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 d’Endocrinologie Moléculaire de la Reproduction (R.M.) UPRES-A CNRS 6026 Université de Rennes I, Campus de Beaulieu 35042 Rennes Cedex, France


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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. 1AGo) and a glutathione-S-transferase (GST)-pull-down assay (Fig. 1BGo). 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/{Delta}COUP-TFI construct was used in conjunction with either Gal4AD/COUP-TFI or Gal4AD/ mutCOUP-TFI (Fig. 1AGo). 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. 1BGo). 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. 1CGo). In vitro transcription/translation reactions were run with a fixed amount of pcDNA/wtCOUP-TFI vector (100 ng - 1X, Fig. 1CGo) 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. 1CGo). 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.



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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-{Delta}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{alpha}, pECE-RXR{alpha}, 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. 1DGo). 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. 1DGo). 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. 1DGo). 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. 1DGo). 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. 1EGo. This was true also for Hela cells, whereas HepG2 cells did not express COUP-TFII (Fig. 1EGo). 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. 1DGo 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. 2AGo) [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. 2AGo), whereas low but detectable levels were seen in the absence of cAMP (Fig. 2AGo). 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. 2Go, 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. 2Go, B and C) or with different combinations of cAMP and RA for 48 h as indicated (Fig. 2EGo). Extracts from P19 control cells (pcDNA) treated with cAMP generated a single complex with the DR-1 probe (noted by an asterisk, Fig. 2BGo) 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. 2BGo) that reflected interaction of the exogenous COUP-TFI with the DR-1 probe, as shown by the antibody supershift (Fig. 2BGo). The specificity of DNA recognition by the overexpressed wtCOUP-TFI was assessed by competition experiments in EMSA (Fig. 2CGo) 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. 2DGo). 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. 2EGo). 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. 2EGo). 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. 2EGo). 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. 2EGo) 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. 2EGo) 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.



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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. 1BGo. 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. 3Go). 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. 3Go). After dissociation of the aggregates, the replated cells were observed during the next 48 h of differentiation (Fig. 3Go). 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. 3Go). 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. 3Go). 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. 3Go). 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. 3Go). 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. 3Go).



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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. 4Go). 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. 3Go). 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. 4Go). Conversely, in the presence of cAMP, RA was not able to inhibit E-cadherin expression in wtCOUP-TFI cells.



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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. 3Go), 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. 5Go). As shown in Fig. 5Go, 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. 5Go). Similar results were obtained when cells were plated from dissociated aggregates. Interestingly, as observed in Fig. 5Go, 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.



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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. 6Go). 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. 6AGo). Cells were then fixed and examined for the presence of neurons by antineurofilament immunohistochemistry (Fig. 6AGo). 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. 6AGo), 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. 6BGo).



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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 {alpha}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. 6AGo 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. 6Go, 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. 7Go). 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. 7Go). wtCOUP-TFI cells showed a much higher rate of migration on all three substrates (Fig. 7Go). 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. 7Go). 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. 8AGo). 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. 8BGo). 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. 8CGo). 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.



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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.

 


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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
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 {alpha}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
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 pAS2–1 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/{Delta}COUP-TFI and GST/{Delta}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 3–52, his 3–200, ade 2–201, lys 2–801, trp 1–901, leu 2–3, 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-{Delta}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-{Delta}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-{Delta}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 3–4 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 10–6 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{alpha}, RXR{alpha}, 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 l’Enseignement 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.


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