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Originally published In Press as doi:10.1074/jbc.M203623200 on May 24, 2002

J. Biol. Chem., Vol. 277, Issue 32, 28683-28689, August 9, 2002
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The Phosphorylation Site Located in the A Region of Retinoic X Receptor alpha  Is Required for the Antiproliferative Effect of Retinoic Acid (RA) and the Activation of RA Target Genes in F9 Cells*

Julie BastienDagger, Sylvie Adam-StitahDagger, Jean-Luc Plassat, Pierre Chambon, and Cécile Rochette-Egly§

From the Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP/Collège de France, BP 163, 67404 Illkirch Cedex, France

Received for publication, April 15, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Mouse F9 embryocarcinoma cells constitute a well established cell autonomous model system for investigating retinoic acid (RA) signaling in vitro. RA induces the differentiation of F9 cells grown as monolayers into endodermal-like cells and decreases their rate of proliferation. Knock-out of the retinoic X receptor alpha  (RXRalpha ) gene abolishes endodermal differentiation and the induction of several endogenous RA-responsive genes. RXRalpha null cells are also drastically impaired in their antiproliferative response to RA. The role of the RXRalpha phosphorylation site located in the N-terminal A region (Ser22) has been investigated here by establishing cell lines re-expressing RXRalpha either wild type or mutated at the phosphorylation site (RXRalpha S22A) in a RXRalpha -null background. We show that Ser22 is dispensable for RA-induced endodermal differentiation but is crucial for the expression of several RA-responsive genes. Ser22 is also indispensable for the antiproliferative effect of RA and necessary for the RA-induced down-regulation of p21CIP and p27KIP CKIs proteins that are known to be involved in the control of cell cycle progression.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Retinoic acid (RA),1 the most potent biologically active metabolite of vitamin A, plays crucial roles in a wide variety of biological processes and influences the proliferation and differentiation of a variety of cell types (for reviews, see Refs. 1-4). RA exerts its effects through two families of nuclear ligand-dependent transcriptional regulators, the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs). There are three RAR (alpha , beta , and gamma ) and three RXR isotypes (alpha , beta , and gamma ), and for each isotype, there are at least two main isoforms that are generated by differential promoter usage and alternative splicing and that differ only in their N-terminal A region (Refs. 5-7 and references therein).

F9 murine embryonal carcinoma cells provide a powerful cell autonomous model system for investigating retinoid signaling in vitro (for review see Ref. 8 and references therein). Upon RA treatment, and depending on culture conditions, F9 cells differentiate into three distinct cell types resembling primitive, parietal, and visceral endodermal extraembryonic cells (9). This RA-induced differentiation is also accompanied by a decrease in the rate of proliferation and the induction of expression of a number of genes. F9 cells express all RAR and RXR isotypes, with RXRalpha 1, RARalpha 1, and RARgamma 2 being the main isoforms. Two strategies have been used to investigate their roles in the response of F9 embryonal carcinoma cells to RA treatment. Firstly, F9 cells lacking one or several RARs or RXRs were engineered through homologous recombination (10-15). Secondly, wild type (WT) and mutant F9 cells were treated with pan-RXR and RAR isotype (alpha , beta , or gamma )-selective retinoids (12, 13, 16-18). These studies demonstrated that RARgamma 2/RXRalpha heterodimers are the functional units transducing most RA-induced events (e.g. primitive and visceral differentiation, growth arrest, and activation of expression of a number of genes), whereas RARalpha /RXRalpha heterodimers mediate some other events such as parietal differentiation.

RARs and RXRs possess two transcriptional activation functions (AFs): AF-1 located in the N-terminal A/B region (19, 20) and AF-2 associated with the ligand-binding domain and activated by the ligand (Refs. 6 and 21 and references therein). The AF-1 domain of RARs is phosphorylated at conserved residues that belong to consensus sites for proline-directed kinases, which include the cyclin-dependent kinases and the mitogen-activated protein kinases (for review see Refs. 22 and 23). In RARalpha 1 and RARgamma 2, the phosphorylated residues have been identified and found to be located in the conserved B region (24, 25). RXRalpha 1 is also phosphorylated, but the phosphorylation site (serine 22) is located in the RXRalpha 1-specific A region (26).

Because the various RA-responses of F9 cells can be restored upon re-expression of WT RARgamma in RARgamma -null cells (27), the role of the activation functions AF-2 and AF-1 and that of the phosphorylation of RARgamma 2 in RA-induced events have been studied by re-expressing a variety of RARgamma 2 mutants in these cells (RARgamma Delta AF-2, RARgamma Delta AF-1, and RARgamma S66/68A "rescue" lines). This strategy allowed us to demonstrate that RARgamma 2 needs the integrity of both its AF-1 and AF-2 domains to efficiently transduce the RA signal (18, 28). RARgamma 2 further requires the phosphorylation site of its AF-1 domain for inducing RA target gene and F9 cell differentiation (18). Phosphorylation is also necessary for the RA-induced degradation of RARgamma 2 by the ubiquitin-proteasome pathway (29).

By contrast, little is known about the mechanisms through which RXR exerts its transcriptional activity. In vitro studies demonstrated that liganded RXR is not active unless its RAR partner is itself liganded (16-18, 30, 31). Phenotypic analysis of mice expressing RXRalpha with its N-terminal A/B region deleted indicated that the RXRalpha AF-1 domain is functionally important for efficiently transducing the retinoid signal during embryonic development (32). However, little is known about the mechanisms through which the N-terminal A region and its phosphorylation site participate in the global activity of RXRalpha under physiological conditions.

Because RXRalpha -null F9 cells are drastically impaired in primitive and parietal endodermal differentiation as well as in their antiproliferative response to RA (14), we functionally dissected the role of RXRalpha Ser22 in these processes by establishing a rescue line expressing RXRalpha S22A in a RXRalpha -null background. Our results demonstrate that in F9 cells Ser22 is dispensable for primitive and subsequent parietal endodermal differentiation but is required for the induction of several RA-responsive genes. This phosphorylation site is also crucial for the antiproliferative effect of RA. In that context, Ser22 is necessary for the RA-induced decrease in the levels of p21CIP and p27KIP proteins that are involved in the control of cell cycle progression.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Plasmid Constructs-- The mouse full-length cDNA of RXRalpha 1 was cloned into the pD402A vector (a gift of D. Lohnes), which is driven by the PGK promoter (33). RXRalpha S22A in PD402A was constructed by subcloning the XhoI-SacI fragment containing the mutation from the pSG5-mRXRalpha construct (26) into the same sites of pD402A RXRalpha WT.

Cell Culture and Establishment of Stable Rescue Lines-- F9 cells were cultured as monolayers on gelatinized surfaces as described previously (10). For differentiation studies, 105 cells were cultured in 10-cm dishes and treated with tRA (100 nM) alone or in combination with 250 µM dibutyryl-cAMP (Sigma) for 96 h with a medium change after 48 h. The control cells were treated with vehicle (final ethanol concentration, 0.1%). To establish the rescue lines, RXRalpha -/- cells (4.5 × 106 cells) were electroporated with the constructs indicated in Fig. 1A linearized with AatII, along with a XhoI-linearized plasmid conferring resistance to hygromycin in a ratio of 10:1. After 24 h, the cells were selected with hygromycin (400 µg/ml) for 10 days (27) and analyzed for the presence and expression of the transgene by Southern and Western blotting.

Antibodies-- Rabbit polyclonal antibodies raised against the A region of RXRalpha 1, RPRXalpha (A), were as described (34). Those against p21CIP (C-19) and p27KIP (Ab-2) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) and NeoMarkers (Lab Vision Corp.), respectively. Goat polyclonal antibodies raised against beta -actin were from Santa Cruz Biotechnology, Inc.

Cell Extracts and Immunoblotting-- Whole cell extracts (WCEs) were prepared as described previously (35). The proteins (40 µg) were resolved by SDS-PAGE (12% acrylamide), electrotransferred onto nitrocellulose filters, immunoprobed, and detected by chemilumiscence according to the manufacturer's protocol (Amersham Biosciences).

RNA Isolation and RT-PCR-- Total RNAs were isolated using the guanidinium thiocyanate method (36), and the aliquots (500 ng) were subjected to real time quantitative RT-PCR, using the SYBR Green Light cycler detection system (Roche Molecular Biochemicals). The transcripts levels were normalized according to 36B4 transcripts, which are unresponsive to retinoids treatment. The RT-PCR oligonucleotides for 36B4, Collagen IV, Laminin B1, HNF3alpha , and HNF1beta were as described (14, 18). The primers were: Stra6, 5'-CTGCAGACCAGCTACTCCGA-3' and 5'-ACAGTAGGCACCACGCTCAC-3'; Hoxa-1, 5'-GAGCTGGAGAAGGAGTTCCA-3' and 5'-CAGAGTTGGGCTGGAGTAGC-3'; Hoxb-1, 5'-CTCGAAGACTTTCCCAAACTTCAC-3' and 5'-TCTCTAAGCTCAAAGGCACTGAAC-3'; CRABPII, 5'-AACCTCCACCACTGTGCGAA-3' and 5'-AGGCAGTTCTTGGACCCGTA-3'; p21CIP, 5'-GCCGTGATTGCGATGCGCTC-3' and 5'-CTCCTGACCCACAGCAGAAG-3'; and p27KIP, 5'-GAGTCAGCGCAAGTGGAATTT-3' and 5'-GCC- TGTAGTAGAACTCGGGCA-3'.

Cell Growth Analysis-- Cell counting experiments were performed in triplicate with untreated and RA-treated cells as follows. The cells were plated at identical densities (2.5 × 103 cells/well) in 6-well plates and fed with fresh medium containing either vehicle or RA (100 nM) every 2 days. At days 3 and 5, the remaining adherent cells were trypsinized and counted with a Coulter particle counter (Coultronics France, SA). The percentage of growth inhibition by RA was calculated as described previously (14).

The cell cycle profiles of F9 WT, RXRalpha -/-, RXRalpha WT, and RXRalpha S22A cells were determined by cell cycle flow cytometry based on cellular DNA content analysis using a FACScan (Beckton Dickinson, Inc.). Subconfluent cultures of control or RA-treated cells were trypsinized and combined with their culture supernatants, pelleted, resuspended in 500 µl of hypotonic buffer (0.1% Triton X-100, 0.1% sodium citrate) containing 50 µg/ml propidium iodide, and incubated for 15 h in the dark at 4 °C. The percentage of cells in the different phases of the cell cycle was determined using the Cell Quest software.

Statistical Analysis-- The data are expressed as the means ± S.E. of three independent experiments unless otherwise indicated. Statistical analysis was performed using the analysis of variance followed by 2 × 2 comparisons based on the Newman-Keul's test.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Generation of Rescue Lines Expressing RXRalpha -- We have previously shown that RXRalpha is "constitutively" (i.e. in the absence of ligand) phosphorylated at serine 22 in COS-1 cells (26) and also in F9 cells.2 To investigate whether this phosphorylation of RXRalpha is involved in primitive and parietal endodermal differentiation of F9 cells, as well as in their antiproliferative response to RA, rescue lines re-expressing wild type RXRalpha (RXRalpha WT line) or RXRalpha mutated at the phosphorylation site (RXRalpha S22A line) were derived from RXRalpha -null cells (Fig. 1A). Two clones were obtained for the RXRalpha WT rescue transgene and one clone for the RXRalpha S22A rescue transgene. The presence of the S22A mutation was verified by sequencing cDNA fragments amplified by RT-PCR from total RNA of the RXRalpha S22A rescue line (data not shown). The expression level of RXRalpha WT and RXRalpha S22A in the derived cell lines was compared with the expression of endogenous RXRalpha in F9 WT cells after immunoblotting. RXRalpha S22A was expressed in the corresponding rescue line at levels similar to that of RXRalpha in F9 WT cells (Fig. 1B, lane 4). The RXRalpha WT rescue lines slightly overexpressed the RXRalpha protein relative to endogenous RXRalpha . Because they yielded similar results in the studies described thereafter, one was selected (Fig. 1B, lane 3).


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  		Fig. 1.   Generation of stable rescue lines re-expressing RXRalpha WT or RXRalpha S22A. A, schematic representation of the constructs used to generate RXRalpha rescue lines in RXRalpha -null cells. Mouse RXRalpha 1 with the DNA-binding domain (DBD) and the AF-1 and AF-2 activation domains, which lie in the A/B and E regions, respectively, are schematically represented (not to scale). The target sequence for phosphorylation by proline-dependent kinases in the A region of RXRalpha 1 is shown, and the serine residue, which has been mutated to alanine (Ser22) is indicated. B, RXRalpha protein in rescue lines. WCEs were prepared from WT F9 cells, RXRalpha -/- cells, and the two rescue lines (RXRalpha WT and RXRalpha S22A). The proteins were resolved by SDS-PAGE, and RXRalpha was detected by Western blotting with a specific rabbit polyclonal antibody, RPRX(A). The presented results correspond to a representative experiment of three.

The RA-induced Endodermal Differentiation of F9 Cells Does Not Require the Phosphorylation Site Located in the A Region of RXRa-- When grown as monolayers in the presence of RA for 96 h, F9 WT cells differentiate into primitive endoderm-like cells (37) exhibiting a characteristic flat triangular morphology (Fig. 2A, panel b). The addition of cAMP along with RA results in the formation of parietal endoderm-like cells (38), which have a rounded and refractile appearance (Fig. 2A, panel c). These two types of differentiation are drastically impaired in RXRalpha -/- cells (14) (Fig. 2A, compare panels e and f with panels b and c), and re-expression of RXRalpha WT (RXRalpha WT rescue line) restores the RA responsiveness (Fig. 2A, panels g-i). Similarly, the RXRalpha S22A rescue line differentiates upon treatment with RA alone or with RA plus cAMP (Fig. 2A, panels j-l), indicating that RXRalpha can efficiently mediate the induction of primitive and parietal endoderm differentiation in the absence of Ser22.


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  		Fig. 2.   The phosphorylation site located in the N-terminal A region of RXRalpha (Ser22) is dispensable for rescuing primitive endodermal differentiation. A, morphological differentiation of F9 WT, RXRalpha -/-, RXRalpha WT, and RXRalpha S22A cells (as indicated) grown for 96 h in presence of 100 nM RA alone or combined with 250 µM cAMP, as viewed under phase contrast microscopy. The control cells treated with 0.1% ethanol (vehicle) or with cAMP alone remained undifferentiated. B and C, relative expression of the differentiation markers laminin B1 and collagen IV(alpha 1). Total RNA (500 ng) from F9 WT, RXRalpha -/-, RXRalpha WT, and RXRalpha S22A cells treated as in A was subjected to quantitative RT-PCR analysis for collagen IV and laminin B1 (see "Experimental Procedures"). The values correspond to the fold induction relative to the amount of RNA transcripts present in ethanol-treated cells. ***, statistically significant differences between WT cells and the other cell lines (p < 0.001).

The differentiation of the various rescue lines was further analyzed by determining the expression of two markers of primitive endodermal differentiation, laminin B1 and collagen IV(alpha 1), using quantitative RT-PCR. RA-induced expression of laminin B1 and collagen IV (Fig. 2, B and C, columns 1), which was impaired in RXRalpha -/- cells (14) (Fig. 2, B and C, columns 2), was restored in the RXRalpha WT rescue line to levels similar to those achieved in F9 WT cells (Fig. 2, B and C, columns 3). In agreement with the morphological differentiation, the expression of these two markers was completely restored in the RXRalpha S22A rescue line (Fig. 2, B and C, columns 4). Altogether, these results indicate that Ser22, located in the N-terminal A region of RXRalpha , is dispensable for RA-induced endodermal differentiation of F9 cells. Similar results were obtained concerning parietal endodermal differentiation as assessed by the expression of a specific marker, thrombomodulin (data not shown) (39).

Role of RXRalpha Ser22 on the Expression of Several RA-responsive Genes-- Knock-out of the RXRalpha gene in F9 cells results in a drastic reduction of the expression of several RA-responsive genes (13, 14), such as Stra6, Hoxa-1, HNF3alpha , CRABPII, Hoxb-1, and HNF1beta (Fig. 3, in each panel, compare lanes 1 and 2; p < 0.001). We investigated the ability of RXRalpha WT and RXRalpha S22A to rescue the expression of these RA target genes, using quantitative real time RT-PCR after treatment of the different cell lines with 100 nM RA for 24 h. Re-expression of RXRalpha WT restored the expression of all genes tested to levels significantly similar to those achieved in F9 WT cells (Fig. 3, compare lanes 1 and 3 in each panel). RXRalpha S22A also restored the expression of Stra6 and Hoxa-1 with the same efficiency as RXRalpha WT (Fig. 3, A and B, compare lanes 1 and 4). However, RXRalpha S22A did not restore the expression of HNF3alpha , CRABPII, Hoxb-1, nor HNF1beta to the levels achieved in WT cells (Fig. 3, C-F, compare lanes 1 and 4; p < 0.001). No responsiveness was observed for up to 96 h of RA treatment, (data not shown), indicating that the RXRalpha S22A mutant does not lead to a delayed activation of these RA target genes.


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  		Fig. 3.   The phosphorylation site of RXRgamma (Ser22) is required for the induction of several RA-responsive genes. The differential RA inducibility of the various RA-responsive genes in WT, RXRalpha -/-, RXRalpha WT, and RXRalpha S22A F9 cells grown in the presence of RA (100 nM) for 24 h was analyzed by quantitative RT-PCR as in Fig. 2. ***, statistically significant differences between the WT cells and the other cell lines (p < 0.001).

Collectively, our results indicate that Ser22 is crucial for the induction of several RA target genes expression and that this process is promoter context-dependent. Note that a difference in the stability of the mutant receptor is ruled out, because RXRalpha WT and RXRalpha S22A levels are not affected within 48 h of RA treatment.2

The Antiproliferative Effect of RA Requires the RXRalpha Phosphorylation Site Located in the A Region-- RA-induced differentiation of F9 cells is also accompanied by a marked decrease in their proliferation rate as determined by counting of the adherent cells with 58 and 84% growth inhibition at 3 and 5 days of RA treatment, respectively (Fig. 4). This antiproliferative response to RA is significantly reduced in RXRalpha -/- cells (14), which exhibit only 32 and 54% growth inhibition upon 3 and 5 days of RA treatment, respectively (Fig. 4). Re-expression of RXRalpha WT restored the antiproliferative response to RA with a growth inhibition similar to that observed in F9 WT cells (Fig. 4). In contrast, the RXRalpha S22A rescue line depicted a different behavior. Indeed, at 3 days of RA treatment, the RXRalpha S22A rescue line retained an antiproliferative response that was not significantly different from that of RXRalpha -/- cells (Fig. 4A; p > 0.05). However, after 5 days of RA treatment, the growth inhibition was slightly rescued but was still significantly different from that of WT cells (Fig. 4B; p < 0.05). Thus, RXRalpha appears to mediate part of the antiproliferative effect of RA through the Ser22 phosphorylation site.


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  		Fig. 4.   The phosphorylation site of RXRalpha (Ser22) is required for the antiproliferative effect of RA. WT F9 cells, RXRalpha -/- cells, and rescue cells were plated in triplicate in 6-well plates at an identical density of 2.5 × 103 cells/well and counted after 3 (A) and 5 (B) days of culture in the absence or presence of RA (100 nM). The number of cells after 3 or 5 days of culture in the presence or absence of RA is indicated. The asterisks indicate statistically significant differences between the RA-treated cell lines (*, p < 0.05; **, p < 0.01; ***, p < 0.001). ns, not significant (p > 0.05). The percentages of growth inhibition are indicated in parentheses.

Previous studies have shown that RA treatment of F9 cells also results in the lengthening of the G1 portion of the cell cycle and that this effect is less pronounced for RXRalpha -/- cells (14). Therefore, we investigated by cell cycle flow cytometry (see "Experimental Procedures") whether RXRalpha Ser22 is also involved in the RA-induced accumulation in the G1 phase. The untreated WT, RXRalpha WT, or RXRalpha S22A cell lines exhibited similar cell cycle profiles, with some insignificant fluctuations reflecting variations in the basal proliferation rate (Table I). Note that the RXRalpha -/- line depicting a lower proportion of cells in the G1 phase may be due to a slight higher basal proliferation rate (14).

                              
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Table I
The phosphorylation site of RXRalpha (Ser22) is indispensable for the RA-induced accumulation of F9 cells into the G1 phase of the cell cycle
F9 wild type RXRalpha -/-, RXRalpha WT, and RXRalpha S22A cells were grown for 5 days in the presence of 100 nM RA. The percentages of cells in the different phases of the cell cycle were analyzed with an fluorescence-activated cell sorter as described under "Experimental Procedures." The values correspond to the percentages of cells in the G0/G1, S, and G2/M phases.

RA treatment of F9 WT cells for 5 days resulted in an accumulation of the cells in the G1 phase of the cell cycle from 36 ± 0.58 to 53 ± 0.88% (Table I). This accumulation was significantly decreased in RXRalpha -/- cells (14) with 36 ± 1.2% of cells in G1 instead of 53% for WT cells (Table I, p < 0.001). The RXRalpha WT rescue cell line recovered a proportion of cells in the G1 phase that was similar to that observed with WT F9 cells (Table I). In contrast, in RA-treated RXRalpha S22A cells, the proportion of cells in the G1 phase remained statistically the same as that of RXRalpha -/- cells (Table I). Collectively, these results indicate that RXRalpha Ser22 plays a crucial role in the antiproliferative effect of RA.

RXRalpha Ser22 Is Required for the RA-induced Down-regulation of the CKI Proteins, p21CIP and p27KIP-- To corroborate the role of RXRalpha Ser22 in the antiproliferative effect of RA, we investigated its contribution to the regulation of some G1 phase-associated molecules that have been shown to be targets for RA action (40-45). We focused upon the cyclin-dependent kinase inhibitors p21CIP and p27KIP.

The expression of p27KIP and p21CIP transcripts did not vary significantly upon RA treatment of F9 WT cells up to 96 h (data not shown). However, p27KIP and p21CIP protein levels were strongly decreased within 48 and 72 h, respectively (Fig. 5), indicating that in F9WT cells, the antiproliferative effect of RA correlates with a down-regulation of these CKIs. Interestingly, in RXRalpha -null cells, the RA-induced down-regulation of p27KIP was delayed and occurred at 96 h instead of 48 h (Fig. 5A), whereas that of p21CIP was completely abolished (Fig. 5B). The down-regulation of both CKIs was fully restored in the RXRalpha WT rescue line (Fig. 5). However, in the RXRalpha S22A line, the decrease in p27KIP was not rescued (Fig. 5A), whereas that in p21CIP was restored with a delay (Fig. 5B). Altogether, these data indicate that, in F9 cells, the RA-induced down-regulation of p27KIP and to a lesser extent of p21CIP requires the phosphorylation site located in the A region of RXRalpha .


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  		Fig. 5.   The phosphorylation site of RXRalpha (Ser22) is required for the RA-induced down-regulation of p27KIP and p21CIP proteins. WCEs were prepared from F9 WT, RXRalpha -/-, RXRalpha WT, and RXRalpha S22A cells left untreated or treated with RA for the indicated times. Equal amounts of WCEs, as assessed by immunoblotting with actin antibodies (data not shown) were resolved by SDS-PAGE and p27KIP (A) and p21CIP (B) proteins were detected by immunoblotting with specific rabbit polyclonal antibodies. The presented results correspond to a similar representative experiment of three.

Because p21CIP and p27KIP proteins are essentially regulated post-transcriptionally by the ubiquitin-proteasome pathway (46-51), we investigated whether in F9 cells, the RA-induced down-regulation of these CKIs involves the activation of this pathway. Treatment of control cells with the proteasome inhibitor MG132 did not significantly affect p21CIP protein levels but markedly increased p27KIP (Fig. 6A), suggesting that in F9 cells, the proteasome-dependent pathway is involved in the turnover of this CKI. In contrast, in RA-treated F9 cells, MG132 abrogated the decrease in p21CIP levels (Fig. 6B) but not that of p27KIP (Fig. 6C). Altogether, these results suggest that the down-regulation of p21CIP induced by RA involves the proteasome pathway, whereas that of p27KIP may occur through an other mechanism.


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  		Fig. 6.   The proteasome and p21CIP and p27KIP protein levels. A, control F9 WT cells were incubated or not with MG132 (40 µM) 15 h before harvesting. Equal WCE amounts, as checked by immunoblotting with actin antibodies, were immunoblotted with p21CIP or p27KIP antibodies. B and C, same as A, but F9 WT cells were treated with RA for the indicated times and left untreated or incubated with MG132 15 h before harvesting. WCEs were immunoblotted with actin, p21CIP (B), or p27KIP (C) antibodies.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The present investigation was designed to analyze the contribution of the constitutively phosphorylated serine residue located in the N-terminal A region of RXRalpha (Ser22), in the control of F9 cells differentiation and growth arrest, and in the expression of RA target genes. To that end, we used rescue RXRalpha -null F9 cells re-expressing RXRalpha either WT or mutated at Ser22. Analysis of the RA response of these cell lines allows us to draw the following conclusions.

First, we demonstrate that the phosphorylation site located in the N-terminal A region of RXRalpha is dispensable for the RA-induced differentiation of F9 cells, because the line rescued with RXRalpha S22A is able to differentiate into primitive endoderm-like cells and subsequently into parietal endodermal cells.

Second, RXRalpha Ser22 is necessary for the induction of certain RA target genes. In F9 cells, the expression of most RA-responsive genes such as Hoxa-1, HNF3alpha , HNF1beta , Stra6, and CRABPII, is known to be controlled by RARgamma /RXR heterodimers, whereas the induction of Hoxb-1 can be mediated by either RARgamma /RXR or RARalpha /RXR heterodimers (8). The activation of these genes is strongly decreased or abrogated in RXRalpha -null cells (13, 14). Our results demonstrate that the N-terminal phosphorylation site of RXRalpha is necessary for the RA-induced expression of some of these genes, such as HNF3alpha , HNF1beta , CRABPII, and Hoxb-1, because RXRalpha S22A is inefficient in restoring their inducibility. This may result from distinct steric conformations of the AF-1 domain of RXRalpha bound to different promoters and therefore from different interactions with putative AF-1 coactivators that could be differentially modulated by phosphorylation. In this respect, we note that the phosphorylation of the A/B domain of some nuclear receptors has been shown to modulate their interaction with coactivators or their ligand affinity. For example, phosphorylation of the estrogen receptor A/B domain promotes recruitment of the SRC-1 coactivator (52), whereas phosphorylation of the PPARgamma A/B domain reduces the ligand binding affinity of the receptor, thus negatively regulating its transcriptional activity (53, 54).

Third, RXRalpha Ser22 is required for the antiproliferative response to RA and the accumulation in the G1 phase, which are severely altered in RXRalpha -null cells (14). In several cell lines, the growth inhibitory effect of RA has been correlated to the expression level of RARbeta 2 (15, 55, 56). However, our results are not consistent with such a mechanism, because RARbeta 2 is similarly induced in F9 WT cells, RXRalpha -null cells (13, 14), and the different rescue lines (data not shown). In fact, progression through the cell cycle is ensured by a number a factors including cyclins, cyclin-dependent kinases, and CKIs (22, 57, 58). Although considerable advances have been made in understanding the role of these factors in G1 progression, how RA controls the coordinated action of these molecules in F9 cells is not completely elucidated. However, according to a number of reports, the antiproliferative effect of RA has been associated with variations in the expression of the CKIs p21CIP and p27KIP (40-44). Initially considered as inhibitors of proliferation, increasing evidence now suggests that CKIs play a complex role and may be also associated with cell cycle progression (59-61). Accordingly, depending on the cell system, either increases or decreases in CKIs levels have been associated with the antiproliferative effect of RA. In the present study performed with F9 cells, we found that RA down-regulates p21CIP and p27KIP levels. The mechanism of this down-regulation remains to be investigated. Similarly, how this down-regulation participates in the antiproliferative effect of RA is still unknown. Nevertheless, the important point of the present investigation is that the phosphorylation site localized in the N-terminal region of RXRalpha 1, which is involved in the antiproliferative effect of RA, is also required for the RA-induced variations in the levels of some proteins engaged in G1 progression. The identification of the RA-responsive genes specifically involved in the regulation of cell cycle progression would provide new insights for understanding cell cycle regulation and the role of RXRalpha in RA signaling.

    ACKNOWLEDGEMENTS

We thank D. Metzger and J. Clifford for kindly providing the RXRalpha WT rescue line. We also thank members of the cell culture facility and of the oligonucleotides facility for help.

    FOOTNOTES

* This work was supported by funds from CNRS, INSERM, the Collège de France, the Association pour la Recherche sur le Cancer, and Bristol-Myers Squibb.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Supported by the Ministère de l'Education Nationale et de la Recherche Scientifique et Technique.

§ To whom correspondence should be addressed: IGBMC, BP 163, 67 404 Illkirch Cedex, CU de Strasbourg, France. Tel.: 33-3-88-65-34-59; Fax: 33-3-88-65-32-01; E-mail: cegly@igbmc.u-strasbg.fr.

Published, JBC Papers in Press, May 24, 2002, DOI 10.1074/jbc.M203623200

2 J. Bastien, S. Adam-Stitah, and C. Rochette-Egly, unpublished results.

    ABBREVIATIONS

The abbreviations used are: RA, retinoic acid; RAR, RA receptor; RXR, retinoid X receptor; WT, wild type; AF, activation function; WCE, whole cell extract; RT, reverse transcription; CKI, cyclin-dependent kinase inhibitor.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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