Title: Phosphorylation of activation functions AF-1 and AF-2 of RARalpha and RARgamma is indispensable for differentiation of F9 cells upon retinoic acid and cAMP treatment
Abstract: Article1 November 1997free access Phosphorylation of activation functions AF-1 and AF-2 of RARα and RARγ is indispensable for differentiation of F9 cells upon retinoic acid and cAMP treatment Reshma Taneja Reshma Taneja R.Taneja and C.Rochette-Egly are equal first authors Search for more papers by this author Cécile Rochette-Egly Cécile Rochette-Egly Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS-INSERM-ULP, Collège de France, BP 163, 67404 Illkirch-Cedex, France R.Taneja and C.Rochette-Egly are equal first authors Search for more papers by this author Jean-Luc Plassat Jean-Luc Plassat Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS-INSERM-ULP, Collège de France, BP 163, 67404 Illkirch-Cedex, France Search for more papers by this author Lucia Penna Lucia Penna Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS-INSERM-ULP, Collège de France, BP 163, 67404 Illkirch-Cedex, France Search for more papers by this author Marie-Pierre Gaub Marie-Pierre Gaub Search for more papers by this author Pierre Chambon Corresponding Author Pierre Chambon Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS-INSERM-ULP, Collège de France, BP 163, 67404 Illkirch-Cedex, France Search for more papers by this author Reshma Taneja Reshma Taneja R.Taneja and C.Rochette-Egly are equal first authors Search for more papers by this author Cécile Rochette-Egly Cécile Rochette-Egly Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS-INSERM-ULP, Collège de France, BP 163, 67404 Illkirch-Cedex, France R.Taneja and C.Rochette-Egly are equal first authors Search for more papers by this author Jean-Luc Plassat Jean-Luc Plassat Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS-INSERM-ULP, Collège de France, BP 163, 67404 Illkirch-Cedex, France Search for more papers by this author Lucia Penna Lucia Penna Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS-INSERM-ULP, Collège de France, BP 163, 67404 Illkirch-Cedex, France Search for more papers by this author Marie-Pierre Gaub Marie-Pierre Gaub Search for more papers by this author Pierre Chambon Corresponding Author Pierre Chambon Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS-INSERM-ULP, Collège de France, BP 163, 67404 Illkirch-Cedex, France Search for more papers by this author Author Information Reshma Taneja2, Cécile Rochette-Egly1, Jean-Luc Plassat1, Lucia Penna1, Marie-Pierre Gaub3 and Pierre Chambon 1 1Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS-INSERM-ULP, Collège de France, BP 163, 67404 Illkirch-Cedex, France 2Department of Medicine, The Mount Sinai School of Medicine, One Gustave L.Levy Place, New York, NY, 10029-6574 USA 3Lab. de Biochimie Biologie Moléculaire, Hôpital de Hautepierre, Avenue Molière BP 48, 67098 Strasbourg Cedex, France *E-mail: [email protected]$#x2013;strasbg.fr The EMBO Journal (1997)16:6452-6465https://doi.org/10.1093/emboj/16.21.6452 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The role of RARα1 and RARγ2 AF-1 and AF-2 activation functions and of their phosphorylation was investigated during RA-induced primitive and parietal differentiation of F9 cells. We found that: (i) primitive endodermal differentiation requires RARγ2, whereas parietal endodermal differentiation requires both RARγ2 and RARα1, and in all cases AF-1 and AF-2 must synergize; (ii) primitive endodermal differentiation requires the proline-directed kinase site of RARγ2-AF-1, whereas parietal endodermal differentiation additionally requires that of RARα1-AF-1; (iii) the cAMP-induced parietal endodermal differentiation also requires the protein kinase A site of RARα-AF-2, but not that of RARγ; and (iv) the AF-1-AF-2 synergism and AF-1 phosphorylation site requirements for RA-responsive gene induction are promoter context-dependent. Thus, AF-1 and AF-2 of distinct RARs exert specific cellular and molecular functions in a cell-autonomous system mimicking physiological situations, and their phosphorylation by kinases belonging to two main signalling pathways is required to enable RARs to transduce the RA signal during F9 cell differentiation. 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 (α, β and γ) and three RXR (α, β and γ) 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 RARγ−/− cells. (A) Mouse RARα1 and RARγ2 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 RARα1 and RARγ2 is shown, and the corresponding serine residues which have been mutated to alanine (S74/77A for RARα, and S66/68A for RARγ2) are indicated. The N-terminal-truncated receptors [RARαΔAB (amino acids 84-462) and RARγΔAB (amino acids 90-458)] as well as the chimeric receptor [RARα1(A-C)γ(D-F), amino acids 1-153 of RARα1 and 156-458 of RARγ1] are also schematically shown. The three additional amino acids in [RARα1(A-C)γ(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 RARα1 and RARγ2. The serine residues at position 369 of RARα1 and at position 360 of RARγ2 were mutated to alanine residues (RARαS369A and RARγS360A, respectively). (C) RARγ protein in AF-1 and AF-2 rescue lines. Whole cell extracts were prepared from WT F9 cells, RARγ−/− cells and each rescue line, and RARγ protein was first immunoprecipitated with specific monoclonal antibodies [Ab2γ(mF)] followed by a Western blot with a specific rabbit polyclonal antibody [RPγ (F)] in F9 WT, RARγ−/−, RARγWT, RARγS66/68A, RARα1(A-C)γ(D-F), RARγΔAB and RARγS360A cell lines (lanes 1-7, respectively, as indicated). (D) RARα protein in AF-1 and AF-2 rescue lines. Whole cell extracts were prepared from WT F9 cells (lanes 1 and 6), RARγ−/− cells (lanes 2 and 7), and the rescue lines RARαWT (clone α53, lane 3; clone 17, lane 8), RARαS74/77A (lane 4), RARαΔAB (lane 5) and RARαS369A (clones 22 and 210, lanes 9 and 10, respectively). RARα was detected by Western blot with specific rabbit polyclonal antibodies [RPα(F)]. Download figure Download PowerPoint 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 RARα. Interestingly, mutation of the serine residue 77 (Ser77) located in the B region of RARα decreases its AF-1 activity in transfected COS cells (Rochette-Egly et al., 1997). In addition, RARα 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 RARα1 and RARγ2 being the main RAR isoforms (Zelent et al., 1989; Wan et al., 1994; Taneja et al., 1995). Knock-out of the RARγ 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 RARα 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 RARγ-specific agonist, but not with an RARα-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 RARγ−/− cells by either re-expressing RARγ2 to wild-type levels or overexpressing RARα1 (Taneja et al., 1995). Here, we have functionally dissected the role of the AF-1 and AF-2 activating domains of RARγ2 and RARα1. Wild-type (WT) and RARγ2 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 RARγ−/− cells to establish stably transformed ‘rescue lines’. Similarly, ‘rescue’ lines were established which overexpressed WT and mutant RARα1. Our results demonstrate that, in a cell-autonomous system, AF-1 and AF-2 of RARγ2 and RARα1 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 RARγ2 and RARα1 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. Results Generation of stable ‘rescue’ lines expressing AF-1 and AF-2 mutants of RARα and RARγ in RARγ−/− cells We have previously shown that re-expression of WT levels of RARγ2 or overexpression of RARα1 in RARγ−/− 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 RARα1 and RARγ2 in these events and responses, stable ‘rescue’ lines overexpressing WT RARα1 (RARαWT line; previously referred to as α53 line in Taneja et al., 1995), re-expressing WT RARγ2 (RARγWT line; referred to as γ51 line in Taneja et al., 1995), expressing deletion mutants of RARα and RARγ lacking the A/B region (RARαΔAB and RARγΔAB lines, respectively), or expressing a chimeric receptor containing the A-C regions of RARα1 fused to the D-F regions of RARγ [RARα1(A-C)γ(D-F) line], were established in RARγ−/− cells (‘AF-1-rescue lines’; see Figure 1A). To investigate whether phosphorylation of RARα1 and RARγ2 could play a role in AF-1 function, stable lines were also established in the RARγ null background, using receptors bearing mutations in conserved putative sites for proline-directed kinases. RARα1 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, RARαS74/77A ‘rescue’ lines). Similarly, the corresponding serine residues in RARγ2 (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, RARγS66/68A ‘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 RARγ or RARα. The serine residue at position 369 in the PKA site of RARα1 was mutated to alanine (Figure 1B, RARαS369A ‘rescue’ line), and a similar mutation was made in RARγ2 at Ser360 (RARγS360A ‘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 RARγWT 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 RARγ in WT F9 cells by immunoprecipitation and Western blotting (Figure 1C, lanes 1-7). RARγWT (γ51 line, Taneja et al., 1995) and RARγΔAB were expressed in the respective rescue lines at similar levels (lanes 3 and 6). The AF-1 phosphorylation mutant line RARγS66/68A (lane 5), the chimeric rescue line RARα1(A-C)γ(D-F) (lane 4; note, however, that this chimera was overexpressed relative to endogenous RARα1), and the AF-2 phosphorylation mutant line RARγS360A (lane 7) exhibited a similar level of RARγ2 protein (which was slightly higher than that in WT F9 cells). The expression of RARαWT, RARαΔAB and RARαS74/77A was detected by Western blotting (Figure 1D, lanes 1-5) in AF-1-rescue lines, and compared with the level of endogenous RARα in WT F9 and RARγ−/− cells. The RARαWT rescue line has been shown previously to overexpress RARα1 (α53 line, Taneja et al., 1995; and Figure 1D, lane 3). The RARαS74/77A mutant was overexpressed when compared with the RARαWT line level (Figure 1D, lane 4, compare with lane 3), and the expression of RARαΔAB was revealed by the detection of faster-migrating species (lane 5). The RARα AF-2-rescue lines (Figure 1B, RARαS369A lines, clones 22 and 210, and their control RARαWT line clone 17) were also analysed for RARα expression (Figure 1D, lanes 6-10). A similar overexpression of RARα1 protein was detected in RARαS369A clone 22 and RARαWT 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 RARγ and RARα in rescuing endodermal differentiation of RARγ−/− cells We first investigated the ability of the AF-1-rescue lines established in the RARγ−/− cell background [RARγWT, RARγΔAB, RARγS66/68A, RARα1(A-C)γ(D-F), RARαWT, RARαΔAB and RARαS74/77A] to restore the differentiation of RARγ−/− cells. The morphological differentiation of each rescue line was analysed upon treatment with 100 nM T-RA either alone or in combination with 250 μM 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 RARγ−/− cells (Figure 2, panels d-f), and re-expression of RARγ2 in the RARγWT 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 RARα1 in the RARαWT rescue line (Figure 2, panels s-u). In contrast, both RARγΔAB line (Figure 2, panels j-l) and RARαΔAB (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 RARγ−/− cells. On the other hand, the RARα1(A-C)γ(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 RARα1(A-C)γ(D-F) at a lower level, similar to that of endogenous RARα1, differentiated very poorly; data not shown]. The RARγS66/68A 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 RARγ2 AF-1 function to participate in the induction of primitive and parietal endoderm differentiation. Interestingly, the rescue line overexpressing RARαS74/77A (Figure 2, panels y-z′) differentiated as efficiently as the RARαWT rescue line to form primitive endoderm in response to T-RA alone. However, in contrast to the RARαWT line, RARαS74/77A cells differentiated poorly into parietal endoderm, indicating that the phosphorylation of RARα1 in AF-1 was required for differentiation in response to RA and cAMP. Since all RARγ−/− rescue lines contain wild-type levels of endogenous RARα, this result suggests that RARαS74/77A behaves as a dominant negative mutant for parietal endodermal differentiation (see below). Figure 2.The A/B region of RARγ and RARα is required for efficient differentiation into primitive and parietal endoderm. Morphological differentiation of WT F9 cells, RARγ−/− 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 μM cAMP for 96 h as viewed under phase-contrast microscopy. Control cells treated with 0.1% ethanol (vehicle) or 250 μM cAMP remained undifferentiated. Download figure Download PowerPoint Table 1. Morphological differentiation and relative levels of the differentiation marker collagen IV (α1) [ColIV (α1)] in WT, RARγ−/− and ‘RARγ−/− rescue’ F9 lines F9 cell lines Cell treatment for 96 h T-RA (100 nM) T-RA (100 nM)/cAMP (250 μM) Col IV (α1) transcript induction Morphological primitive endoderm differentiation Col IV (α1) transcript induction Morphological parietal endoderm differentiation WT (Figure 2a-c) 14 +++ 27 +++ RARγ−/− (Figure 2d-f) 3 ± 5 ± RARγWT (Figure 2g-i) 16 +++ 32 +++ RARγΔAB (Figure 2j-l) 6 + 10 ± RARγS66/68A (Figure 2m-o) 7 + 12 ± RARα1(A-C)γ(D-F) (Figure 2p-r) 12 ++ 28 ++ RARαWT [53] (Figure 2s-u) 15 +++ 32 +++ RARαΔAB (Figure 2v-x) 4 ± 7 ± RARαS74/77A (Figure 2y-z′) 13 +++ 25 + RARγS360A [7] (Figure 4m-p) 12 +++ 25 +++ RARαWT [17] (Figure 4q-t) 13.5 +++ 21 +++ RARαS369A [22] (Figure 4v-x) 16 +++ 26 ± Cell lines were grown in the presence of all-trans RA (T-RA) or a combination of T-RA and cAMP for 96 h, as indicated. The levels of collagen type IV (α1) transcripts were estimated by semi-quantitative RT-PCR, and expressed relative to those of ethanol-treated cells (undifferentiated), which was given a value of 1. The extent of morphological cell differentiation of a given rescue line is indicated as follows as a percentage of total cells examined: +++, 70-90%; ++, 40-70%; +, <10%; ±, <5% of the cells appeared differentiated. In all cases these values are representative of at least five independent experiments. The differentiation of the various rescue lines was further analysed by determining the expression of collagen type IV (α1), 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 (α1) transcripts (Table I) was up-regulated in RARγWT and RARαWT 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 (α1) transcripts was markedly decreased in both RARγΔAB and RARαΔAB lines treated with T-RA. A low level of collagen transcripts was also induced in the RARγS66/68A line. On the other hand, despite poor parietal endodermal differentiation, the RARαS74/77A line showed a high level of collagen type IV (α1) transcripts, in agreement with an efficient rescue of primitive endodermal differentiation. As expected, the RARα1(A-C)γ(D-F) line also expressed increased levels of the differentiation-specific marker. Role of the AF-1 activating domain of RARγ and RARα in the expression of several RA-responsive genes Knock-out of the RARγ gene in F9 cells was shown to result in a marked reduction of the expression of several RA-responsive genes, such as Hoxa-1, HNF1β, Stra6, Stra4 and HNF3α (Boylan et al., 1993; Taneja et al., 1995). Thus, we investigated the ability of RARγ2 or RARα1 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, RARγ−/− cells, and the rescue cell lines RARγWT, RARγΔAB, RARγS66/68A, RARα1(A-C)γ(D-F), RARαWT, RARαΔAB and RARαS74/77A, 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. Download figure Download PowerPoint Table 2. Role of the AF-1 activating domain and of its phosphorylation on the RA-inducibility of responsive genes RA-responsive genes Level of induction of RA-responsive genes (Figure 3) WT F9 cells RARγ−/− cells RARγ−/− cells rescued by RARγWT RARγAB RARγS66/68A RARαWT RARαΔAB RARαS74/77A RARα1 (A-C)γ(D-F) Hoxa-1 8.0 2.0 7.8 7.2 6.0 7.8 4.0 8.0 6.0 HNF1β 8.0 1.0 8.0 1.0 1.0 9.0 2.0 8.0 9.0 HNF3α 10 1.0 7.5 3.0 6.0 8.0 2.0 3.0 2.5 Stra4 8.0 3.5 8.2 7.5 8.5 8.5 7.8 8.5 7.5 Stra6 25 2.0 15 2.0 18 13 4.0 6.0 6.0 The relative level of induction of Hoxa-1, HNF1β, HNF3α, Stra4 and Stra6 transcripts in WT F9 cells, RARγ−/− cells, and each of the AF-1-rescue lines grown in the absence or presence of all-trans RA (100 nM) for 24 h was estimated by semi-quantitative RT-PCR (see Figure 3) followed by quantitation of the blots on a phosphorimager. The numbers 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 of at least three independent experiments which agreed within ±15%. Responsiveness of RA target genes in rescue lines re-expressing RARγWT, RARγΔAB or RARγS66/68A. Re-expression of RARγ2 (RARγWT) reactivated the expression of all genes tested (Figure 3; Table II; see also Taneja et al., 1995). In contrast, RARγΔAB did not restore HNF1β and Stra6 expression, whereas that of HNF3α expression was partially rescued. The expression of HNF1β only was not induced in the RARγS66/68A 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 RARαWT, RARαΔAB and RARαS74/77A. The expression of all RA-responsive genes was restored by overexpression of RARα1 (RARαWT) (see also Taneja et al., 1995). In contrast, all genes except Stra4 were not efficiently induced in the RARαΔAB rescue line. Interestingly, the RARα phosphorylation mutant RARαS74/77A did not efficiently restore Stra6 and HNF3α expression, indicating that phosphorylation of region B contributes to AF-1 activity. However, the same RARαS74/77A mutation did not affect the induction of Hoxa-1, HNF1β or Stra4 expression, showing that this contribution is also promoter context-dependent. Responsiveness of RA target genes in the RARα1(A-C)γ(D-F) rescue line. The induction of HNF1β was fully restored, and that of Stra6 partially rescued in the RARα1(A-C)γ(D-F) line (Figure 3; Table II), indicating a cooperativity between AF-1 of RARα1 and AF-2 of RARγ 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 HNF3α, whereas, as expected those of Hoxa-1 and Stra4 were completely restored, as in the case of RARγΔAB 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 RARα or RARγ (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 RARγ−/− cells (panels e-h). As expected, both primitive and parietal endoderm differentiation were restored in RARγWT (Figure 4, panels i-l) and RARαWT (Figure 4, panels q-t) rescue cell lines after 96 h and 120 h of trea