remarkably, several of the genes
cloned from P19 cells induced to differentiate along the
neuroectodermal pathway are exclusively expressed in the nervous system
(38, 39, 67). The data presented here are thus likely to reflect
physiological functions of COUP-TFs. Our experiments clearly indicate
that early expression of COUP-TFI in P19 cells specifically affects
neuronal cell fate. Indeed, neuronal cells were absent from wtCOUP-TFI
cells treated with RA and cAMP, even if these cells did express Brn-2.
Monolayers of the same cell line did differentiate into nonneural cell
types and expressed high amounts of Stra8 transcripts in response to RA
(data not shown). These results suggest that COUP-TFI modulates the
expression of RA-regulated genes in a differentiation-dependent manner.
Hence, the timing of COUP-TF expression needs to be tightly controlled
during development to avoid inappropriate gene induction in cells
normally committed to a neural fate. In this respect, it must be
mentioned that RA treatment of Xenopus embryos induces an
earlier expression of the xCOUP-TF-A gene, when compared with control
embryos (33). It is then tempting to speculate that part of the
teratogenic effects of RA are mediated by COUP-TF orphan receptors
since injection of COUP-TF mRNA in Xenopus embryos leads to
developmental defects in anterior neural structures that mimic those
induced by an early RA exposure (68).
By inhibiting the activity of endogenous COUP-TFs, we were able to
confirm the involvement of these orphan receptors in the regulation of
cell adhesion and migration. The clear differences between the
phenotypes of our stable cell lines could be partly explained by
changes in the expression levels of ECM molecules. Our data suggest
that COUP-TFI could favor neuron migration on glial cells by a
RGD-dependent mechanism. Vitronectin is recognized by integrin through
an RGD motif and is involved in neural development (56, 69). The
integrin receptor
vß1, which recognizes vitronectin, is required
for efficient migration of embryonic cortical neurons along radial
glial fibers (55). Perturbation experiments using antibodies against
vitronectin would certainly shed light on the potential involvement of
vitronectin, and indirectly COUP-TFI, in neuronal migration. A close
relationship between vitronectin and COUP-TFs is also suggested by
other observations. First, as it is the case for COUP-TFII, vitronectin
is expressed in developing motoneurons and P19 cells under the control
of the morphogen sonic hedgehog (23, 70, 56). Second, in
perturbation experiments, antibodies against vitronectin inhibit
motoneuron differentiation in vitro and in vivo
(56). Third, vitronectin has also been shown to be expressed at the
surface of neural crest cells (NCCs) and required for the migration of
these cells (71), and COUP-TFI is expressed in early mouse embryo in
some premigratory and migratory NCCs (14). And fourth, vitronectin
supports axogenesis in PC12 cells (69). The experiments described here
are in favor of a direct control of the vitronectin gene by COUP-TF and
suggest that COUP-TF could transduce a sonic hedgehog signal
to the extracellular environment, at least in differentiating
motoneurons.
The present report provides evidence that COUP-TFs are regulators of
cell adhesion mechanisms required for the differentiation of embryonal
carcinoma cells. That COUP-TFs could, as a result, modify the migratory
behavior of cells is an appealing hypothesis in view of their
expression patterns during development. Indeed, these transcription
factors are not exclusively expressed in the nervous system, but are
also found in a number of other tissues at the time when tissue
modeling through cell movements is a widespread event (19). We propose
that COUP-TFs, which are the most conserved members of the nuclear
receptor family throughout evolution (19), modulate cell
adhesion-dependent morphogenetic processes in metazoan.
 |
MATERIALS AND METHODS
|
---|
Plasmid Construction
The human COUP-TFI cDNA (a gift from M. Pfafhl, La Jolla, CA)
was transferred from Bluescript to pcDNA3 (Invitrogen, San
Diego, CA) by BamHI/XhoI double digest. The
mutant C141
S hCOUP-TFI cDNA (41) was also
inserted in pcDNA. This mutant has a serine instead of the fourth
cysteine in the second zinc finger of hCOUP-TFI DBD and thus has lost
DNA binding ability (41). For yeast two-hybrid and GST pull-down
assays, a SmaI/EcoRI fragment of the wtCOUP-TFI
cDNA was cloned in frame with the Gal4DBD coding sequence of the
pAS21 vector (CLONTECH Laboratories, Inc., Palo Alto,
CA) and the GST coding sequence of the pGEX-2T (Pharmacia Biotech, Piscataway, NJ) vector, respectively. The corresponding
fusion proteins lack the first 56 N-terminal residues of COUP-TFI
and were called Gal4DBD/
COUP-TFI and GST/
COUP-TFI. The
full-length wtCOUP-TFI and mutCOUP-TFI cDNAs were inserted in frame
with the Gal4 activation domain coding sequence of the pACT2
vector (CLONTECH Laboratories, Inc.) by use of the
EcoRI site.
Two-Hybrid Assay
The yeast strain used in this study was Y190 (MATa,
ura 352, his 3200, ade 2201,
lys 2801, trp 1901, leu 23, 112,
gal 4?, gal 80?,
cyhr 2, LYS2::
GAL1UAS- HIS3TATA- HIS3,
URA3:: GAL1UAS-
GAL1TATA- LacZ). Yeast cells were transformed
using a lithium acetate method (72), and transformants were selected by
growth on complete minimum medium lacking uracil, histidine, and
tryptophan and, when necessary, leucine. The slight endogenous activity
of the his 3 gene was inhibited by 25
mM 3-aminotriazole. Reconstitution of Gal4
transactivation activity via interaction of the fusion proteins was
first tested by growth on the selective media. To confirm the
interaction, Lac Z activity was then tested by a filter-lift
assay by transferring colonies on filter paper (Whatman,
Clifton, NJ) . The filters were frozen for 15 sec in liquid nitrogen,
thawed at room temperature, and incubated on Whatman 3MM
soaked with Z buffer (60 mM
Na2HPO4, 40
mM
NaH2PO4, 10
mM KCl, 1 mM
MgSO4, and 50 mM
2-mercaptoethanol) containing 0.33 mg/ml X-Gal for 4 h at 30 C.
Quantification of the ß-galactosidase activity was assayed as
previously described (72), using O-nitrophenyl
ß-D-galactopyranoside as a substrate. Activity
was expressed in Miller units (73).
GST Pull-Down Assay
GST-
COUP-TFI fusion protein and GST expressed from pGEX2-T in
Escherichia coli were prepared according to protocols
supplied by Pharmacia Biotech. Concentration of the fusion
protein or the GST in crude lysates was estimated by separation on a
10% polyacrylamide SDS-PAGE, followed by a quantification of the total
protein concentration by a Bradford test. Crude bacterial extracts
containing the same amounts of GST or GST-
COUP-TFI were incubated
overnight at 4 C with 50 µl of glutathione-agarose beads
(Sigma, St. Louis, MO) in NENT buffer (20
mM Tris, pH 8; 100 mM NaCl;
1 mM EDTA, 0.5% Nonidet). Beads were then washed
five times in NENT and resuspended in 300 µl of NENT plus protease
inhibitors (10 µg/ml leupeptin, pepstatin, aprotinin, and 1
mM phenylmethylsulfonylfluoride). Four
microliters of 35S-labeled mutCOUP-TFI expressed
in TNT rabbit reticulocyte lysate system (Promega Corp.,
Madison, WI) were incubated with 20 µl (
2.5 µg) of
GST-
COUP-TFI or GST bound to the agarose matrix for 3 h at 4 C
in binding buffer (50 mM Tris, pH 8, 50
mM NaCl, 0.02% Tween 20, 0.02% BSA, and
protease inhibitors). Beads were then washed 10 times with washing
buffer (50 mM Tris, pH 8, 150
mM NaCl, 0.02% Tween 20, and protease
inhibitors). Bound proteins were eluted by boiling in SDS-sample buffer
and resolved by SDS-PAGE.
Cell Culture and Transfections
P19 EC cells (a gift from H. Gronemeyer, IGBMC, Strasbourg,
France) were maintained as already described (37). Cells were grown in
DMEM supplemented with 10% FCS. For RA-induced differentiation in
monolayer, P19 cells were plated at a density of
104 cells per well of 24-well plates. For
RA-induced neuronal differentiation, cells were grown at a density of
106 cells/100-mm bacteriological grade petri dish
and allowed to aggregate for 3 days. Aggregates were then treated with
trypsin/EDTA and dissociated cells were replated in tissue culture
grade six-well plates and further cultured for 34 days in the absence
of drugs. Drugs were used at 1 µM for
all-trans-retinoic acid, or 1 mM for
8-Br-cAMP (Sigma) unless indicated. For stable
transfections, 106 cells were plated in 100-mm
dishes and transfected as described (74) with 10 µg of expression
vector. After 24 h, cells were grown in the presence of 400
µg/ml of G 418 (Roche Molecular Biochemicals,
Indianapolis, IN). After 10 days of selection, clones of resistant
cells were dissociated and pooled under the names "pcDNA,"
"wtCOUP-TF," and "mutCOUP-TF" for cells transfected,
respectively, with the empty expression vector or the different nuclear
receptor expression vectors. Cells were then systematically grown in
the presence of G 418 (400 µg/ml) to avoid loss of transgene. For
growth on different substrates (all three from Sigma),
"easygrip" untreated plastic dishes (Falcon, Becton Dickinson and Co., Combourg, France) were coated for 1 h with
either poly D-lysine (0.1 mg/ml), laminin (10
µg/ml), or fibronectin (10 µg/ml). After 3 days of incubation in
DMEM/10% FCS and 106 M RA, the aggregates were
pelleted and resuspended in Neurobasal medium (Life Technologies, Inc., Gaithersburg, MD) supplemented with the N2 complement
(Life Technologies, Inc.), before seeding in the coated
plates. For transient transfections assays, we used 800 ng of a
reporter gene containing base pairs -528 to +47 of the mouse
vitronectin gene linked to the luciferase coding sequence (60),
together with 150 ng of a ß-galactosidase expression vector (pCH110)
and various amounts of wt or mutant COUP-TFI expression vectors. Cells
were transfected with the calcium phosphate procedure (74) and allowed
to express the reporter gene for 36 h before luciferase activity
was assayed with luciferin (Promega Corp.). The raw
luciferase activities were divided by the corresponding
ß-galactosidase activities to correct for transfection efficiencies.
CHO and Hela cells were transiently transfected in 12-well plates with
1 µg of ApoA1-tk-CAT reporter gene and various expression vectors.
The resulting CAT activity was assayed as previously described (45) and
normalized for ß-galactosidase expression.
Peptide Interference Assay
For peptide interference assays, a RGDS peptide, or the control
peptide SDGRG (both from Sigma), were added 24 h
after plating the RA-treated aggregates onto tissue-culture plastic
dishes. Antineurofilament immunohistochemistry was run 24 h later
on paraformaldehyde-fixed cells. The percentage of aggregates that
showed neuronal outgrowth was then determined by scoring each time at
least 50 aggregates per cell line and in three independent
experiments.
EMSA
In vitro translated proteins were made with the TNT
reticulocyte lysate system (Promega Corp.). Whole-cell
extracts (WCEs) from P19 EC-transfected cells were prepared and used
for EMSA as described (21). Briefly, 4 µg of WCEs were preincubated
with 1 µg poly(dI-dC), and eventually with competitors, in 20 µl of
binding buffer (20 mM HEPES, pH 7.9, 1
mM dithiothreitol, 50 mM
KCl, 10% glycerol, 2.5 mM
MgCl2) at room temperature for 15 min. For
supershifting experiments, 1 µl of antibodies against COUP-TFI or
COUP-TFII (Santa Cruz Biotechnology, Inc., Santa Cruz, CA)
were preincubated for 30 min with WCEs before addition of binding
buffer. The samples were then incubated with
32P-labeled probes (15,000 dpm) for 20 min at
room temperature. Protein-DNA complexes were separated from the free
probe by nondenaturing electrophoresis in 4% polyacrylamide
gels in 0.5 x TBE (45 mM Tris base, 45
mM boric acid, 1 mM EDTA)
at 4 C. The oligonucleotides were synthesized by Eurogentec (Seraing,
Belgium) and used double stranded in gel-shift experiments:
DR-1: 5'-TCGAGGGTCAGAGGTCACGA-3'; DR-5 (ß-RARE):
5'-GATGGGTTCACCGAAAGTTCACTC-3'; DR-7 (hARR; 21):
5'-GATCTAGGTTGACATTTCTCCTTGACCTTTTAGATC-3'; ERE (rtER gene, 75):
5'-TTGCTGTGTCATGTTGACCTGCTCTAGAGA-3'; AP-1:
5'-TCGACGCTTGATGACTCAGCCGGAA-3'; GRE:
5'-CCAGAACACAGTGTTCTGAG-CTAAAATAACACATTCAG-3'.
Immunohistochemistry
Immunohistochemistry procedures were run directly in six-well
tissue culture plates. Cells were fixed with a 4% paraformaldehyde
solution in PBS for 12 min and then incubated for 10 min in a 4%
paraformaldehyde-0.2% Triton X-100 solution in PBS. After washing and
blocking, cells were incubated with antineurofilament 200 (NF-200,
Sigma) diluted 1:100 for 1 h. After incubation with
an antirabbit IgG/peroxidase conjugate (Sigma), the
presence of NF-200 was revealed with diaminobenzidine.
Western Blot Analysis
COUP-TFI and COUP-TFII expression levels were assayed by Western
blot with anti-COUP-TFI and anti-COUP-TFII from Santa Cruz Biotechnology, Inc.. For these assays, 80 µg of whole cell
extracts were run and the gels were treated as described previously
(41).
Semiquantitative RT-PCR Analysis
Total RNA from P19 cells was purified using the Trizol reagent
(Life Technologies, Inc.). Two micrograms of RNA were used
for reverse transcription with 5 µM random hexamer
oligonucleotides for 30 min at 37 C and 15 min at 42 C. For PCR
reactions, 1/20 of the reverse transcription reaction mixture was used.
Number of cycles (indicated for each primer set) and annealing
temperatures varied according to the set of primer used. The following
upstream (up) and downstream (down) primers were used (numbering is
made with the +1 being the A of the initiation codon except when
indicated): Brn-2 (accession number: X66602) up,
5'-TGCAAGCTGAAGCCTTTGTTG-3' (1137/1157); Brn-2 down,
5'-CCTTTTCTCTTTCTGTCTCCTG-3' (1406/1385) [32 cycles]; E-cadherin up,
5'-CTATGATGAAGAAGGAGGTGG-3' (+2268/+2288); E-cadherin down,
5'-CACTGCCCTCGTAATCGAAC-3'
(+2521/+2502) [32 cycles]; COUP-TFI up,
5'-AAGCACTA-CGGCCAATTCAC-3' (+283/+302); COUP-TFI down,
5'-AGCTCGCAGATGTTCTCGAT-3' (+662/+643) [27 cycles]; PO (36B4) up,
5'-CAGCTCTGGAGAAACTGCTG-3' (+217/+236); PO (36B4) down,
5'-GTGTACTCAGTCTCCACAGA-3' (+772/+753) [26 cycles]; Bmp4 up,
5'-GTAACCGAATGCTGATGGTC-3' (+11/+30); Bmp4 down,
5'-TTTTCTGGGATGCTGGTGAG-3' (+452/+433) [32 cycles]; Fibronectin up,
5'-CCAGGACAACAGCATCAG-3' (+2237/+2254); Fibronectin down,
5'-TAGGTCACCCTGTACCTG-3' (+2575/+2558) [32 cycles]; Vitronectin up,
5'-TACTATCAGAGCTGCTG-3' (+136/+152); Vitronectin down,
5'-AGTTGATGCGAGTGAAG-3' (+640/+624) [32 cycles]. The acidic ribosomal
phosphoprotein (PO) gene (76), also called 36B4 (77), was used as a
control gene since its expression has been shown to be invariant upon
RA-induced differentiation of P19 EC cells (78).
 |
ACKNOWLEDGMENTS
|
---|
We are grateful to Hinrich Gronemeyer (IGBMC, Strasbourg,
France) for the gift of P19 cells. We thank Magnus Pfahl (Sidney Kimmel
Cancer Center, La Jolla, CA) and David Loskutoff (Scripps
Research Institute, La Jolla, CA) for the generous gift of hCOUP-TFI,
RAR
, RXR
, ApoA1-tk-CAT, and the vitronectin promoter/luciferase
plasmids. We would also like to thank Yves Pichon, François
Tiaho, and Pascal Benquet for helpful discussions, and
Marie-Hélène Salmon for technical assistance. We also thank
Jean-Loup Duband for his invaluable help.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. Gilles Salbert, Equipe Information et Programmation Cellulaire, Université de Rennes I, UPRES-A CNRS 6026, Campus de Beaulieu, 35042 Rennes Cedex, France.
This work was supported by funds from the Centre Nationale de la
Recherche Scientifique and Direction de la Recherche et des Etudes
Doctorales and from the Ministère de lEnseignement et de
la Recherche to T.S. and R.M.
Received for publication January 26, 2000.
Revision received July 24, 2000.
Accepted for publication August 22, 2000.
 |
REFERENCES
|
---|
-
Mangelsdorf DJ, Thummel C, Beato M, Herrlich P,
Schütz G, Umesono K, Blumberg B, Kastner P, Mark M, Chambon P,
Evans RM 1995 The nuclear receptor superfamily: the second decade. Cell 83:835839[Medline]
-
Mangelsdorf DJ, Borgmeyer U, Heyman RA, Zhou JY, Ong ES, Oro
AE, Kakizuka A, Evans RM 1992 Characterization of three RXR genes that
mediate the action of 9-cis retinoic acid. Genes Dev 6:329344[Abstract]
-
Lala DS, Rice DA, Parker KL 1992 Steroidogenic factor 1, a
key regulator of steroidogenic enzyme expression, is the mouse homolog
of fushi tarazu factor 1. Mol Endocrinol 6:12491258[Abstract]
-
Miyajima N, Kadowaki Y, Fukushige SI, Semba SK, Yamanashi Y,
Matsubara KI, Toyoshima K, Yamamoto T 1988 Identification of two novel
members of erbA superfamily by molecular cloning: the gene
products of the two are highly related to each other. Nucleic Acids Res 16:1105711074[Abstract]
-
Wang LH, Tsai SY, Cook RG, Beattie WG, Tsai MJ, OMalley BW 1989 Coup transcription factor is a member of the steroid receptor
superfamily. Nature 340:163166[CrossRef][Medline]
-
Hoshizaki DK, Blackburn T, Price C, Ghosh M, Miles K, Ragucci
M, Sweis R 1994 Embryonic fat-cell lineage in Drosophila
melanogaster. Development 120:24892499[Abstract/Free Full Text]
-
Hoshizaki DK, Lunz R, Ghosh M, Johnson W 1995 Identification
of fat-cell enhancer activity in Drosophila melanogaster
using P-element enhancer traps. Genome 38:497506[Medline]
-
Ikeda Y, Luo X, Abbud R, Nilson JH, Parker KL 1995 The
nuclear receptor steroidogenic factor 1 is essential for the formation
of the ventromedial hypothalamic nucleus. Mol Endocrinol 9:478486[Abstract]
-
Kastner P, Grondona J, Mark M, Gransmuller A, LeMeur M,
Décimo D, Vonesh JL, Dollé P, Chambon P 1994 Genetic
analysis of RXR
developmental function: convergence of RXR and
RAR signaling in heart and eye morphogenesis. Cell 78:9871003[Medline]
-
Kramer S, West SR, Hiromi Y 1995 Cell fate control in the
Drosophila retina by the orphan receptor seven-up: its role
in the decisions mediated by the ras signaling pathway. Development 121:13611372[Abstract/Free Full Text]
-
Lavorgna G, Karim FD, Thummel CS, Wu C 1993 Potential role for
a FTZ-F1 steroid receptor superfamily member in the control of
Drosophila metamorphosis. Proc Natl Acad Sci USA 90:30043008[Abstract]
-
Luo J, Ikeda Y, Parker KL 1994 A cell-specific nuclear
receptor is essential for adrenal and gonadal development and sexual
differentiation. Cell 77:481490[Medline]
-
Mlodzik M, Hiromi Y, Weber U, Goodman CS, Rubin GM 1990 The
Drosophila seven-up gene, a member of the steroid receptor
gene superfamily, controls photoreceptor cell fates. Cell 60:211224[Medline]
-
Qiu Y, Pereira FA, De Mayo FJ, Lydon JP, Tsai SY, Tsai MJ 1997 Null mutation of mCOUP-TFI results in defects in morphogenesis of the
glossopharyngeal ganglion, axonal projection, and arborization. Genes
Dev 11:19251937[Abstract/Free Full Text]
-
Sucov HM, Dyson E, Gumeringer CL, Price J, Chien KR, Evans RM 1994 RXR
mutant mice establish a genetic basis for vitamin A
signaling in heart morphogenesis. Genes Dev 8:10071018[Abstract]
-
Escriva H, Safi R, Hänni C, Langlois M-C,
Saumitou-Laprade P, Stehelin D, Capron A, Pierce R, Laudet V 1997 Ligand binding was acquired during evolution of nuclear receptors. Proc
Natl Acad Sci USA 94:68036808[Abstract/Free Full Text]
-
Sagami I, Tsai SY, Wang H, Tsai MJ, OMalley BW 1986 Identification of two factors required for transcription of the
ovalbumin gene. Mol Cell Biol 6:42594267[Medline]
-
Ladias JAA, Karathanasis SK 1991 Regulation of the
apolipoprotein A1 gene by ARP-1, a novel member of the steroid receptor
superfamily. Science 251:561565[Medline]
-
Tsai SY, Tsai MJ 1997 Chicken ovalbumin upstream
promoter-transcription factor (COUP-TFs): coming of age. Endocr Rev 18:229240[Abstract/Free Full Text]
-
Fjose A, Nornes S, Weber U, Mlodzik M 1993 Functional
conservation of vertebrate seven-up related genes in neurogenesis and
eye development. EMBO J 12:14031414[Abstract]
-
Lu X-P, Salbert G, Pfahl M 1994 An evolutionary conserved
COUP-TF binding element in a neural-specific gene and COUP-TF
expression patterns support a major role for COUP-TFI in neural
development. Mol Endocrinol 8:17741788[Abstract]
-
Lutz B, Kuratani S, Cooney AJ, Wawersik S, Tsai SY, Eichele G,
Tsai MJ 1994 Developmental regulation of the orphan receptor COUP-TFII
gene in spinal motor neurons. Development 120:2536[Abstract/Free Full Text]
-
Pereira FA, Qiu Y, Tsai MJ, Tsai SY 1995 Chicken ovalbumin
upstream promoter transcription factor (COUP-TF): expression during
mouse embryogenesis. J Steroid Biochem Mol Biol 53:503508[CrossRef][Medline]
-
Qiu Y, Cooney AJ, Kuratani S, DeMayo FJ, Tsai SY, Tsai M-J 1994 Spatiotemporal expression patterns of chicken ovalbumin upstream
promoter-transcription factors in the developing mouse central nervous
system: evidence for a role in segmental patterning of the
diencephalon. Proc Natl Acad Sci USA 91:44514455[Abstract]
-
Vlahou A, Gonzales-Rimbau M, Flytzanis CN 1996 Maternal mRNA
encoding the orphan steroid receptor SpCOUP-TFI is localized in sea
urchin eggs. Development 122:521526[Abstract/Free Full Text]
-
Connor H, Nornes H, Neuman T 1995 Expression screening reveals
an orphan receptor chick ovalbumin upstream promoter transcription
factor I as a regulator of neurite/substrate-cell contacts and
aggregation. J Biol Chem 270:1506615070[Abstract/Free Full Text]
-
Zhou C, Qiu Y, Pereira FA, Crair MC, Tsai SY, Tsai MJ 1999 The
nuclear orphan receptor COUP-TFI is required for differentiation of
subplate neurons and guidance of thalamocortical axons. Neuron 24:847859[Medline]
-
Pereira FA, Qiu Y, Zhou G, Tsai MJ, Tsai SY 1999 The orphan
nuclear receptor COUP-TFII is required for angiogenesis and heart
development. Genes Dev 13:10371049[Abstract/Free Full Text]
-
Kastner P, Mark M, Chambon P 1995 Nonsteroid nuclear
receptors: what are genetic studies telling us? Cell 83:859869[Medline]
-
Brubaker K, McMillan M, Neuman T, Nornes HO 1996 All-trans
retinoic acid affects the expression of orphan receptors COUP-TFI and
COUP-TFII in the developing neural tube. Dev Brain Res 93:198202[Medline]
-
Fjose A, Weber U, Mlodzick M 1995 A novel vertebrate
svp-related nuclear receptor is expressed as a step gradient in
developing rhombomeres and is affected by retinoic acid. Mech Dev 52:233246[CrossRef][Medline]
-
Jonk LJC, de Jonge MEJ, Pals CE, Wissink S, Vervaart JMA,
Schoorlemmer J, Kruijer W 1994 Cloning and expression during
development of three murine members of the COUP family of nuclear
orphan receptors. Mech Dev 47:8197[CrossRef][Medline]
-
Van der Wees J, Matharu PJ, de Roos K, Destrée OHJ,
Godsave SF, Durston AJ, Sweeney GE 1996 Developmental expression and
differential regulation by retinoic acid of Xenopus
COUP-TF-A and COUP-TF-B. Mech Dev 54:173184[CrossRef][Medline]
-
Jones-Villeneuve EMV, McBurney MW, Rogers KA, Kalnins VI 1982 Retinoic acid induces embryonal carcinoma cells to differentiate into
neurons and glial cells. J Cell Biol 94:253262[Abstract]
-
McBurney MW, Jones-Villeneuve EMV, Edwards MKS, Anderson PJ 1982 Control of muscle and neuronal differentiation in a cultured
embryonal carcinoma cell line. Nature 299:165167[Medline]
-
Mummery CL, Feijen A, Moolenaar WH, van den Brink CE, de Laat
SW 1986 Establishment of a differentiated mesodermal line from P19 EC
cells expressing functional PDGF and EGF receptors. Exp Cell Res 165:229242[Medline]
-
MacPherson PA, McBurney MW 1995 P19 embryonal carcinoma cells:
a source of cultured neurons amenable to genetic manipulation. Methods:
Companion Methods Enzymol 7:238252[CrossRef]
-
Imai Y, Suzuki Y, Tohyama M, Wanaka A, Takagi T 1994 Cloning
and expression of a neural differentiation-associated gene, p205, in
the embryonal carcinoma cell line P19 and in the developing mouse.
Brain Res Mol Brain Res 24:313319[Medline]
-
Imai Y, Suzuki Y, Matsui T, Tohyama M, Wanaka A, Takagi T 1995 Cloning of a retinoic acid-induced gene, GT1, in the embryonal
carcinoma cell line P19: neuron-specific expression in the mouse brain.
Brain Res Mol Brain Res 31:19[CrossRef][Medline]
-
Jonk LJ, de Jonge ME, Vervaart JM, Wissink S, Kruijer W 1994 Isolation and developmental expression of retinoic acid-induced genes.
Dev Biol 161:604614[CrossRef][Medline]
-
Lazennec G, Kern L, Valotaire Y, Salbert G 1997 The nuclear
receptors COUP-TF and ARP-1 positevely regulate the trout estrogen
receptor gene through enhancing autoregulation. Mol Cell Biol 17:50535066[Abstract]
-
Kadowaki Y, Toyoshima K, Yamamoto T 1992 Ear3/COUP-TF binds
most tightly to a response element with tandem repeat separated by one
nucleotide. Biochem Biophys Res Commun 183:492498[Medline]
-
Neuman K, Soosaar A, Nornes HO, Neuman T 1995 Orphan receptor
COUP-TFI antagonizes retinoic acid-induced neuronal differentiation.
J Neurosci Res 41:3948[Medline]
-
Cooney AJ, Tsai SY, OMalley BW, Tsai MJ 1992 Chicken
ovalbumin upstream promoter transcription factor (COUP-TF) dimers bind
to different GGTCA response elements, allowing COUP-TF to repress
hormonal induction of the vitamin D3, thyroid hormone, and retinoic
acid receptors. Mol Cell Biol 12:41534163[Abstract]
-
Tran P, Zhang XK, Salbert G, Hermann T, Lehmann J, Pfahl M 1992 COUP orphan receptors are negative regulators of retinoic acid
response pathways. Mol Cell Biol 12:46664676[Abstract]
-
Remboutsika E, Lutz Y, Gansmuller A, Vonesch J-L, Losson R,
Chambon P 1999 The putative nuclear receptor mediator TIF1
is
tightly associated with euchromatin. J Cell Sci 112:16711683[Abstract/Free Full Text]
-
Fujii H, Hamada H 1993 A CNS-specific POU transcription
factor, Brn-2, is required for establishing mammalian neural cell
lineages. Neuron 11:11971206[Medline]
-
Suzuki T, Kim H-S, Kurabayashi M, Hamada H, Fujii H, Aikawa M,
Watanabe M, Sakomura Y, Yazaki Y, Nagai R 1996 Preferential
differentiation of P19 mouse embryonal carcinoma cells into smooth
muscle cells. Circ Res 78:395404[Abstract/Free Full Text]
-
Schmidt JW, Brugge JS, Nelson WJ 1992 pp60 src tyrosine kinase
modulates P19 embryonal carcinoma cell fate by inhibiting neuronal but
not epithelial differentiation. J Cell Biol 116:10191033[Abstract]
-
Hynes RO 1992 Integrins: versatility, modulation, and
signaling in cell adhesion. Cell 69:1125[Medline]
-
Galileo DS, Majors J, Horwitz AF, Sanes JR 1992 Retrovirally
introduced antisense integrin RNA inhibits neuroblast migration
in vivo. Neuron 9:11171131[Medline]
-
Calof AL, Lander AD 1991 Relationship between neuronal
migration and cell-substratum adhesion: laminin and merosin promote
olfactory neuronal migration but are anti-adhesive. J Cell Biol 115:779794[Abstract]
-
Perris R, Paulsson M, Bronner-Fraser M 1989 Molecular
mechanisms of avian neural crest cell migration on fibronectin and
laminin. Dev Biol 136:222238[Medline]
-
Tomaselli KJ, Reichardt LF, Bixby JL 1986 Distinct molecular
interactions mediate neuronal process outgrowth on non-neuronal cell
surfaces and extracellular matrices. J Cell Biol 103:26592672[Abstract]
-
Anton ES, Kreidberg JA, Rakic P 1999 Distinct function of
3 and
v integrin receptors in neuronal migration and laminar
organization of the cerebral cortex. Neuron 22:277289[Medline]
-
Martinez-Morales JR, Barbas JA, Marti E, Bovolenta P, Edgar D,
Rodriguez-Tébar A 1997 Vitronectin is expressed in the ventral
region of the neural tube and promotes the differentiation of motor
neurons. Development 124:51395147[Abstract/Free Full Text]
-
Neugebauer KM, Emmett CJ, Venstrom KA, Reichardt LF 1991 Vitronectin and thrombospondin promote retinal neurite outgrowth:
developmental regulation and role of integrins. Neuron 6:345358[Medline]
-
Legraverand C, Eguchi H, Ström A, Lahuna O, Mode A,
Tollet P, Westin S, Gustafsson J-A 1994 Transactivation of the rat
CYP2C13 gene promoter involves HFN-1, HNF-3, and members of the orphan
receptor subfamily. Biochemistry 33:98899897[Medline]
-
Rohr O, Aunis D, Schaeffer E 1997 COUP-TF and Sp1 interact and
cooperate in the transcriptional activation of the human
immunodeficiency virus type 1 long terminal repeat in human microglial
cells. J Biol Chem 272:3114931155[Abstract/Free Full Text]
-
Seiffert D, Curriden SA, Jenne D, Binder BR, Loskutoff DJ 1996 Differential regulation of vitronectin in mice and humans in
vitro. J Biol Chem 271:54745480[Abstract/Free Full Text]
-
Liu Y, Yang N, Teng CT 1993 COUP-TF acts as a competitive
repressor for estrogen -mediated activation of the mouse lactoferrin
gene. Mol Cell Biol 13:18361846[Abstract]
-
Cooney AJ, Leng X, Tsai SY, OMalley BW, Tsai MJ 1993 Multiple mechanisms of chicken ovalbumin upstream promoter
transcription factor-dependent repression of transactivation by the
vitamin D, thyroid hormone, and retinoic acid receptors. J Biol
Chem 268:41524160[Abstract/Free Full Text]
-
Kliewer SA, Umesono K, Heyman RA, Mangelsdorf DJ, Dyck JA,
Evans RM 1992 Retinoid X receptor-COUP-TFI interactions modulate
retinoic acid signaling. Proc Natl Acad Sci USA 89:14481452[Abstract]
-
Bain G, Ray WJ, Yao M, Gottlieb DI 1994 From embryonal
carcinoma cells to neurons: the P19 pathway. Bioessays 16:343348[Medline]
-
Bain G, Kitchens D, Yao M, Huettner JE, Gottlieb DI 1995 Embryonic stem cells express neuronal properties in vitro.
Dev Biol 168:342357[CrossRef][Medline]
-
Bouillet P, Oulad-Abdelghani M, Vicaire S, Garnier J-M,
Schuhbauer B, Dollé P, Chambon P 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 170:420433[CrossRef][Medline]
-
Bouillet P, Sapin V, Chazaud C, Messaddeq N, Décimo D,
Dollé P, Chambon P 1997 Developmental expression pattern of
Stra6, a retinoic acid-responsive gene encoding a new type of membrane
protein. Mech Dev 63:173186[CrossRef][Medline]
-
Schuh TJ, Kimelman D 1995 COUP-TFI is a potential regulator of
retinoic acid-modulated development in Xenopus embryo. Mech
Dev 51:3949[CrossRef][Medline]
-
Grabham PW, Gallimore PH, Grand RJ 1992 Vitronectin is the
major serum protein essential for NGF-mediated neurite outgrowth from
PC12 cells. Exp Cell Res 202:337344[Medline]
-
Krishnan V, Elberg G, Tsai M-J, Tsai SY 1997 Identification of
a novel sonic hedgehog response element in the chicken ovalbumin
upstream promoter-transcription factor II promoter. Mol Endocrinol 11:14581466[Abstract/Free Full Text]
-
Delannet M, Martin F, Bossy B, Cheresh DA, Reichardt LF,
Duband J-L 1994 Specific roles of the
Vß1,
Vß3, and
Vß5 integrins in avian neural crest cell adhesion and migration on
vitronectin. Development 120:26872702[Abstract/Free Full Text]
-
Petit F, Le Goff P, Cravedi JP, Valotaire Y, Pakdel F 1997 Two
complementary bioassays for screening the estrogenic potency of
xenobiotics: recombinant yeast for trout estrogen receptor and trout
hepatocyte cultures. J Mol Endocrinol 19:321335[Abstract/Free Full Text]
-
Sambrook J, Fritsch EF, Maniatis T 1989 Monitoring expression
of ß-galactosidase activity. In: Molecular Cloning: A Laboratory
Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, p
17.35
-
Pfahl M, Tzukerman M, Zhang X-K, Lehmann JM, Hermann T, Wills
K, Graupner G 1990 Rapid procedures for retinoic acid receptor cloning
and their analysis. Methods Enzymol 153:256270
-
Lazennec G, Kern L, Salbert G, Saligaut D, Valotaire Y 1996 Cooperation between the human estrogen receptor (ER) and MCF-7
cell-specific transcription factors elicits high activity of an
estrogen-inducible enhancer from the trout ER gene. Mol Endocrinol 10:1161126
-
Krowczynska AM, Coutts M, Makrides S, Brawerman G 1989 The
mouse homologue of the human acidic ribosomal phosphoprotein PO: a
highly conserved polypeptide that is under translational control.
Nucleic Acids Res 17:6408[Medline]
-
Clifford J, Chiba H, Sobieszczuk D, Metzger D, Chambon P 1996 RXR
-null F9 embryonal carcinoma cells are resistant to the
differentiation, anti-proliferative and apoptotic effects of retinoids.
EMBO J 15:41424155[Abstract]
-
Boudjelal M, Taneja R, Matsubara S, Bouillet P, Dollé P,
Chambon P 1997 Overexpression of Stra13, a novel retinoic
acid-inducible gene of the basic helix-loop-helix family, inhibits
mesodermal and promotes neuronal differentiation of P19 cells. Genes
Dev 11:20522065[Abstract/Free Full Text]