Article

• The EMBO Journal (1997) 16, 6452 - 6465
• doi:10.1093/emboj/16.21.6452

Phosphorylation of activation functions AF-1 and AF-2 of RAR and RAR is indispensable for differentiation of F9 cells upon retinoic acid and cAMP treatment

Reshma Taneja^2,4, Cécile Rochette-Egly^1,4, Jean-Luc Plassat¹, Lucia Penna¹, Marie-Pierre Gaub³ and Pierre Chambon¹

1. Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS-INSERM-ULP, Collège de France, BP 163, 67404 Illkirch-Cedex, France
2. Present address: Department of Medicine, The Mount Sinai School of Medicine, One Gustave L.Levy Place, New York, NY 10029-6574, USA
3. Present address: Lab. de Biochimie Biologie Moléculaire, Hôpital de Hautepierre, Avenue Molière BP 48, 67098 Strasbourg Cedex, France
4. R.Taneja and C.Rochette-Egly are equal first authors

Correspondence to:

Pierre Chambon, E-mail: igbmc@igbmc.u-strasbg.fr

Received 16 July 1997; Revised 19 August 1997

• Keywords:

  • parietal endoderm,
  • primitive endoderm,
  • proline-directed kinases,
  • protein kinase A,
  • signal transduction

Introduction

It is well established that retinoids (the active derivatives of vitamin A) play a crucial role in a wide variety of biological processes involved in vertebrate morphogenesis, organogenesis and cell differentiation (Blomhoff, 1994; Gudas et al., 1994; Sporn et al., 1994; Kastner et al., 1995). Genetic analyses in the mouse (Kastner et al., 1995) have shown that the retinoid signal is transduced by retinoic acid (RA) receptors (RARs) and the retinoid X receptors (RXRs), which are ligand-dependent transcriptional regulators belonging to the superfamily of nuclear receptors characterized by the presence of several modular domains designated A to F (see Figure 1). There are three RAR (, and ) and three RXR (, and ) isotypes, and for each isotype there are at least two main isoforms which are generated by differential promoter usage and alternative splicing, and differ only in their N-terminal A region (Leid et al., 1992; Blomhoff, 1994; Chambon, 1994, 1996; Sporn et al., 1994; Gronemeyer and Laudet, 1995; Mangelsdorf and Evans, 1995; Brocard et al., 1996; and references therein).

Figure 1.

Schematic representation of the constructs used to generate AF-1- and AF-2-rescue lines in RAR^-/- cells. (A) Mouse RAR1 and RAR2 with the functional domains AF-1 and AF-2 which lie in the A/B region and the E region respectively are schematically represented (not to scale), and the DNA-binding domain (DBD) as well as the ligand-binding domain (LBD) are depicted. The target sequence for phosphorylation by proline-directed kinases in the B domain of RAR1 and RAR2 is shown, and the corresponding serine residues which have been mutated to alanine (S74/77A for RAR, and S66/68A for RAR2) are indicated. The N-terminal-truncated receptors [RARAB (amino acids 84–462) and RARAB (amino acids 90–458)] as well as the chimeric receptor [RAR1(A–C)(D–F), amino acids 1–153 of RAR1 and 156–458 of RAR1] are also schematically shown. The three additional amino acids in [RAR1(A–C)(D–F)] which have been introduced (Nagpal et al., 1992) are indicated. Numbers refer to amino acid positions. (B) Schematic representation of the protein kinase A (PKA) phosphorylation sites in AF-2 activating domain of RAR1 and RAR2. The serine residues at position 369 of RAR1 and at position 360 of RAR2 were mutated to alanine residues (RARS369A and RARS360A, respectively). (C) RAR protein in AF-1 and AF-2 rescue lines. Whole cell extracts were prepared from WT F9 cells, RAR^-/- cells and each rescue line, and RAR protein was first immunoprecipitated with specific monoclonal antibodies [Ab2(mF)] followed by a Western blot with a specific rabbit polyclonal antibody [RP (F)] in F9 WT, RAR^-/-, RARWT, RARS66/68A, RAR1(A–C)(D–F), RARAB and RARS360A cell lines (lanes 1–7, respectively, as indicated). (D) RAR protein in AF-1 and AF-2 rescue lines. Whole cell extracts were prepared from WT F9 cells (lanes 1 and 6), RAR^-/- cells (lanes 2 and 7), and the rescue lines RARWT (clone 53, lane 3; clone 17, lane 8), RARS74/77A (lane 4), RARAB (lane 5) and RARS369A (clones 22 and 210, lanes 9 and 10, respectively). RAR was detected by Western blot with specific rabbit polyclonal antibodies [RP(F)].

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As other members of the superfamily (Gronemeyer and Laudet, 1995), RARs and RXRs contain two transcriptional activation functions (AFs): AF-1, located in the A/B region, is ligand-independent, whereas AF-2, present within the C-terminal E region which also contains the ligand-binding domain (LBD), is ligand-dependent (Nagpal et al., 1992, 1993; Folkers et al., 1993; Durand et al., 1994; Chambon, 1996). In vitro studies performed in cultured cells co-transfected with artificial reporter genes and vectors expressing AF-1 or AF-2 of the various retinoid receptors have shown that the AF-1 and AF-2 activity of a given isotype can be cell type- and promoter context-dependent, at least to some extent. Furthermore it has been established, for both RARs and RXRs, that the AF-1s of isoforms of a given isotype could synergize with the AF-2s of the same or different isotypes, in a response element- and promoter context-dependent manner (Nagpal et al., 1992, 1993; Durand et al., 1994).

Several members of the nuclear receptor superfamily, including RARs, are phosphoproteins, and the role of some of the phosphorylation sites, has been examined by site-directed mutagenesis (Kuiper and Brinkmann, 1994; Weigel, 1996). The presence of Ser-Pro motifs, some of which are located in the A/B region, has suggested an involvement of proline-dependent kinases, which include cyclin-dependent kinases, mitogen-activated (MAP) kinases and stress-activated kinases (Davis, 1994; Hunter, 1995; Marshall, 1995; Morgan, 1995), in nuclear receptor phosphorylation. Recently the epidermal growth factor (EGF) has been shown to activate AF-1 of the oestrogen receptor (ER) (Kato et al., 1995; Bunone et al., 1996) through phosphorylation by MAP kinase of a serine residue located in the B region (Ali et al., 1993). Several putative sites for proline-dependent kinases are also located in the B region of RAR. Interestingly, mutation of the serine residue 77 (Ser77) located in the B region of RAR decreases its AF-1 activity in transfected COS cells (Rochette-Egly et al., 1997). In addition, RAR has been shown to be phosphorylated by protein kinase A (PKA) at Ser369 located in the LBD region E, and mutation of this site was reported to alter the response of RAR reporter genes in transfected cells treated with cAMP (Rochette-Egly et al., 1995). These transfection studies suggested that phosphorylation might also modulate the activity of RAR AF-1 and AF-2 under physiological conditions. Gene knock-outs in the mouse have provided genetic evidence that the different RARs and RXRs are, at least to some extent, specifically involved in one or several of the many events which are controlled by RA during development and post-natal life. However, the interpretation of these genetic studies is often equivocal due to: (i) the difficulty in discriminating between cell-autonomous and non-cell-autonomous events; and (ii) functional redundancies between receptor isotypes, which may be artefactually generated, at least in part, by the gene knock-outs (see Kastner et al., 1995, 1997 for further discussion of these points).

The embryonal carcinoma (EC) F9 cells offer a RA-responsive cell-autonomous 'developmental' system in which the functional specificity of the different retinoid receptors, as well as that of their phosphorylated or unphosphorylated AF-1 and AF-2 activating domains, can be studied under conditions which are much closer to in vivo situations than those generated in in vitro transiently transfected cells (containing overexpressed receptors and uncontrolled amounts of artificial RA-responsive reporter genes). RA induces the differentiation of F9 EC cells in monolayer culture, resulting in the formation of primitive endoderm-like cells, whereas a combination of RA and dibutyryl cAMP (cAMP) leads to parietal endodermal differentiation (Strickland et al., 1980; Hogan et al., 1983). These two cell types are characterized by their distinct morphology (Strickland et al., 1980), and by the expression of several differentiation-marker genes (Gudas et al., 1994). F9 cells contain all RAR and RXR isotypes with RAR1 and RAR2 being the main RAR isoforms (Zelent et al., 1989; Wan et al., 1994; Taneja et al., 1995). Knock-out of the RAR gene (all isoforms) in F9 cells drastically impairs primitive and parietal endoderm differentiation and affects the induction of many endogenous RA-responsive genes (Boylan et al., 1993; Taneja et al., 1995), whereas RAR gene knock-out (all isoforms) was reported to have milder and more restricted effects (Boylan et al., 1995). Moreover, the differentiation of F9 cells into primitive endoderm can be brought about by a RAR-specific agonist, but not with an RAR-specific agonist (Taneja et al., 1996). The specific role played by the various RAR and RXR isotypes in mediating the effects of RA on F9 cell differentiation and RA-responsive gene expression is increasingly clear (Boylan et al., 1993, 1995; Taneja et al., 1995, 1996; Clifford et al., 1996; Chiba et al., 1997). In contrast, the role of the individual AF-1 and AF-2 activating domains, as well as the possible control of their activity through phosphorylation, is still unknown

We have previously shown that the various RA-responses of F9 cells to RA treatment can be restored in RAR^-/- cells by either re-expressing RAR2 to wild-type levels or overexpressing RAR1 (Taneja et al., 1995). Here, we have functionally dissected the role of the AF-1 and AF-2 activating domains of RAR2 and RAR1. Wild-type (WT) and RAR2 mutants lacking the AF-1 activating domain or bearing mutations in the AF-1 or AF-2 phosphorylation sites were re-expressed to WT levels in RAR^-/^- cells to establish stably transformed 'rescue lines'. Similarly, 'rescue' lines were established which overexpressed WT and mutant RAR1. Our results demonstrate that, in a cell-autonomous system, AF-1 and AF-2 of RAR2 and RAR1 exert specific, often synergistic functions, with respect to both RA-induced differentiation events and induction of expression of RA-responsive genes. Most importantly, the present study shows that the phosphorylation sites of the AF-1 and AF-2 activating domains of RAR2 and RAR1 are differentially required for the differentiation and target gene responses to RA treatment, thus demonstrating that RARs are sophisticated transducers integrating signals from several major signalling pathways.

Generation of stable 'rescue' lines expressing AF-1 and AF-2 mutants of RAR and RAR in RAR^-/- cells

We have previously shown that re-expression of WT levels of RAR2 or overexpression of RAR1 in RAR^-/- F9 cells fully restores the differentiation events and responsiveness of target genes which occur in WT F9 cells upon RA treatment (Taneja et al., 1995). To investigate the role played by the transactivation domain AF-1 of RAR1 and RAR2 in these events and responses, stable 'rescue' lines overexpressing WT RAR1 (RARWT line; previously referred to as 53 line in Taneja et al., 1995), re-expressing WT RAR2 (RARWT line; referred to as 51 line in Taneja et al., 1995), expressing deletion mutants of RAR and RAR lacking the A/B region (RARAB and RARAB lines, respectively), or expressing a chimeric receptor containing the A–C regions of RAR1 fused to the D–F regions of RAR [RAR1(A–C)(D–F) line], were established in RAR^-/- cells ('AF-1-rescue lines'; see Figure 1A). To investigate whether phosphorylation of RAR1 and RAR2 could play a role in AF-1 function, stable lines were also established in the RAR null background, using receptors bearing mutations in conserved putative sites for proline-directed kinases. RAR1 serine residues 74 and 77 [of which Ser77 has been shown to be phosphorylated and involved in AF-1 activity in COS cells (Rochette-Egly et al., 1997)] were mutated to alanine (Figure 1A, RARS74/77A 'rescue' lines). Similarly, the corresponding serine residues in RAR2 (residues 66 and 68), which have been found to be phosphorylated in F9 cells (C.Rochette-Egly and P.Chambon, unpublished observations), were mutated to alanine (Figure 1A, RARS66/68A 'rescue' lines). The role of phosphorylation in the transcriptional activity of AF-2 was assessed by making 'AF-2-rescue lines' carrying a mutation in the conserved PKA phosphorylation site (Rochette-Egly et al., 1995) located in the E region of either RAR or RAR. The serine residue at position 369 in the PKA site of RAR1 was mutated to alanine (Figure 1B, RARS369A 'rescue' line), and a similar mutation was made in RAR2 at Ser360 (RARS360A 'rescue line').

Several clones were obtained for each 'rescue' transgene, and the level of transgene expression was determined in the derived cell lines. The expression level of RARWT and of its deletion or mutant derivatives in each of the AF-1- and AF-2-rescue line, was compared with the endogenous expression of RAR in WT F9 cells by immunoprecipitation and Western blotting (Figure 1C, lanes 1–7). RARWT (51 line, Taneja et al., 1995) and RARAB were expressed in the respective rescue lines at similar levels (lanes 3 and 6). The AF-1 phosphorylation mutant line RARS66/68A (lane 5), the chimeric rescue line RAR1(A–C)(D–F) (lane 4; note, however, that this chimera was overexpressed relative to endogenous RAR1), and the AF-2 phosphorylation mutant line RARS360A (lane 7) exhibited a similar level of RAR2 protein (which was slightly higher than that in WT F9 cells).

The expression of RARWT, RARAB and RARS74/77A was detected by Western blotting (Figure 1D, lanes 1–5) in AF-1-rescue lines, and compared with the level of endogenous RAR in WT F9 and RAR^-/- cells. The RARWT rescue line has been shown previously to overexpress RAR1 (53 line, Taneja et al., 1995; and Figure 1D, lane 3). The RARS74/77A mutant was overexpressed when compared with the RARWT line level (Figure 1D, lane 4, compare with lane 3), and the expression of RARAB was revealed by the detection of faster-migrating species (lane 5). The RAR AF-2-rescue lines (Figure 1B, RARS369A lines, clones 22 and 210, and their control RARWT line clone 17) were also analysed for RAR expression (Figure 1D, lanes 6–10). A similar overexpression of RAR1 protein was detected in RARS369A clone 22 and RARWT clone 17 'rescue' lines (Figure 1D, compare lanes 8 and 9 with lanes 6 and 7), whereas a lower overexpression was seen for the clone 210 rescue line (Figure 1D, lane 10).

For each of the AF-1 and AF-2 'rescue' transgenes, two cell lines derived from two independent clones expressing the transgene at comparable levels (data not shown) yielded similar results in the studies described thereafter.

Involvement of the AF-1 activating domain and phosphorylation site in the B region of RAR and RAR in rescuing endodermal differentiation of RAR^-/- cells

We first investigated the ability of the AF-1-rescue lines established in the RAR^-/- cell background [RARWT, RARAB, RARS66/68A, RAR1(A–C)(D–F), RARWT, RARAB and RARS74/77A] to restore the differentiation of RAR^-/- cells. The morphological differentiation of each rescue line was analysed upon treatment with 100 nM T-RA either alone or in combination with 250 M cAMP for 96 h (Figure 2; Table I). When grown as monolayers in the presence of RA alone, WT F9 cells differentiated into primitive endodermal-like cells (Figure 2, compare panels a and b) exhibiting a characteristic flat triangular morphology with cytoplasmic granules (Strickland and Mahdavi, 1978). Addition of cAMP along with RA resulted in the formation of parietal endoderm-like cells (Figure 2, panel c) which, in contrast to primitive endodermal cells, have a rounded and refractile appearance (Strickland et al., 1980; Hogan et al., 1983). As previously shown (Boylan et al., 1993; Taneja et al., 1995), these two types of differentiation were drastically impaired in RAR^-/- cells (Figure 2, panels d–f), and re-expression of RAR2 in the RARWT rescue line restored the RA-responsiveness of these cells to form primitive and parietal endoderm (see Figure 2, panels g–i), as did the overexpression of RAR1 in the RARWT rescue line (Figure 2, panels s–u). In contrast, both RARAB line (Figure 2, panels j–l) and RARAB (Figure 2, panels v–x) lines mostly retained a stem cell morphology, indicating that a cooperativity between AF-1 and AF-2 was required to rescue the differentiation defects of RAR^-/- cells. On the other hand, the RAR1(A–C)(D–F) rescue line responded to both T-RA alone, or T-RA and cAMP, to differentiate into primitive endoderm, and to a large extent into parietal endoderm [Figure 2, panels p–r; note, however, that lines expressing RAR1(A–C)(D–F) at a lower level, similar to that of endogenous RAR1, differentiated very poorly; data not shown]. The RARS66/68A line (Figure 2, panels m–o) also differentiated poorly both upon T-RA and T-RA plus cAMP treatment, indicating that phosphorylation of the B region is important for RAR2 AF-1 function to participate in the induction of primitive and parietal endoderm differentiation. Interestingly, the rescue line overexpressing RARS74/77A (Figure 2, panels y–z') differentiated as efficiently as the RARWT rescue line to form primitive endoderm in response to T-RA alone. However, in contrast to the RARWT line, RARS74/77A cells differentiated poorly into parietal endoderm, indicating that the phosphorylation of RAR1 in AF-1 was required for differentiation in response to RA and cAMP. Since all RAR^-/- rescue lines contain wild-type levels of endogenous RAR, this result suggests that RARS74/77A behaves as a dominant negative mutant for parietal endodermal differentiation (see below).

Figure 2.

The A/B region of RAR and RAR is required for efficient differentiation into primitive and parietal endoderm. Morphological differentiation of WT F9 cells, RAR^-/- cells and AF-1-rescue lines (as indicated) grown in the presence of 100 nM T-RA alone, or a combination of 100 nM RA and 250 M cAMP for 96 h as viewed under phase-contrast microscopy. Control cells treated with 0.1% ethanol (vehicle) or 250 M cAMP remained undifferentiated.

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Table 1: Morphological differentiation and relative levels of the differentiation marker collagen IV (1) [ColIV (1)] in WT, RAR^-/- and 'RAR^-/- rescue' F9 lines

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The differentiation of the various rescue lines was further analysed by determining the expression of collagen type IV (1), which is induced during both primitive and parietal endodermal differentiation of F9 cells (Strickland and Madhavi, 1978) (Table I; also data not shown). In keeping with the observed morphological differentiation, the expression of collagen type IV (1) transcripts (Table I) was up-regulated in RARWT and RARWT rescue lines to levels similar to that achieved in F9 WT cells upon T-RA treatment (see also Taneja et al., 1995). In contrast, the expression of collagen type IV (1) transcripts was markedly decreased in both RARAB and RARAB lines treated with T-RA. A low level of collagen transcripts was also induced in the RARS66/68A line. On the other hand, despite poor parietal endodermal differentiation, the RARS74/77A line showed a high level of collagen type IV (1) transcripts, in agreement with an efficient rescue of primitive endodermal differentiation. As expected, the RAR1(A–C)(D–F) line also expressed increased levels of the differentiation-specific marker.

Role of the AF-1 activating domain of RAR and RAR in the expression of several RA-responsive genes

Knock-out of the RAR gene in F9 cells was shown to result in a marked reduction of the expression of several RA-responsive genes, such as Hoxa-1, HNF1, Stra6, Stra4 and HNF3 (Boylan et al., 1993; Taneja et al., 1995). Thus, we investigated the ability of RAR2 or RAR1 and of their AF-1 mutant derivatives to restore the expression of these RA target genes, using semi-quantitative RT–PCR after treatment of the rescue lines with 100 nM T-RA for 24 h (Figure 3; Table II), ensuring that for each gene, the determination was carried out in the linear range of the PCR-amplification reaction.

Figure 3.

Differential RA-inducibility of RA-responsive genes in AF-1-rescue lines. RNA was isolated from WT F9 cells, RAR^-/- cells, and the rescue cell lines RARWT, RARAB, RARS66/68A, RAR1(A–C)(D–F), RARWT, RARAB and RARS74/77A, with or without treatment of each cell line with 100 nM T-RA (RA) for 24 h, as indicated and transcripts from each gene were analysed by semi-quantitative RT–PCR, using transcripts of the 36B4 gene as an internal control to normalize the amounts of RNA.

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Table 2: Role of the AF-1 activating domain and of its phosphorylation on the RA-inducibility of responsive genes

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Responsiveness of RA target genes in rescue lines re-expressing RARWT, RARAB or RARS66/68A. Re-expression of RAR2 (RARWT) reactivated the expression of all genes tested (Figure 3; Table II; see also Taneja et al., 1995). In contrast, RARAB did not restore HNF1 and Stra6 expression, whereas that of HNF3 expression was partially rescued. The expression of HNF1 only was not induced in the RARS66/68A rescue line, indicating that phosphorylation of region B could modulate the AF-1 activity in a promoter context-dependent manner. On the other hand, the induction of both Hoxa-1 and Stra4 expression was unaffected by either the deletion of the A/B region or the mutation of the region B phosphorylation site, indicating that these inductions do not require AF-1.

Responsiveness of RA target genes in rescue lines overexpressing RARWT, RARAB and RARS74/77A. The expression of all RA-responsive genes was restored by overexpression of RAR1 (RARWT) (see also Taneja et al., 1995). In contrast, all genes except Stra4 were not efficiently induced in the RARAB rescue line. Interestingly, the RAR phosphorylation mutant RARS74/77A did not efficiently restore Stra6 and HNF3 expression, indicating that phosphorylation of region B contributes to AF-1 activity. However, the same RARS74/77A mutation did not affect the induction of Hoxa-1, HNF1 or Stra4 expression, showing that this contribution is also promoter context-dependent.

Responsiveness of RA target genes in the RAR1(A–C)(D–F) rescue line. The induction of HNF1 was fully restored, and that of Stra6 partially rescued in the RAR1(A–C)(D–F) line (Figure 3; Table II), indicating a cooperativity between AF-1 of RAR1 and AF-2 of RAR for activation of these promoters by RA. On the other hand, AF-1 and AF-2 of two receptor types could not cooperate to restore the induced expression of HNF3, whereas, as expected those of Hoxa-1 and Stra4 were completely restored, as in the case of RARAB line.

Role of the cAMP-induced phosphorylation of the AF-2 activating domain in F9 cell responsiveness to RA

Activation of the PKA pathway by cAMP is required for RA-treated F9 cells to differentiate into parietal endoderm-like cells (Strickland et al., 1980; Hogan et al., 1983). To investigate the possible contribution of phosphorylation of the PKA site present in the LBD of either RAR or RAR (Rochette-Egly et al., 1995; and our unpublished observations) to the ligand-induced activation function-2 (AF-2), 'rescue' lines bearing mutation in these PKA sites (Figure 1B) were analysed for their ability to differentiate upon RA-treatment for 96 and 120 h, and their differentiation patterns were compared with those of WT F9 (Figure 4, panels a–d) and RAR^-/- cells (panels e–h). As expected, both primitive and parietal endoderm differentiation were restored in RARWT (Figure 4, panels i–l) and RARWT (Figure 4, panels q–t) rescue cell lines after 96 h and 120 h of treatment with RA alone or RA and cAMP, respectively (see also Figure 2 and Table I).

Figure 4.

A mutation in the PKA site of RAR1, but not of RAR2 results in defective parietal endoderm differentiation upon RA and cAMP treatment. WT F9 cells (a–d), RAR^-/- cells (e–h), and the rescue cell lines RARWT (i–l); RARS360A (clone 7; m–p); RARWT (clone 17; q–t), RARS369A (clone 22; u–x), cells were treated with either T-RA (100 nM) alone, or with a combination of T-RA (100 nM) and cAMP (250 M), as indicated. Control cells were treated with 0.1% ethanol (vehicle), and cells were photographed under a phase-contrast microscope after 96 h and 120 h.

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Mutation of the serine residue of the PKA phosphorylation site of RAR (RARS360A), did not alter its potential to rescue the differentiation of RAR^-/- cells into either primitive endoderm or parietal endoderm-like cells (Figure 4, panels m–p). Mutation of the corresponding serine residue in RAR also did not affect differentiation of the corresponding rescue line (RARS369A; clone 22) into primitive endoderm-like cells in the presence of T-RA alone (Figure 4, panel v). However, when treated with T-RA and cAMP, this rescue line retained the primitive endoderm-like morphology, resulting at 96 h in defective parietal endoderm differentiation (Figure 4, panel w), whereas some parietal endodermal differentiation could be observed upon continued treatment of the cells for 120 h (Figure 4, panel x; see also Table I). Similar results were obtained with a second rescue line expressing the same mutant receptor (RARS369A clone 210; data not shown). These results suggest that phosphorylation of the PKA site of RAR (but not of RAR) plays an important role in the differentiation of F9 cells into parietal endoderm. Note that the RARWT and RARS369A lines express similar levels of RAR protein, including the endogenous F9 cell RAR (Figure 1D). Thus, the ability of the RARS369A cell line to differentiate as efficiently as the RARWT cell line into primitive, but not into parietal endoderm, indicates that, as in the case of the RARS74/77A mutation (see above), the PKA site S369A mutation generates a RAR dominant negative mutant which selectively prevents the endogenous RAR present in RAR^-/- cells from promoting parietal endodermal differentiation.

The extent of differentiation of the RARWT, RARS360A, RARWT and RARS369A rescue cell lines was also estimated by the expression of transcripts of two markers of endodermal differentiation, collagen IV (1) and laminin B1. RT–PCR analysis showed that the induction of collagen IV (1) and laminin B1 transcripts, which is much reduced in RAR^-/- cells (see also Boylan et al., 1993; Taneja et al., 1995), was restored in all four rescue cell lines (Table I, and data not shown). Note that, as seen above in the case of the RARS74/77A line and despite its impaired parietal endodermal differentiation, the RARS369A line efficiently expressed collagen IV (1) transcripts, in agreement with its primitive endodermal differentiation. The expression of several RA-responsive genes whose induction was strongly reduced in RAR^-/- cells (Taneja et al., 1995; see Table II) was similarly analysed in these rescue lines upon 24 h treatment with T-RA either alone or in combination with cAMP. Wild-type transcript levels were restored for all genes tested in both RARWT and RARWT lines, as well as in the mutant rescue lines (RARS360A, RARS369A clones 22 and 210; data not shown).

A RAR-specific retinoid together with cAMP cannot trigger the parietal differentiation of F9 WT cells, unless combined with a RAR-specific ligand

To investigate further the respective functions of RAR and RAR in primitive and parietal endodermal differentiation, WT F9 cells were treated for 96 h with 100 nM T-RA or with synthetic retinoids selective for RAR (BMS753; Taneja et al., 1996), RAR (BMS961; Taneja et al., 1996), either individually or in combination with a pan-RXR-selective retinoid BMS649 [Roy et al., 1995; also referred to as SR11237 (Lehmann et al., 1992)], in the presence or absence of 250 M cAMP (Figure 5). At a high concentration (100 nM), the RAR-selective ligand BMS961 on its own was almost as efficient as T-RA in inducing primitive endoderm-like differentiation (Figure 5, compare panels b and d; see also Taneja et al., 1996). However, unlike the parietal endodermal differentiation achieved with T-RA and cAMP (Figure 5, panel c), addition of cAMP along with BMS961 did not trigger any parietal endodermal differentiation, and the cells retained a primitive endoderm-like morphology (Figure 5, panel e), even after 120 h of retinoid treatment (not shown). Interestingly, the concomitant addition of the pan RXR-specific agonist BMS649, which is inactive on its own but can synergize with BMS961 to promote primitive endodermal differentiation (Roy et al., 1995; Taneja et al., 1996) did not result in the formation of parietal endoderm either, even up to 120 h (Figure 5, panel f and data not shown).

Figure 5.

Activation of RAR is required, in addition to activation of RAR, for parietal endodermal differentiation in presence of cAMP. Morphological differentiation of WT F9 cells after treatment with various retinoids either singly or in combination, as indicated: 100 nM T-RA (panels b and c) or 10 nM (panels m–o); 100 nM BMS961 (RAR-selective; panels d–f and j–l); 100 nM BMS753 (RAR-selective; panels g–l); 1 M BMS649 (pan-RXR; panels f, i and l), and 1 M BMS614 (RAR antagonist; panel o) in the absence or presence of cAMP (250 M, as indicated). Control cells (panel a) were treated with 250 M cAMP alone, and retained an undifferentiated stem cell morphology. Cells were photographed under a phase-contrast microscope after 96 h (panels a–l) or 120 h (panels m–o).

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The RAR agonist BMS753 on its own or along with cAMP, did not induce primitive or parietal endodermal differentiation of F9 cells (Figure 5, panels g and h; also Taneja et al., 1996), but when combined with the pan-RXR agonist BMS649, could synergize to weakly induce primitive, but not parietal endodermal differentiation, even up to 120 h (Figure 5, panel i; also data not shown). Activation of both RAR and RAR by a combination of suboptimal concentrations of BMS961 and BMS753 still resulted in primitive endodermal differentiation only (Figure 5, panel j). However, the addition of cAMP, along with the same concentration of both BMS961 and BMS753, triggered only a weak differentiation of F9 cells into parietal endoderm (Figure 5, panel k). This synergism between the RAR- and RAR-selective ligands for parietal endodermal differentiation was further enhanced (to achieve full differentiation) upon concomitant activation of RXRs with the agonist BMS649 (Figure 5, panel l). Note that full parietal endodermal differentiation was also obtained with a BMS961/BMS753 combination at optimal concentration (1 M; data not shown).

Taken together, the above results strongly suggest that activation of RAR is essential for triggering the differentiation of WT F9 cells into primitive endoderm-like cells, whereas the activation of RAR is necessary for parietal endodermal differentiation in the presence of cAMP. To support this suggestion, we used the RAR-selective antagonist BMS614 (Chen et al., 1996) to determine whether its addition would prevent the formation of parietal, but not of primitive endoderm-like cells, when used along with RA and cAMP. WT F9 cells treated with 10 nM RA alone differentiated into primitive endoderm (Figure 5, panel m), and parietal endodermal cells were seen upon addition of cAMP (panel n). The addition of BMS614 along with 10 nM T-RA alone did not affect the differentiation of F9 cells into primitive endoderm (data not shown). In contrast, when added along with a combination of 10 nM T-RA and 250 M cAMP, the RAR antagonist suppressed the appearance of parietal endodermal cells brought about by T-RA and cAMP, and the primitive endoderm-like morphology was fully retained at 120 h (Figure 5, compare panels n and o).

The role of RAR in the parietal differentiation of F9 cells was definitely confirmed using RAR^-/- cells. As WT F9 cells, these cells did differentiate into primitive endoderm, when treated with either 100 nM T-RA or RAR-selective ligand BMS961 (Figure 6, compare panel a with panels b and e; see also Boylan et al. 1995; Taneja et al., 1996). However, no parietal endodermal differentiation of RAR^-/- cells could be triggered at 96 h by either of these two ligands in presence of cAMP (Figure 6, panels c and f), whereas, upon continued treatment (120 h), these cells could differentiate into parietal endoderm-like cells with a combination of either T-RA and cAMP or BMS961 and cAMP (Figure 6, panels d and g). Thus, a delayed morphological differentiation of RAR^-/- cells into parietal endoderm could be brought about by RAR activation in RAR^-/- cells, but not in WT F9 cells (see above), which supports our previous conclusion that gene knock-outs can create artefactual conditions leading to unphysiological functional redundancies between RARs.

Figure 6.

RAR^-/- cells exhibit a delay in parietal endoderm differentiation. Morphological differentiation of RAR^-/- cells (a) after treatment with 100 nM T-RA or 100 nM BMS961 either alone (b and e, respectively), or in combination with 250 M cAMP for 96 h (c and f, respectively), or 120 h (d and g, respectively).

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RAR^-/- cells are severely impaired in RA-induced primitive and parietal endodermal differentiation (Boylan et al., 1993, 1995), and we previously reported that re-expression of RAR2 or overexpression of RAR1 in these cells could restore primitive endodermal differentiation. This restoration requires the AF-2 activation function, as dominant negative (dn) receptors in which AF-2 has been abrogated by mutation of the AF-2 AD core (Chambon, 1996) are unable to restore differentiation (our unpublished results), in agreement with the lack of differentiation of the RAC 65 P19 EC cell mutant which expresses a dn form of RAR (Pratt et al., 1990; Kruyt et al., 1992). In the present study, we have analysed the contribution of the RAR N-terminal AF-1 activating domain to F9 cell differentiation, and examined whether the activity of AF-1 and AF-2 is modulated by phosphorylation.

The RAR AF-1 activating domain and its proline-directed kinase phosphorylation site are indispensable for RA-induced primitive endodermal differentiation

The observation that the re-expression of RAR2 lacking its AF-1-containing N-terminal A/B region is unable to restore RA-induced primitive endodermal differentiation, clearly indicates a requirement for AF-1 and a synergism between AF-1 and AF-2 of RAR to mediate the RA-induced differentiation of F9 cells. Interestingly, a similar AF-1–AF-2 synergism also occurs when differentiation is restored by overexpression of RAR1 or a chimeric receptor in which activating domains of different RAR isotypes (RAR1 AF-1 and RAR AF-2) are associated [RAR1(A–C)(D–E)] (see Table I).

Mutations of serine residues, which are phosphorylation sites for proline-directed kinases in the AF-1-containing region of RAR2 (RARS66/68A mutation), drastically reduced the ability of this receptor to restore primitive endodermal differentiation. However, the counterpart mutations in overexpressed RAR1 (RARS74/77A) had no effect on this differentiation. Thus, a synergism between AF-1 and AF-2 is required for both RAR2 and overexpressed RAR1 to restore primitive endodermal differentiation, but phosphorylation of the AF-1 activating domain is required for RAR2 only, indicating that the unphosphorylated forms of the AF-1 activating domain of these two receptors differ in their ability to synergize with their cognate AF-2.

In contrast, the phosphorylation of the AF-2 activating domain of both RAR2 and RAR1 by PKA appears dispensable for primitive endodermal differentiation, which could be efficiently restored by either expression of RARS360A or overexpression of RARS369A which are mutated in their PKA phosphorylation sites.

Promoter context-dependent requirement of the AF-1 activating domains of RAR1 and RAR2 and of their phosphorylation sites for expression of RA-responsive genes

We have previously reported the promoter context-dependence of the synergistic activation of transcription which can be brought about by the AF-1 and AF-2 of a given RAR or distinct RAR isotypes (Nagpal et al., 1992, 1993). As these promoter context dependencies were observed under non-physiological conditions, with respect to both receptor and RA-responsive reporter gene levels, it was important to investigate whether similar dependencies could also exist under the present conditions, where more physiological receptor levels mediate the activation of endogenous RA-responsive genes in their normal chromatin environment.

The analysis (see Table II) of the expression of the RA-responsive genes, Hoxa-1, HNF1, HNF3, Stra4 and Stra6, clearly shows that, not only can such promoter context dependencies be observed in the present rescue lines, but also that AF-1 and AF-2 of different RAR isotypes differ to some extent in their capacity to homo- and to hetero-synergize, as: (i) a synergism between AF-1 and AF-2 of re-expressed RAR2 is required for efficient expression of HNF3, HNF3 and Stra6, whereas AF-1 is dispensable in the case of Hoxa-1 and Stra4; (ii) a synergism between AF-1 and AF-2 of overexpressed RAR1 is required for efficient expression of all of these genes, with the exception of Stra4; and (iii) in the chimeric rescue line RAR1(A–C)(D–F), the expression of HNF1 and Stra6 is induced to higher levels than in RARAB (which lacks AF-1 activity), albeit to a lesser extent in the case of Stra6, whereas there was no synergism between RAR1 AF-1 and RAR AF-2 in the case of HNF3.

Similarly, the requirement for the proline-directed kinase phosphorylation sites of the AF-1 activating domain is also promoter- and receptor isotype-dependent. The phosphorylation site of the AF-1 activating domain of re-expressed RAR2 is specifically required for efficient induction of HNF-1 expression, whereas that of the AF-1 activating domain of overexpressed RAR1 is indispensable for the induction of HNF3 and Stra6. On the other hand, as in the case of primitive endodermal differentiation, mutation of the PKA phosphorylation site of the AF-2 domain of either RAR or RAR does not affect the expression of the RA-responsive genes analysed in the present study.

Parietal endodermal differentiation requires RAR, in addition to RAR, and is dependent on phosphorylation of both AF-1 and AF-2 activating domains of RAR1

Activation of the PKA pathway with cAMP is known to modify the differentiation response of F9 cells, which form parietal endoderm cells that are morphologically distinct from the primitive endoderm cells formed in presence of RA alone (Strickland et al., 1980; Hogan et al., 1983). Furthermore, the formation of parietal endodermal cells is apparently achieved in two steps, with an initial RA-induced differentiation into primitive endoderm, followed by a cAMP-induced differentiation switch from primitive to parietal endoderm (Strickland et al., 1980). Thus, differentiation into primitive endoderm could be a prerequisite for parietal endoderm formation. This notion is supported by the present observation that restoring primitive endodermal differentiation, also restores parietal endodermal differentiation, whereas rescue lines that are defective for primitive endodermal differentiation (RARAB, RARAB and RARS66/68A) are also defective for parietal endodermal differentiation.

Several lines of evidence support the conclusion that RAR is indeed required for parietal, but not for primitive endodermal differentiation, and also that primitive endodermal differentiation mediated by RAR is required for parietal endodermal differentiation to occur: (i) treatment of WT F9 cells with a RAR-specific synthetic retinoid (BMS961) in the presence of cAMP promotes the formation of primitive, but not of parietal endodermal cells, which occurs only upon additional treatment with a RAR-specific retinoid (BMS753); (ii) a RAR-specific antagonist (BMS614) impairs parietal, but not primitive endodermal differentiation upon WT F9 cell treatment with RA and cAMP; (iii) RAR^-/- F9 cells differentiate as efficiently as WT cells into primitive endodermal cells, whereas they are markedly delayed in parietal endodermal differentiation (Taneja et al., 1996; and the present study); and (iv) re-expression of RAR1 in RAR^-/- cells restores efficient parietal endodermal differentiation upon RA and cAMP treatment (our unpublished results). On the other hand, RAR cannot mediate primitive endodermal differentiation of WT F9 cells, as no differentiation occurs upon treatment with the RAR-specific agonist BMS753, irrespective of the presence of the pan-RXR-agonist BMS649 (Taneja et al., 1996).

It is noteworthy that although the formation of primitive endodermal cells precedes the appearance of parietal endodermal cells, the formation of the latter cells does not appear to require a strict temporal order of activation of RAR and RAR. Both F9 cells first treated with the RAR agonist BMS961 and the pan-RXR agonist BMS649 for 48 h, and subsequently with the RAR agonist BMS753 and cAMP, as well as F9 cells treated in the reverse order, i.e. first with BMS753 and BMS649 and subsequently with BMS961 and cAMP, differentiate into parietal endoderm (our unpublished results). This process is also independent of the continued presence of BMS961 or BMS753, as F9 cells treated for 48 h with the RAR-specific ligand and then with the RAR ligand, or subjected to the reverse treatment, still form parietal endodermal cells (our unpublished results). We therefore conclude that: (i) RAR/RXR heterodimers mediate events required for RA-induced differentiation of WT F9 cells into primitive endoderm, whereas activation of RAR/RXR heterodimers mediate additional events necessary for differentiation into parietal endoderm in the presence of RA and cAMP; and (ii) even though these events do not have to occur in a fixed temporal order, the 'primitive endodermal events' must occur to allow the 'parietal endodermal events' to become morphologically manifest.

Most interestingly, the RARS74/77A rescue line differentiates as efficiently as the RARWT line into primitive endoderm, but not into parietal endoderm, indicating that the AF-1 activating domain phosphorylation site of RAR is critical for parietal endodermal differentiation. As RAR^-/- cells do contain functional endogenous RAR, the differentiation of the RARS74/77A line into primitive, but not into parietal endoderm must reflect a dominant negative effect of the RARS74/77A mutant on the endogenous RAR which is required for parietal endoderm formation (see above).

Thus, phosphorylation of the proline-directed kinase site of the AF-1 activating domain of RAR does not appear to be required for allowing overexpressed RAR1 to substitute functionally for RAR during primitive endodermal differentiation, but is mandatory for parietal endodermal differentiation. That RAR plays a specific role in parietal endodermal differentiation, is further supported by the observation that the PKA phosphorylation site of the AF-2 activating domain of RAR is also not required for RAR1 to substitute functionally for RAR during primitive endodermal differentiation, whereas it is indispensable for the appearance of parietal endodermal cells. As in the case of the RARS74/77A mutation (see above), the lack of parietal endodermal differentiation in the RARS369A rescue line can be accounted for by a dominant negative effect of the overexpressed RAR mutant, which prevents the endogenous RAR present in RAR^-/- cells from promoting the formation of parietal endodermal cells. The observation that upon prolonged treatment with RA and cAMP, some parietal endodermal cells appear in the RARS369A cell line, may reflect an incomplete dominant negative effect. We conclude that, both the proline-directed kinase phosphorylation site of the AF-1 activating domain and the PKA phosphorylation site of the AF-2 activating domain are indispensable for RAR1 specifically to mediate parietal endodermal differentiation.

Functional redundancies can be artefactually generated by RAR knock-outs in F9 cells

We have previously reported several observations indicating that the effects of knock-outs of given RAR and/or RXR isotypes in F9 cells could be functionally compensated by other RAR and/or RXR isotypes, whereas there was no indication that these redundancies occur in WT F9 cells (Taneja et al., 1996; Chiba et al., 1997). Similarly, we show here that RAR activation in RAR^-/- cells can bring about an artefactual delayed morphological differentiation of these cells into parietal endodermal cells, whereas RAR activation in WT F9 cells does not result in any parietal endodermal differentiation. Thus, in WT F9 cells, the presence of RAR hinders the less efficient RAR to mediate parietal endodermal differentiation. Along these same lines, note that overexpression of RAR1 in RAR^-/- cells allows primitive endodermal differentiation and expression of some RA-responsive genes to occur, whereas these events are selectively mediated by RAR in WT F9 cells (Taneja et al., 1995; and the present study).

In conclusion, the use of F9 cells which differentiate into primitive and parietal endodermal cells in the presence of RA and RA plus cAMP, respectively, together with the use of RAR^-/- and RAR^-/- mutant cells, has allowed us to demonstrate unequivocally that different RAR isotypes exert specific developmental functions in an in vivo-like cell-autonomous system. Furthermore, the use of RAR^-/- cells rescued with either RAR1, RAR2 or their mutants in AF-1 and AF-2 activating domains, has permitted establishment of the individual and synergistic contributions of activation functions AF-1 and AF-2 on specific differentiation events and activation of expression of RA-responsive genes.

Most importantly, we demonstrate the crucial requirement of the proline-directed kinase phosphorylation sites present in the AF-1 activating domains of RAR1 and RAR2, as well as of the PKA phosphorylation site present in the AF-2 activating domain of RAR, in distinct differentiation events and individual expression of RA-responsive genes. It is particularly noteworthy that the specific contribution of RAR1 in parietal endodermal differentiation requires the integrity of both the AF-1 proline-directed kinase phosphorylation site, which has been recently shown to be phosphorylated by cdk7 (Rochette-Egly et al., 1997), and the AF-2 PKA phosphorylation site. Our study also reveals that, although overexpressed RAR can replace RAR for the formation of primitive endodermal cells in RAR knock-out cells, and RAR can substitute (albeit inefficiently at physiological levels) for RAR (in RAR knock-out cells) during the formation of parietal endodermal cells, these RAR and RAR redundant functions are not exerted under physiological conditions in WT F9 cells. Thus, the multiple RARs are sophisticated integratory transducers unable to mediate retinoid signals for cell differentiation, unless their activation functions are phosphorylated by kinases belonging to two other major signalling pathways. This certainly explains, at least in part why, in the course of vertebrate evolution, RARs have been enrolled to control so many diverse and important developmental and post-natal events (see Kastner et al., 1995).

Plasmid constructs

All constructs containing either the mouse full-length receptors RAR1 and RAR2, A/B region truncated receptors, or those harbouring mutations in the phosphorylation sites were cloned into pD402A (a gift of D.Lohnes) which is driven by the PGK promoter (Adra et al., 1987). The expression constructs pD403A and pD405A which have mouse RAR1 and RAR2 cDNAs respectively cloned in pD402A, have been described previously (Taneja et al., 1995). RARAB, RARAB and RAR1(A–C)(D–F) were isolated as EcoR1 fragments from pSG5 constructs (Nagpal et al., 1992) and subcloned in the same site of pD402A. Mutations in the phosphorylation sites in the B domain of RAR1 and RAR2 were carried out as follows: mRARS66/68A was constructed by a double PCR amplification reaction (Ho et al., 1989), to generate a NaeI–MscI fragment containing the appropriate mutations. The external nucleotides were 5'-CCGGGCCCGGCTTACTACGC-3' and 5'-GCATTTGGTGGCCAGCTCGC-3' comprising the NaeI and MscI sites, respectively. Internal nucleotides used in the PCR reaction encoded alanine (A) instead of serine (S) at positions 66 and 68. The NaeI–MscI fragment containing the mutations was inserted into pD405 restricted with NaeI and MscI. mRARS74/77A in pD402A was constructed by subcloning the RARS74/77A from pSG5 (Rochette-Egly et al., 1997) into the EcoR1 site of pD402A. Site-directed mutagenesis of the PKA phosphorylation site of RAR in pSG5 converting Ser369 to alanine (RARS369A) has been described (Rochette-Egly et al., 1995). The WT RAR1 cDNA in pD403A was excised with EcoR1 and replaced by the above mutant in the same sites. RARS360A was constructed by double PCR reaction generating a MscI–SacI fragment containing the mutation. The external oligonucleotides were 5'-GCGAGCTGGCCACCAAATGC-3' and 5'-GGCAAAATAACGAGCTCATT-3' encompassing the MscI and SacI sites respectively. The MscI–SacI fragment containing the mutation was inserted into MscI–SacI-digested pD405A.

Cell culture and establishment of stable rescue lines

F9 cells were cultured in monolayer on gelatinized surfaces as described (Boylan et al., 1993). For differentiation studies, 10⁵ cells were cultured in a 10 cm dish, and treated with retinoids alone or in combination with cAMP for 96 h and 120 h, with a change of media after 48 h. Retinoids (T-RA, BMS753, BMS961, BMS614 and BMS649) were dissolved in ethanol and used at concentrations indicated in the figure legends; dibutyryl cAMP (cAMP) was dissolved in water and used at a concentration of 250 M; control cells were treated with 0.1% ethanol (final concentration) alone. To establish the rescue lines, RAR^-/- cells were electroporated with each of the constructs indicated in Figure 1A and B along with a plasmid conferring resistance to puromycin (pD503; a gift of D.Lohnes), in a ratio of 10:1. After 24–36 h, cells were selected with 0.8 mg/ml of puromycin for 10 days as described (Taneja et al., 1995) and analysed for the presence and expression of the transgene by Southern and Western blotting.

Extract preparation, immunoprecipitation and immunoblotting of RAR and RAR

Whole cell extracts were prepared (Rochette-Egly et al., 1991) from rescue lines grown as monolayers in the absence of RA, and RAR was immunoprecipitated with protein A–Sepharose crosslinked with monoclonal antibodies directed to the F region of RAR [(Ab2(F); Rochette-Egly et al., 1991)]. Proteins were resolved by SDS–PAGE (10% acrylamide), electrotransferred onto nitrocellulose filters and immunoprobed with polyclonal antibodies against the F region of RAR [RP(F); Ghyselinck et al., 1997], followed by peroxidase-labelled protein A and chemiluminescence detection. RAR was detected by immunoblotting (without prior immunoprecipitation) with polyclonal antibodies against the F region of RAR [RP(F); Gaub et al., 1992)].

RNA isolation and RT–PCR

RNA was isolated using the guanidinium thiocyanate method and conditions for semi-quantitative RT–PCR were as described (Bouillet et al., 1995). The quantity of RNA used for RT–PCR in each reaction was normalized with 36B4 transcripts (Krowczynska et al., 1989; Bouillet et al., 1995) which is unresponsive to retinoid treatment. The RT–PCR oligonucleotides used for the various genes were as follows: Hoxa-1, 5'-CTACTTACCAGACTTCTGGA-3' and 5'-CAAAGGTCTGCGCTGGAGAA-3'; HNF1, 5'-CTTCGACAATCAGTCACCAT-3' and 5'-AGCCACACTGTTAATGACCG-3'; Stra4, 5'-GCTCTACACAACTCCATAGA-3' and 5'-GTCCTGACTAGGTAGTACTT-3'; Stra6, 5'-CTTGTGCAGAGTCTCCGTCA-3' and 5'-GGACTAGACCAGACGTGAGA-3'; HNF3, 5'-TGGCGTAGGACATGTTGAAG-3' and 5'-GCATGAGAGCAACGACTGGA-3'; laminin B1, 5'-TGATTCACCAGACGGGCCTT-3' and 5'-TGTCAGGACCATCAGGACAA-3'; collagen type IV (1) 5'-ACAACAGATGACCCACTGTG-3' and 5'-GTG- TGCATCACGAAGGAATA-3'; and 36B4 5'-ATGTGAAGTCACTGTGCCAG-3' and 5'-GTGTAATCCGTCTCCACAGA-3'. For each gene, several samples harvested at various time points of the PCR-amplification reaction were analysed to ascertain that the RNA transcript determination was performed in the linear range. The RT–PCR blots were probed with cognate ³²P-labelled cDNA fragments, and the signals were quantified using a Bio-Imaging Analyser (BAS 2000, Fuji Ltd).

We are grateful to N.Chartoire, I.Scheuer and V.Pfister for technical assistance, to Dr P.Reczek for the gifts of the synthetic retinoids BMS961, BMS753, BMS614 and BMS649, to Dr L.Gudas for the gift of laminin B1 and collagen type IV (1) cDNAs, and to Dr P.Bouillet for the gifts of several plasmids and probes. We also thank the cell culture facility, the oligonucleotide and sequencing facilities, as well as B.Boulay and J.M.Lafontaine for photography, C.Werlé and S.Metz for artwork and secretarial staff for help with the preparation of the manuscript. This work was supported by funds from the Centre National de la Recherche Scientifique, the Institut National de la Santé et de la Recherche Médicale, the Collège de France, the Centre Hospitalier Universitaire Régional, the Association pour la Recherche sur la Cancer, the Fondation pour la Recherche Médicale, the Human Frontier Science Program and Bristol-Myers Squibb. R.T. was supported by fellowships from the CNRS, ULP and Fondation pour la Recherche Médicale.

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