The AF-1 and AF-2 Activating Domains of Retinoic Acid Receptor-{alpha} (RAR{alpha}) and Their Phosphorylation Are Differentially Involved in Parietal Endodermal Differentiation of F9 Cells and Retinoid-Induced Expression of Target Genes

Cécile Rochette-Egly, Jean-Luc Plassat, Reshma Taneja1 and Pierre Chambon

Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP/Collège de France BP 163, 67404 ILLKIRCH Cedex CU de Strasbourg, France


    ABSTRACT
 TOP
 ABSTRACT
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Retinoic acid (RA) induces the differentiation of F9 cells cultured as monolayers into primitive endodermal-like cells, whereas a combination of RA and cAMP leads to parietal endodermal differentiation. In RA receptor {alpha}-null F9 cells (RAR{alpha}-/- cells), RA still efficiently triggers RAR{gamma}-mediated primitive endodermal differentiation, but parietal endodermal differentiation is markedly delayed. To investigate the role of RAR{alpha}1 activation functions AF-1 and AF-2 and of their phosphorylation sites during RA- and cAMP-induced parietal differentiation, cell lines reexpressing WT or mutated RAR{alpha}1 were established in RAR{alpha}-/- cells. We have found that the protein kinase A (PKA) phosphorylation site and the AF-2AD core (helix 12) of RAR{alpha}1 are required for efficient parietal endodermal differentiation, whereas the AF-1 proline-directed kinase phosphorylation site is dispensible. Interestingly, deletion of the AF-1 activating domain (the A/B region), but not of the AF-2AD core, generates a dominant negative mutant that abrogates primitive endodermal differentiation when expressed in RAR{alpha}-/- cells. We also show that the RAR{alpha} AF-1 and AF-2 activation functions, but not their phosphorylation sites, are involved in the induction of RA-responsive genes in a differential promoter context-dependent manner. is the most potent biologically active form of vitamin A. RA exhibits a wide range of activities and influences the proliferation and differentiation of a variety of cell types. In that context, F9 embryonal carcinoma (EC) cells represent a well established cell-autonomous model system for investigating retinoid signaling in vivo, as upon RA treatment and depending on culture conditions, they differentiate into three distinct cell types resembling primitive, parietal, and visceral endodermal extraembryonic cells (1–4). RA- induced differentiation of F9 EC cells is accompanied by an apoptotic response and a decrease in the rate of proliferation, as well as by the induction of expression of a number of genes (3, 5–7).

RA exerts its pleiotropic effects through two classes of nuclear ligand-dependent transregulators: the retinoic acid receptors (RAR{alpha}, RARß, and RAR{gamma} isotypes and their isoforms) activated by either all-trans-RA or its 9-cis-isomer and the retinoid X receptors (RXR{alpha}, RXRß and RXR{gamma}) activated by 9-cis-RA only (8–11). F9 cells express all RARs and RXRs (12–14), and two strategies have been used to investigate their roles in the response of F9 EC cells to RA treatment. Firstly, using homologous recombination, we engineered F9 cells in which either the RAR{alpha}, the RAR{gamma}, or the RXR{alpha} gene, both the RAR{gamma} and RXR{alpha} genes or both the RAR{alpha} and RXR{alpha} genes, are knocked out (6, 7, 15, 16). Secondly, wild-type (WT) and mutant F9 cells were treated with pan-RXR- and RAR isotype ({alpha}, ß, or {gamma})-selective retinoids (7, 17–20). It was established that RAR{gamma} is indispensible for RA-induced differentiation of F9 EC cells into primitive endoderm-like cells, whereas RAR{alpha} is additionally required for efficient parietal endodermal differentiation that occurs in the presence of RA and cAMP (20), and for which primitive endodermal differentiation is a prerequisite. These studies also demonstrated that RAR/RXR heterodimers are the functional units transducing the retinoid signal in vivo (7, 19).

RARs and RXRs possess two transcriptional activation functions (AFs): AF-1, located in the N-terminal A/B region, and AF-2, associated with the ligand binding domain (LBD) (region E) and activated by agonistic ligands (8, 21–24). The integrity of a conserved amphipatic {alpha}-helix, referred to as the AF-2AD core, is required for AF-2 activity (23, 25–28, and references therein). The AF-2AD core is located in the C-terminal {alpha} helix of the LBD (helix 12) that is indispensible for the formation of the coactivator-binding surface generated during the LBD transconformation triggered by ligand binding (8, 29–31).

In addition, RARs and RXRs are phosphorylated in their AF-1 domain that contains sites for proline- directed kinases (20, 32, 33). In RAR{alpha}1, the phospho-rylated residue in the AF-1domain has been identified as serine 77 (32). Furthermore, RAR{alpha}1 can be phosphorylated by PKA at serine 369 that is located in the LBD/AF-2 domain (34). Interestingly, RAR{alpha}1 is phosphorylated at these two residues in F9 cells, as well as in transfected COS cells (20, 34). Similar phospho-rylation sites are present in other RARs and have been shown to be phosphorylated in RAR{gamma}2, which is the major RAR{gamma} isoform in F9 cells (34, 35). The role of RAR{gamma} phosphorylation in retinoid-induced events has been studied in RAR{gamma}-/- F9 cells, by establishing rescue cell lines reexpressing either RAR{gamma}WT or RAR{gamma} mutated at its phosphorylation sites (20). The RAR{gamma} AF-1 domain and the proline-directed phosphorylation sites located in this domain were found to be required for rescuing the differentiation of F9 cells into primitive endoderm-like cells, whereas the PKA phosphorylation site was dispensible (20). As we had previously established that overexpression of RAR{alpha} in RAR{gamma}-/- cells could also restore their primitive and parietal endodermal differentiation (14), the role of RAR{alpha} phosphorylation was similarly studied using overexpressed RAR{alpha} mutants. We concluded that both the RAR{alpha} AF-1 and AF-2 phosphorylation sites were not required for allowing overexpressed RAR{alpha} to functionally replace RAR{gamma} for primitive endodermal differentiation, but they were apparently mandatory for parietal differentiation (20).

Thus, RAR{alpha} and its phosphorylation sites could be selectively required for parietal endodermal differentiation. However, an unequivocal demonstration of this RAR{alpha}-selective function requires to establish rescue cell lines reexpressing RAR{alpha} (either WT or mutated) in RAR{alpha}-/- F9 EC cells that can still differentiate into primitive endodermal cells (16) but whose parietal endodermal differentiation is greatly delayed (20), thus providing a model for directly analyzing the contribution of RAR{alpha} AF-1 and AF-2 and of their phospho-rylation sites in parietal endodermal differentiation. Furthermore, such lines also offer the possibility to study which RAR{alpha} mutations may generate dominant negatives, preventing endogenous RAR{gamma} from mediating primitive endodermal differentiation. Rescue lines were therefore established with RAR{alpha} mutated at either one of its phosphorylation sites, as well as with AF-1 or AF-2AD core deletion mutants. We demonstrate here that the PKA phosphorylation site and the AF-2AD core (helix 12 of the LBD) of RAR{alpha} are required for parietal endodermal differentiation, whereas the AF-1 proline-directed kinase phosphorylation site is dispensible. None of the corresponding mutants behaved as dominant negatives, preventing primitive endodermal differentiation, whereas deletion of the AF-1 activating domain (the A/B region) generated such a dominant negative mutant. The present study also shows that RAR{alpha} AF-1 and AF-2 are differentially implicated in the induction of RA-responsive genes, whereas their phosphorylation sites are not involved.


    RESULTS
 TOP
 ABSTRACT
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Generation of Stable Rescue Lines Expressing RAR{alpha}1 Lacking Either AF-1 or the AF-2AD Core or Mutated at Either the AF-1 or the AF-2 Phosphorylation Sites
To investigate whether RAR{alpha} AF-1 and AF-2 are involved in parietal endodermal differentiation, rescue lines stably expressing wild-type RAR{alpha} (RAR{alpha}WT line) and RAR{alpha} lacking the A/B region (RAR{alpha}{Delta}AB line) or the AF-2AD core (RAR{alpha}{Delta}AF2 line) were derived from RAR{alpha}-/- cells (see Fig. 1AGo). Similarly, to investigate the role played in this differentiation by phosphorylation of the AF-1 domain at the proline-directed kinases sites (serines 74 and 77) and of the AF-2 domain at the PKA site (serine 369), rescue lines stably expressing RAR{alpha} mutated at these phosphorylation sites (RAR{alpha}S74/77A and RAR{alpha}S369A lines, respectively, see Fig. 1AGo), were also established.



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Figure 1. Schematic Representation of the Constructs Used to Generate the Various RAR{alpha} Rescue Lines in RAR{alpha}-/- Cells

A, Mouse RAR{alpha}1 with the functional domains AF-1 and AF-2, which lie in the A/B region and the E region respectively, and the DNA binding domain (DBD), are schematically represented (not to scale). The target sequences for phosphorylation by proline-directed kinases in the B domain and by protein kinase A (PKA) in the AF-2 activating domain of RAR{alpha}1 are shown. The corresponding serine residues, which have been mutated to alanine (S74/77A and S369A, respectively), are indicated. The N-terminal truncated (RAR{alpha}{Delta}AB) and the AF-2AD core (amino acids 408–416) deleted (RAR{alpha}{Delta}AF2) receptors are also schematically shown. B, RAR{alpha} protein in the rescue lines. Whole cell extracts were prepared from WT F9 cells, RAR{alpha}-/- cells and each rescue line [RAR{alpha}WT, RAR{alpha}S74/77A (clones 3 and 14), RAR{alpha}{Delta}AB, RAR{alpha}{Delta}AF2, and RAR{alpha}S369A]. RAR{alpha} was immunoprecipitated with specific monoclonal antibodies [Ab9{alpha}(F)] and subsequently detected by Western blotting with a specific rabbit polyclonal antibody [RP{alpha} (F)]. The presented results correspond to a representative experiment among three.

 
Several clones were obtained for each transgene, and in each case the presence of the mutations was checked by sequencing complementary DNA (cDNA) fragments amplified by RT-PCR from total RNA of the corresponding line (data not shown). The expression level of RAR{alpha}WT and of its mutant derivatives was determined after immunoprecipitation and Western blotting, and compared with the expression of endogenous RAR{alpha} in WT F9 cells (Fig. 1BGo). RAR{alpha}{Delta}AB and RAR{alpha}S369A were expressed in the selected rescue lines at levels similar to that of RAR{alpha} in WT F9 cells (Fig. 1BGo, lanes 1, 6 and 8). The RAR{alpha}WT and RAR{alpha}{Delta}AF2 rescue lines overexpressed the RAR{alpha} protein relative to endogenous RAR{alpha} (Fig. 1BGo, lanes 3 and 7). Two RAR{alpha}S74/77A rescue cell lines were selected that expressed the RAR{alpha} protein at a level either similar (clone 3) or higher (clone 14) than that of RAR{alpha} in WT F9 cells (Fig. 1BGo; lanes 4 and 5).

Role of RAR{alpha} AF-1 and AF-2 and of Their Phosphorylation Sites in F9 Cell Primitive and Parietal Endodermal Differentiation
When grown as monolayers in the presence of all-trans-RA alone, WT F9 cells differentiate at 96 h into primitive endoderm-like cells exhibiting a characteristic flat triangular morphology (4, 20). Addition of cAMP along with RA, results in the formation of parietal endoderm-like cells that have a rounded and refractile appearance (1, 20).

WT F9 cells also differentiated into primitive endoderm-like cells when treated for 96 h with a combination of a RAR{gamma} agonist (BMS961) and a pan-RXR agonist (BMS649) (36) (Fig. 2bGo). Parietal endodermal differentiation occurred upon subsequent addition at 48 h of the RAR{alpha} agonist BMS753 (36), the pan-RXR agonist BMS649, and cAMP (Fig. 2cGo). No parietal differentiation occurred at 96 h (or even later at 120 h) in the absence of BMS753, confirming that RAR{alpha} is required for parietal endodermal differentiation of WT F9 cells (Ref. 20 and data not shown).



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Figure 2. The AF-2AD Core and the PKA Phosphorylation Site of RAR{alpha} Are Indispensable for Rescuing Parietal Endodermal Differentiation, Whereas the Proline Kinase Site Is Not, and RAR{alpha}{Delta}AB Generates a Dominant Negative

Morphological differentiation of WT F9 cells, RAR{alpha}-/- cells and rescue lines (as indicated) grown for 96 h in the presence of 100 nM BMS961 and 1 µM BMS649 without or with a subsequent addition at 48 h of 100 nM BMS753, 1 µM BMS649, and 250 µM cAMP as viewed under phase contrast microscopy. Control cells treated with either 0.1% ethanol (vehicle) or 250 µM cAMP remained undifferentiated.

 
As previously reported, under these conditions, primitive endodermal differentiation was not affected in RAR{alpha}-/- cells (Fig. 2eGo) (16), but no parietal endodermal differentiation could be seen at 96 h (Fig. 2fGo) (20). In fact, parietal differentiation was markedly delayed and did not occur before 120 h (Ref. 20 , and data not shown). Similar results were obtained with the RAR{gamma} agonist and cAMP, in the absence of the RAR{alpha} agonist (data not shown). Thus, in RAR{alpha}-/- cells, RAR{gamma} can substitute at least to some extent for RAR{alpha}, leading to a delayed parietal endodermal differentiation.

Reexpression of RAR{alpha}WT (RAR{alpha}WT cell line) restored parietal endoderm differentiation at 96 h (Fig. 2iGo). The two RAR{alpha}S74/77A lines also differentiated efficiently into parietal endoderm-like cells (Fig. 2lGo, and data not shown), indicating that RAR{alpha} can efficiently mediate this differentiation in the absence of AF-1 phosphorylation. In contrast, the RAR{alpha}S369A rescue line did not differentiate into parietal endoderm at 96 h (Fig. 2oGo). As in the case of the parental RAR{alpha}-/- cell line, parietal endodermal differentiation was delayed and occurred only at 120 h (data not shown). The same observation was made with the RAR{alpha}{Delta}AF2 rescue line (Fig. 2uGo). Interestingly, the RAR{alpha}{Delta}AB rescue line retained a stem cell morphology in response to addition of BMS961 and BMS649. Only a few differentiated cells were observed upon subsequent addition of BMS753, BMS649, and cAMP (Fig. 2Go, p–r), thus suggesting that deletion of the A/B region generates a RAR{alpha} dominant negative mutant that prevents endogenous RAR{gamma} from mediating primitive endodermal differentiation and subsequently suppresses parietal endodermal differentiation.

The extent of differentiation of the various rescue lines was also assessed by the expression of transcripts of two markers of primitive endodermal differentiation, collagen IV ({alpha}1), and laminin B1 (4, 37), and of one marker specific for parietal endodermal differentiation, thrombomodulin (2, 38). Semiquantitative RT-PCR analysis showed that the induction of collagen IV and laminin B1, which was not affected in RAR{alpha}-/- cells (Fig. 3AGo, lanes 4–6), was similar in all rescue cell lines, except the RAR{alpha}{Delta}AB line where it was significantly reduced (Fig. 3AGo, lanes 16–18), in agreement with the absence of obvious morphological differentiation (see above).



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Figure 3. Expression of the Differentiation Markers Collagen IV ({alpha}1), Laminin B1, and Thrombomodulin

A, Total RNA (2 µg) from WT cells, RAR{alpha}-/- cells and the rescue lines treated as in Fig. 2Go for 96 h was subjected to RT-PCR analysis for collagen IV (13 amplification cycles) and laminin B1 (15 amplification cycles) using transcripts of the 36B4 gene (15 amplification cycles) as an internal control to normalize the amounts of RNA. B, Total RNA (2 µg) from WT and RAR{alpha}-/- F9 cells treated for 96 h with either vehicle (lanes 1 and 6), 250 µM cAMP (lane 3), BMS961, and BMS649 without (lanes 2 and 7) or with cAMP (lanes 4 and 8), and the BMS961/649/753 and cAMP combination (lanes 5 and 9), was subjected to RT-PCR analysis for Thrombomodulin (20 cycles) and 36B4. (C) Total RNA (2 µg) from WT, RAR{alpha}-/- and the rescue lines treated for 96 h with vehicle or the combination of BMS961 and BMS649 and the subsequent addition at 48 h of BMS753, BMS649 and cAMP were subjected to RT-PCR for thrombomodulin and 36B4. The results presented in A, B, and C correspond to a representative experiment among three.

 
No expression of thrombomodulin was seen in WT F9 cells treated with BMS961(selective for RAR{gamma}) and BMS649 (selective for all RXRs) either in the absence or presence of cAMP (Fig. 3BGo, lanes 2–4), thus corroborating previous reports (2, 39). However, thrombomodulin was induced upon subsequent addition of BMS753 (selective for RAR{alpha}), BMS649, and cAMP (Fig. 3BGo, lane 5), indicating that its expression is indeed specific for parietal endodermal differentiation. Note that there was no induction by BMS753 in the absence of the RAR{gamma} agonist (data not shown). The induction of thrombomodulin was only mildly reduced in the RAR{alpha}-/- cell line, in agreement with the delayed morphological parietal endodermal differentiation (Fig. 3BGo, lanes 8 and 9 and Fig. 3CGo, lane 4), and also confirming that RAR{alpha} can be, to some extent, functionally replaced by RAR{gamma} (20). The induction of thrombomodulin was restored in the RAR{alpha}WT and RAR{alpha}S74/77A lines (Fig. 3CGo, lanes 6 and 8). Interestingly, in the RAR{alpha}S369A and RAR{alpha}{Delta}AF-2 lines, the induction of this gene was mildly reduced, as in the case of the RAR{alpha}-/- line (Fig. 3CGo, lanes 12 and 14) and in agreement with the delayed parietal endodermal differentiation. In contrast, the induction of thrombomodulin was completely abrogated in the RAR{alpha}{Delta}AB rescue line (Fig. 3CGo, lane 10), thus corroborating the lack of differentiation.

Effect of RAR{alpha} AF-1 and AF-2 Activating Domains and of Their Phosphorylation Sites on Expression of RA-Responsive Genes
Whereas knockout of the RAR{gamma} gene in F9 cells results in a marked reduction of the expression of several RA-responsive genes such as Hoxa-1, HNF3{alpha}, HNF1ß, Stra6, and Stra8 (19, 20), knockout of RAR{alpha} had no effect on their expression (Fig. 4Go, compare lanes 2 and 4; Fig. 5Go, compare lanes 6 and 7 and Fig. 6Go, lane 2). In fact, only Hoxb-1 and CRABP II gene expression was significantly reduced in RAR{alpha}-/- cells (Fig. 4Go, compare lanes 2 and 4; Fig. 5Go, compare lanes 6 and 7 and Fig. 6Go, lane 2) (16, 19). The expression of these RA-responsive genes was investigated in the various rescue cell lines using semiquantitative RT-PCR (for each gene, in the linear range of the PCR amplification reaction) after treatment for 24 h with 100 nM RA or a synthetic retinoid selective for RAR{gamma} (BMS961) at 100 nM. The results that are presented in Figs. 4Go and 5Go are summarized in Fig. 6Go.



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Figure 4. The Expression or RA-Target Genes Is Not Affected in the Rescue Cell Lines Reexpressing RAR{alpha} Mutated at Its Phosphorylation Sites

Total RNA was isolated from WT F9 cells, RAR{alpha}-/- cells, and the rescue cell lines RAR{alpha}WT, RAR{alpha}S74/77A (clones 3 and 14) and RAR{alpha}S369A with or without treatment of each cell line with 100 nM RA for 24 h as indicated. Transcripts from each gene were analyzed by semiquantitative RT-PCR, using transcripts of the 36B4 gene as an internal control to normalize the amounts of RNA. For each gene, the amount of RNA and the numbers of amplification cycles are described in Materials and Methods. The results correspond to a representative experiment among three.

 


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Figure 5. Differential RA-Inducibility of RA-Target Genes in the Rescue Lines Reexpressing RAR{alpha}{Delta}AB or RAR{alpha} Deleted for Its AF-2AD Core

Total RNA was isolated from WT F9 cells, RAR{alpha}-/- cells, and the rescue cell lines RAR{alpha}WT, RAR{alpha}{Delta}AB and RAR{alpha}{Delta}AF2 without (lanes 1–5) or with a 24 h treatment with 100 nM RA (lanes 6–10) or 100 nM BMS961 (lanes 11–15) as indicated. Transcripts from each gene were analyzed as in Fig. 4Go. The results correspond to a representative experiment among five.

 


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Figure 6. Role of the AF-1 and AF-2 Activating Domains of RAR{alpha} and of Their Phosphorylation in Retinoid-Induced Expression of Target Genes

The relative level of induction of RA responsive genes in WT F9 cells, RAR{alpha}-/- cells and each of the rescue lines grown in the absence or presence of RA (100 nM, stippled bars) or the RAR{gamma} selective agonist (BMS961 100 nM, solid bars) for 24 h was estimated by semi quantitative RT-PCR as in Figs. 4Go and 5Go, followed by quantification of the signals with a Bio-Imaging Analyzer. The values correspond to the fold-induction relative to the amount of RNA transcripts present in ethanol-treated cells, which was given an arbitrary value of 1. The values are an average ± SEM of duplicates from five independent experiments, except in the case of HNF1ß where they are an average of seven experiments.

 
The expression of Hoxb-1 and CRABPII was restored after reexpression of RAR{alpha}WT (Fig. 6Go, lane 3). The phosphorylation site mutants for proline-directed kinases and PKA (RAR{alpha}S74/77A and RAR{alpha}S369A, respectively) were as efficient as RAR{alpha}WT (Fig. 6Go, lanes 4 and 5). RAR{alpha} deleted for either the AF-2AD core or the A/B region also rescued the RA-induced expression of Hoxb-1, but not that of CRABPII (Fig. 6Go, lanes 6 and 7).

We previously concluded that the retinoid-induced expression of Hoxb-1 and CRABPII could be mediated by both RAR{alpha}-RXR and RAR{gamma}-RXR heterodimers (19). However, the expression of both genes, in WT F9 cells, was less efficiently induced by a retinoid selective for RAR{gamma} (BMS961) than by RA (Fig. 5Go, compare lanes 6 and 11 and Fig. 6Go, compare lanes 1 and 8). Interestingly, this latter retinoid was more efficient in RAR{alpha}-/- cells (Fig. 6Go, compare lanes 8 and 9), indicating that the presence of RAR{alpha} was to some extent preventing RAR{gamma} to mediate the induction of Hoxb-1 (19), and CRABPII. Note the down-effect of reexpressing RAR{alpha}WT (Fig. 6Go, lane 10), which was not observed with the two deletion mutants (Fig. 6Go, lanes 11 and 12).

Genes whose expression was not affected in RAR{alpha}-/- cells, e.g. Hoxa-1, HNF3{alpha}, HNF1ß, Stra6, and Stra8, were also analyzed in the rescue lines. Their induction was not significantly affected in RAR{alpha}WT, RAR{alpha}S74/77A, and RAR{alpha}S369A rescue lines (Fig. 6Go, lanes 3–5). Note that HNF3{alpha} expression was mildly reduced in the RAR{alpha}S74/77A line corresponding to clone 14 (Fig. 4Go, lane 10), which expressed higher RAR{alpha} levels than the WT rescue line (Fig. 1BGo, lane 5). The RAR{alpha}{Delta}AB and RAR{alpha}{Delta}AF2 deletion mutants did not affect the RA-induced expression of Hoxa-1 but inhibited markedly those of HNF3{alpha} and HNF1ß (Fig. 6Go, lanes 6 and 7). On the other hand, only RAR{alpha}{Delta}AF2 mildly decreased the RA-inducibility of Stra6 and Stra8 (Fig. 6Go, lanes 6 and 7).

The RAR{gamma} selective agonist (BMS961) was almost as efficient as RA in inducing the expression of Hoxa-1, HNF3{alpha} and Stra8 in WT and RAR{alpha}-/- cells (Fig. 5Go, compare lanes 6 and 11; Fig. 6Go, compare lanes 1 and 8). Whereas Hoxa-1 expression was unaffected by reexpressed RAR{alpha}{Delta}AB and RAR{alpha}{Delta}AF2, that of HNF3{alpha} was reduced and that of Stra8 was reduced only by RAR{alpha}{Delta}AF2 (Fig. 6Go, lanes 11 and 12). Note that the basal level of expression of Stra8 was also decreased by this mutant (Fig. 5Go, lane 5). In contrast, the RAR{gamma}-selective retinoid was more efficient in RAR{alpha}-/- cells than in WT F9 cells for inducing not only the expression of Stra6 (19), but also that of HNF1ß (Fig. 6Go, lanes 8 and 9), confirming that maximal induction of some genes by RAR{gamma} may be competed for by RAR{alpha} (see above). Note, in that respect, the effect of reexpressing RAR{alpha}WT (Fig. 6Go, lane 10). Note also that in some experiments, the basal levels of HNF1ß expression were mildly up-regulated in RAR{alpha}-/- cells (Fig. 5Go, lane 2). RAR{alpha}{Delta}AF2 was as efficient as RAR{alpha}WT in preventing maximal induction of Stra6 by RAR{gamma} but completely abrogated the activation of HNF1ß by BMS961 (Fig. 6Go, lane 12). RAR{alpha}{Delta}AB also abrogated the activation of HNF1ß but did not inhibit the activation of Stra6 by the RAR{gamma} agonist (Fig. 6Go, lane 11).

We also analyzed the expression of another RA-inducible gene, CYP26, whose expression has been shown to be mediated by RAR{gamma}/RXR{alpha} heterodimers (40). Accordingly, it was only slightly decreased in RAR{alpha}-/- cells (Fig. 7Go, compare lanes 1–4). The induction of CYP26, which was maximal at 96 h, was not affected in the various rescue lines with the exception of the RAR{alpha}{Delta}AB line where it was markedly reduced (Fig. 7Go, lane 10).



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Figure 7. The RA-Inducibility of CYP26 Is Abrogated in the RAR{alpha}{Delta}AB Rescue Cell Line

Total RNA (2 µg) from WT F9 cells, RAR{alpha}-/- cells, and the rescue cell lines RAR{alpha}WT, RAR{alpha}S74/77A, RAR{alpha}{Delta}AB, RAR{alpha}S369A, and RAR{alpha}{Delta}AF2 treated for 96 h with 100 nM RA was subjected to RT-PCR analysis for CYP26 (20 amplification cycles) using transcripts of the 36B4 gene as an internal control to normalize the amounts of RNA. The results correspond to a representative experiment among three.

 

    DISCUSSION
 TOP
 ABSTRACT
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The aim of the present study was to analyze the contribution of the AF-1 and AF-2 domains of RAR{alpha} to F9 cell parietal endodermal differentiation and the activation of RA-responsive genes, and also to examine whether the activity of RAR{alpha} could be modulated by phosphorylation of these activating domains. To that end, RAR{alpha}1 mutants lacking either the AF-1 domain or the AF-2AD core (helix 12), or bearing mutations in either the AF-1 or AF-2 phosphorylation sites, were reexpressed to WT levels in RAR{alpha}-/- cells. We demonstrate that both the AF-1 and AF-2 activating domains of RAR{alpha} contribute to parietal endodermal differentiation of F9 cells and to the expression of RA-responsive genes. We also demonstrate that, whereas the phosphorylation sites located in the AF-1 activating domain are not involved in these processes, the PKA phosphorylation site located in the LBD is required for parietal endodermal differentiation. Indeed, mutation of the PKA site abrogates parietal endodermal differentiation at 96 h even in the presence of cAMP.

The RAR{alpha} AF-2 Activating Domain and Its PKA Phosphorylation Site Are Required for Efficient Parietal Endodermal Differentiation
The AF-2AD core motif of RARs belongs to the C-terminal {alpha}-helix of nuclear receptor ligand binding domains (helix 12), which is indispensible for the formation of the coactivator-binding surface during the LBD transconformation triggered by ligand binding, thus generating transcriptionally active receptors (8, 31). Numerous studies have demonstrated that the integrity of the AF-2AD core is required for efficient ligand-dependent interactions with coactivators in vitro (for reviews, see Refs. 8, 41, 42). In the present study, we demonstrate that the AF-2AD core of RAR{alpha} and therefore its AF-2 activation function, is required to mediate the effects of RA and cAMP on F9 cells differentiation in vivo, as RAR{alpha} deleted for this domain is unable to restore parietal endodermal differentiation. Note that this AF-2 activation function was also found to be required for RAR{gamma} and overexpressed RAR{alpha} to restore primitive endodermal differentiation in RAR{gamma}-/- cells (see Table 1Go).


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Table 1. Morphological Differentiation and Relative Expression of RA-Responsive Genes in F9 WT Cells, RAR{gamma}-/-, and RAR{alpha}-/- Cells and Mutant "Rescue" Lines

 
Mutation of the PKA phosphorylation site in the AF-2 domain (RAR{alpha}S369A mutation) also abrogates the ability of RAR{alpha} to efficiently restore parietal endodermal differentiation, demonstrating that this cAMP-dependent phosphorylation of RAR{alpha} is additionally required for this differentiation process. This is in accordance with our previous report (20) showing that overexpression of RAR{alpha}S369A in RAR{gamma}-/- cells was also unable to restore parietal endodermal differentiation (see Table 1Go), which suggested that this mutant exerts a dominant negative effect on endogenous RAR{alpha}, preventing parietal differentiation. However, it is clear from our present results that RAR{alpha}S369A has no dominant negative (dn) effect on RAR{gamma}, as the RAR{alpha}S369A line normally differentiates into primitive endoderm-like cells.

Collectively these results demonstrate that the contribution of RAR{alpha} to parietal endodermal differentiation of F9 cells requires the integrity of the AF-2 surface interacting with coactivators, as well as the integrity of the PKA phosphorylation site located in the AF-2 domain. The effect of this cAMP-dependent RAR{alpha} LBD phosphorylation is unknown, but it may modulate the efficiency of ligand-dependent coactivator/corepressor binding to RAR{alpha}, and/or its dimerization with RXR and thus its DNA binding properties to RA-response elements (34). Interestingly, PKA-mediated phosphorylation of certain coactivators may also modulate their interaction with RAR/RXR heterodimers (Refs. 41, 42, 43, 44 and references therein).

Phosphorylation of the AF-1 Activating Domain Is Not Required for Parietal Endodermal Differentiation, but Deletion of this Domain Generates a Dominant Negative Mutant for F9 Cell Differentiation
When reexpressed in RAR{alpha}-/- cells, RAR{alpha} mutated at its proline-directed kinase phosphorylation site in the N-terminal AF-1 domain (RAR{alpha}S74/77A) is as efficient as RAR{alpha}WT at restoring parietal endodermal differentiation, indicating that phosphorylation of the RAR{alpha} AF-1 domain is not required for this F9 cells differentiation. In contrast, we concluded in our previous study (see Table 1Go) that although the RAR{alpha} AF-1 phosphorylation site was apparently not required to allow overexpressed RAR{alpha} to restore, at the morphological level, the primitive endodermal differentiation of RAR{gamma}-/- cells, it was indispensible for subsequent parietal endodermal differentiation. However, the expression of several RA-responsive genes was not restored to WT levels in RAR{gamma}-/- cells rescued with RAR{alpha}S74/77A (see Table 1Go). It is therefore possible that the phosphorylation of RAR{alpha} AF-1 is in fact indispensible in the RAR{gamma}-/- rescued cells to efficiently rescue primitive endodermal differentiation at the molecular level, which is a prerequisite for subsequent parietal differentiation, whereas this phosphorylation is not required for parietal differentiation per se.

Expression of RAR{alpha} lacking the A/B region in RAR{alpha}-/- cells (RAR{alpha}{Delta}AB rescue line) abrogates their differentiation into primitive endoderm-like cells. This must reflect a ligand-independent (see Fig. 2Go) dn effect of RAR{alpha}{Delta}AB on the endogenous RAR{gamma}, which is required for primitive endodermal differentiation. This dn effect of RAR{alpha}{Delta}AB was also exerted on the expression of certain RA-responsive genes (see below and Fig. 6Go). Note in this respect that we have previously shown (Table 1Go) that RAR{alpha}{Delta}AB overexpressed in RAR{gamma}-/- cells could not restore RA-induced primitive endodermal differentiation and expression of most RA-induced genes, and thus could not functionally replace RAR{gamma}.

We conclude that the RAR{alpha} AF-1 proline-directed phosphorylation site is dispensible for parietal endodermal differentiation of F9 cells, whereas deletion of the A/B region (see Fig. 2Go, RAR{alpha}{Delta}AB), but not of the AF-2AD core (RAR{alpha}{Delta}AF2), results in a dominant negative receptor that blocks the RAR{gamma}-mediated primitive endodermal differentiation.

Differential Promoter Context-Dependent Effect of the AF-1 and AF-2 Activating Domains of RAR{alpha} and of Their Phosphorylation Sites on Expression of RA-Responsive Genes
Among the F9 cell RA-responsive genes that we have studied here and previously (16, 19), the expression of only two of them (Hoxb-1 and CRABPII) is reduced in RAR{alpha}-/- cells, whereas that of the others is not affected. In the present study, we have investigated the role played by RAR{alpha} AF-1 and AF-2 activating domains and their phosphorylation sites in the expression of these RA-responsive genes. Our results, summarized in Fig. 6Go and in Table 1Go, lead to the following conclusions.

Firstly, the PKA site located in the AF-2 domain and the proline-directed kinase site located in the AF-1 domain are not required for restoring the RA-induced expression of Hoxb-1 and CRABPII genes, which is decreased in RAR{alpha}-/- cells. This is in contrast with our previous observation showing that in RAR{gamma}-/- cells overexpressing RAR{alpha}, the phosphorylation of RAR{alpha}1 AF-1 (RAR{alpha}S74/77A) is required for RA-induced expression of some other RA-responsive genes (see Table 1Go). Note in this respect that phosphorylation of RAR{gamma} AF-1 (RAR{gamma}S66/68A) is also required for RA-induced expression of a subset of responsive genes (see Table 1Go).

Secondly, the AF-1 domain and the AF-2AD core of RAR{alpha} are differentially required for RA-induced expression of target genes. Indeed, RAR{alpha} deleted for either of these two domains restores the induction of Hoxb-1, but not of CRABPII. Note that a similar promoter-context dependent requirement of the AF-1 activating domains of RAR{alpha} and RAR{gamma} was also reported for RA-induced expression of other responsive genes in RAR{gamma}-/- rescue cells (see Table 1Go).

Thirdly, both AF-1 and AF-2 deletion mutants of RAR{alpha} behave as dominant negatives in a promoter context-dependent manner, as the induction of RA-responsive genes whose expression is not altered in RAR{alpha}-/- cells (Hoxa-1, HNF3{alpha}, HNF1ß, Stra6, Stra8, and CYP26) was differentially decreased upon reexpression of RAR{alpha} mutants deleted for either the A/B region or the AF-2AD core (see RAR{alpha}{Delta}AB and RAR{alpha}{Delta}AF2 in Fig. 6Go). Note that promoter context-dependent dn effects have also been observed with reexpressed RAR{gamma}{Delta}AF2 and overexpressed RAR{alpha}{Delta}AF2 in RAR{gamma}-/- cells (see Table 1Go and Ref. 46), in keeping with the previous suggestion that AF-2 deletion mutants may behave as dominant negatives for the transcription of RA-responsive genes (23, 45).

Fourthly, depending also on the promoter context, the presence of RAR{alpha} differentially hinders the potential ability of RAR{gamma} to mediate RA-induced expression of a number of responsive genes. Indeed, in several cases, the RAR{gamma}-selective agonist BMS961 was found to be a more efficient inducing ligand in RAR{alpha}-/- cells than in F9 WT cells (Fig. 6Go). These observations confirm that functional redundancies between RARs can be artefactually generated upon gene knockout (18, 20), and furthermore clearly show that the extent of these redundancies is variable, being dependent on the promoter context of the RA-responsive genes.

Conclusion
Using the cell autonomous RA-responsive F9 EC cell differentiation system, we have demonstrated in our present and previous studies (see Table 1Go) that the AF-1 and AF-2 activation functions of both RAR{alpha} and RAR{gamma}, as well as their proline-directed kinase and PKA phosphorylated forms, play important and distinct roles in primitive and parietal endodermal differentiation and RA-induced expression of responsive genes. Moreover, our data show that RAR{alpha} and RAR{gamma} AF-1 and AF-2, and their phosphorylated forms exhibit distinct functions that are differentially required, in a promoter context-dependent manner, to elicit the induction of RA-responsive genes expression. Interestingly, the dominant negative effect of some AF-1 and AF-2 deletion mutants on RAR{alpha}- and RAR{gamma}-mediated induction of expression of RA-responsive genes also exhibits a promoter-context dependency. Finally, a similar promoter-context dependency is also characteristic of the artefactual functional redundancy generated between RAR{gamma} and RAR{alpha} upon RAR{alpha} knockout in F9 cells. All of these promoter-context dependencies certainly reflect the diversity of the responsive complexes that can be combinatorially assembled on polymorphic control regions with different receptor isoforms that are differentially phosphorylated, interact with different coactivators, and synergize with a variety of transregulators (41, 42).


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Plasmid Constructs
All constructs containing the cDNA of either mouse (m) full-length RAR{alpha}1 (RAR{alpha}WT), A/B region-truncated RAR{alpha} (RAR{alpha}{Delta}AB), AF-2AD core-deleted RAR{alpha} (RAR{alpha}{Delta}AF2), or RAR{alpha} harboring mutations in phosphorylation sites were cloned into the pD402A vector (gift of D. Lohnes, Institut de Recherches Cliniques de Montréal, Montreal, Canada) which is driven by the PGK promoter (47). The expression construct pD403A containing mRAR{alpha}1 cDNA cloned in pD402A, has been described (14). RAR{alpha}{Delta}AB, RAR{alpha}S74/77A, and RAR{alpha}S369A were isolated as EcoRI fragments from the corresponding pSG5 constructs (22, 32, 34) and subcloned into the same site of pD402A. RAR{alpha} deleted for the AF-2AD core (amino acids 408–416) was constructed in pD402A, by replacing the WT SacI/BamHI fragment in pD403A by the corresponding fragment containing the deletion in the described pSG5 construct (23).

Cell Culture and Establishment of Stable Rescue Lines
Fig 9 cells were cultured as monolayers on gelatinized surfaces as described (15). For differentiation studies, 105 cells were cultured in a 10-cm dish, and treated with RA (100 nM) alone or in combination with cAMP (250 µM) for 96 h, with a change of media after 48 h. Cells were also treated with a combination of a RAR{gamma} agonist (BMS961, 100 nM) and a pan-RXR agonist (BMS649, 1 µM), followed by the subsequent addition at 48 h of a RAR{alpha} agonist (BMS753, 100 nM), the pan-RXR agonist and cAMP. Control cells were treated with vehicle alone (0.1% ethanol final concentration). To establish rescue lines, RAR{alpha}-/- cells were electroporated with each of the constructs displayed in Fig. 1AGo along with a plasmid conferring resistance to puromycin (pD503; gift of D. Lohnes), in a ratio of 10:1. After 24–36 h, cells were selected with 0.8 µg/ml of puromycin for 10 days as described (14) and analyzed for the presence and expression of the transgene by Southern and Western blotting.

Cell Extracts, Immunoprecipitation and Immunoblotting of RAR{alpha}
Whole cell extracts were prepared (48) from rescue lines grown as monolayers in the absence of RA, and RAR{alpha} was immunoprecipitated with Protein A-Sepharose cross-linked with monoclonal antibodies directed to the F region of RAR{alpha} [(Ab9{alpha}(F)] (49). Proteins were resolved by SDS-PAGE (10% acrylamide), electrotransferred onto nitrocellulose filters and immunoprobed with polyclonal antibodies against the F region of RAR{alpha} [RP{alpha}(F)] (49), followed by peroxidase-labeled protein A and chemiluminescence detection, according to manufacturer’s protocol (Amersham Pharmacia Biotech Europe GmbH, Saclay, France).

RNA Isolation and RT-PCR
RNA was isolated using the guanidinium thiocyanate method and conditions for semiquantitative RT-PCR were as described (5). The quantity of RNA used for RT-PCR in each reaction (2 µg) was normalized with 36B4 transcripts (5, 50) which are unresponsive to retinoid treatment. The RT-PCR oligonucleotides used for Hoxa-1, HNF3{alpha}, HNF1ß, Stra8, laminin B1, collagen type IV ({alpha}1), Hoxb-1, CRABPII, and 36B4 were as described (6, 20). The RT-PCR primers for throm-bomodulin (51) were as follows: 5'-TGGAGCATGAGTGCTTCGC-3' and 5'-GGTGTTGTAGGTACTAGAGA-3'. The primers for CYP26 (52) were 5'-GCTCAAGCTCTGGGACCTGT-3' and 5'-CGATCACGAGCACGTAGCAC-3'. The amplifications were done for 13 cycles (collagen IV), 15 cycles (laminin B1, Stra8, and 36B4), 18 cycles (Hoxb-1, Hoxa-1, and HNF3{alpha}), 20 cycles (thrombomodulin, CRABPII, Stra6, and CYP26) or 24 cycles (HNF1ß). Aliquots were electrophoresed on a 1.5% agarose gel and samples were transferred onto Hybond N membranes. The RT-PCR blots were probed with cognate [32P]-labeled cDNA fragments or with end-labeled oligonucleotide probes for thrombomodulin and CYP26. The signals were quantified using a Bio-Imaging Analyzer (BAS 2000, Fuji Photo Film Co., Ltd., Tokyo, Japan).


    ACKNOWLEDGMENTS
 
We are grateful to Dr. P. Reczek for the gifts of the synthetic retinoids BMS961, BMS753 and BMS649, to Dr. L. Gudas for the gift of laminin B1 and Collagen type IV ({alpha}1) cDNAs and to P. Bouillet and D. Lohnes for the gift of several plasmids and probes. We thank D. Metzger for critically reading the manuscript. We also thank S. Vicaire for DNA sequencing, the cell culture group for maintaining and providing cells, the staff of oligonucleotide synthesis, and C. Werlé, S. Metz, B. Boulay, and J. M. Lafontaine for preparing the figures.


    FOOTNOTES
 
Address requests for reprints to: Dr. Cécile Rochette-Egly, Institut de Génétique et de Biologie Moléculaire et Cellulaire. BP 163, 67404 Illkirch Cedex, CU de Strasbourg, France.

This work was supported by funds from the Centre National de la Recherche Scientifique (CNRS), the Institut National de la Santé et de la Recherche Médicale (INSERM), the Collège de France, the Hôpital Universitaire de Strasbourg, the Association pour la Recherche sur le Cancer (ARC), the Comité Départemental du Haut-Rhin de la Ligue Nationale Contre le Cancer, and Bristol-Myers Squibb.

1 Present address: Department of Biochemistry and Molecular Biology, Box 1126, Mount Sinai School of Medicine, New York, New York 10029-6574. Back

Received for publication March 27, 2000. Revision received June 2, 2000. Accepted for publication June 8, 2000.


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