The Nuclear Receptor Superfamily: A Personal Retrospect on the First Two Decades

Pierre Chambon

Institut Clinique de la Souris and Institut de Génétique et de Biologie Moléculaire et Cellulaire, Collège de France, 67404 Illkirch Cedex, France

Address all correspondence and requests for reprints to: Pierre Chambon, Institut de Génétique et de Biologie Moléculaire et Cellulaire, Collège de France, BP 10142, 67404 Illkirch Cedex, France. E-mail: chambon{at}igbmc.u-strasbg.fr.

THE BIRTH OF THE NUCLEAR RECEPTOR (NR) SUPERFAMILY

Although lipophilic hormones (steroids, thyroid hormones, and vitamin D3) were isolated in the first part of the 20th century, their mediators remained elusive until the synthesis of radiolabeled estradiol by Jensen and Jacobsen in 1960, which allowed the identification of one or more estradiol-binding proteins. The finding that this protein was translocated from cytoplasm to nucleus upon hormone administration suggested that there was a link between control of transcription and the physiological action of the hormone (1). This concept was supported by the work of the Clever and Ashburner laboratories (2) showing that chromosomal puffing in insect polytene chromosomes was triggered by ecdysteroids. The subsequent identification of transcriptionally responsive genes in vertebrate steroid hormone target tissues, together with the finding that the glucocorticoid receptor (GR), could exhibit a modular structure with separable hormone- and DNA-binding sites (3), and also that a MMTV (mouse mammary tumor virus) GR response element (GRE) had the characteristic properties of a transcriptional enhancer element, led to a model of steroid hormone action by postulating that steroid hormone receptors are ligand-regulated enhancer-binding transcriptional factors (Refs. 4 and 5 and references therein). At this stage, the cloning of steroid receptors became an imperious necessity, because reverse genetic studies were obviously required to elucidate the molecular basis of this model.

Twenty years have passed since the human GR cDNA was cloned by Ron Evans and his collaborators (6, 7); concomitantly, using antibodies prepared by Geoffrey Greene and Elwood Jensen against purified human estrogen receptor (ER), together with oligonucleotide probes derived from its amino acid sequence, we isolated and sequenced ER cDNA clones (8) and showed that one of them contained the entire open reading frame of the ER now known as ER{alpha} (9, 10). The GR and the ER, which incidentally were the first RNA polymerase II transcription factors to be cloned, exhibited a surprising homology with the v-erbA viral oncogene whose cellular counterpart, the c-erbA protein, was shown shortly afterward to be the thyroid hormone receptor (TR) (11, 12). This provided the first indication that steroid receptors could belong to a superfamily of structurally related receptors interacting with chemically unrelated ligands (13).

A comparison of the above receptors with the chicken ER revealed six regions of homology (A to F, from the N-terminal to the C-terminal end), two of which (C and E) being highly conserved (14). In vitro studies showed that regions E and C were the ligand binding domain (LBD) and the DNA binding domain (DBD), respectively (15). This was unequivocally confirmed by the end of 1986, when Stephen Green demonstrated in our laboratory that a chimeric receptor, in which he had swapped region C of ER for that of GR, could bind estradiol but not activate a chimeric estradiol-responsive gene (vit-tk-CAT), whereas it activated a glucocorticoid-responsive gene (MMTV-CAT) in the presence of estradiol, but not in the presence of a glucocorticoid (16). Using a reciprocal construct, Vijay Kumar et al. (17) demonstrated that, in the presence of a glucocorticoid, the chimeric receptor could activate the expression of the vit-tk-CAT reporter gene, but not of MMTV-CAT.

These experiments not only unequivocally demonstrated the modular structure of NRs, in which the DBD and the LBD can function independently (see also Ref. 18), but even more importantly they indicated how such chimeric receptors could be used to characterize the ligand requirement of a novel putative member of the NR superfamily: hooking the LBD of a novel receptor candidate to the DBD of either the ER or the GR would result, upon addition of a cognate agonistic ligand, in activation of an estrogen- or a glucocorticoid-responsive reporter gene, respectively, unless this ligand is endogenously produced by the transfected cultured cells, or the LBD of the novel receptor is constitutively transcriptionally active. The value of this approach was illustrated shortly afterward by the independent discovery of the first RAR [retinoic acid (RA) receptor] {alpha} by Martin Petkovitch in our laboratory (19) and Vincent Giguère in Ron Evans’ laboratory (20), which was followed by the subsequent finding of two additional RAR isotypes (subtypes), RARß (21), and RAR{gamma} (22, 23).

Clearly, in 1987, the stage was set to clone all members of the NR superfamily, based on sequence homology of their DBDs and LBDs. This was achieved in 1999: 48 human and 49 mouse NRs have been cloned (examination of the human and mouse genome sequences did not reveal any additional member of the superfamily), among which 24 are orphan receptors for which no ligand have yet been discovered (24, 25). Which ones will remain true orphans needs to be established (25, 26).

NR ACTIVATION FUNCTIONS, COREGULATORS, AND MECHANISM OF THE NR MOLECULAR SWITCH

The functional dissection of the ER demonstrated that it is a ligand-regulated transcription factor that modulates target gene transcription. Two distinct synergistic transcriptional activation functions (AFs) were unveiled: the ligand-independent AF-1 located in the N-terminal A/B region, and the ligand-dependent AF-2 encompassing region E (the LBD) (27, 28). Both ER AF-1 and AF-2 were found to act in promoter context- and cell-specific fashions (29). Similarly, the dissection of the activation functions of the six RAR and RXR isotypes ({alpha}, ß, and {gamma}) and, for each of them, of their isoforms that differ in their N-terminal A regions, revealed the existence of isotype-specific AF-2 and isoform-specific AF-1, the synergism of which was dependent on both the promoter context and the nature of the retinoic acid response element (RARE). Moreover, the AF-1 and AF-2 of a given RAR isoform (e.g. RAR{alpha}) appeared to be significantly different from those of the two other RAR isotypes (RARß and {gamma}), as the AF-1 from a given RAR isoform synergized with the AF-2 of its cognate RAR isotype, but not with the AF-2 of the two other RAR isotypes (Refs. 30, 31, 32, 33 and references therein). A very weak, autonomous and constitutively active, activation domain (AF-2 AD) was identified within the C-terminal end of the homology region E of RARs and RXRs (34), and shown to contain an amphipathic {alpha}-helix core motif (the AF-2 AD core), which was also present in ER and TR (Refs. 24 , 33 , and 34 and references therein). Its integrity was required for AF-2 activity, but in general not for ligand and DNA binding, which initially suggested that it may belong to an interactive surface for the binding of factors mediating AF-2 (24, 33, 34, 35).

The observation that AFs acted in a promoter context- and/or cell-specific fashion suggested to us that NRs could cooperate with cell-specific promoter-bound transcription factors and/or interact with cell-specific factors mediating their activity. The latter assumption was supported by the results of transcriptional interference/squelching experiments in cultured cells, in which the activity of a given receptor was inhibited "off the DNA," and in an agonist- and AF-2 integrity-dependent manner, by an excess (relative to its cotransfected cognate-responsive reporter gene) of the same receptor (autosquelching) or by addition of a different receptor (heterosquelching). These squelching data (36, 37, 38) were interpreted as resulting from the sequestration of transcriptional intermediary factors (TIFs) (also named mediators/coregulators) that, acting as coactivators, may mediate the AF-1 and AF-2 activities of NRs on the transcription machinery and chromatin template. To substantiate this concept, searches for proteins that would interact with the LBD in the presence of agonistic ligands, but not in the presence of antagonists, were undertaken using both yeast two hybrid-based and Far-Western blotting-based screens.

The cDNAs of several such proteins were cloned using human and mouse expression libraries in 1995–1996 (33). Two of these proteins, steroid receptor coactivator (SRC)-1 (39) and TIF-2 (40), isolated by O’Malley and colleagues and our own group, respectively, had the expected properties of bona fide coactivators. For instance, TIF-2 was shown 1) to interact directly, in vitro and in cultured cells, with the LBDs of several NRs in an agonist- and AF-2 AD-core-integrity-dependent manner; 2) to harbor an autonomous activation function; 3) to relieve NR autosquelching; and 4) to enhance the activity of NR AF-2s when overexpressed in mammalian cells (40). Moreover, sequence similarities between SRC-1 and TIF-2 (40) unequivocally supported the existence of a p160 gene family of NR coactivators (41).

In our search for additional TIFs/mediators/coregulators that would bind in a ligand-dependent fashion to LBDs of various NRs, we isolated TIF1{alpha}, which did not exhibit all of the above bona fide characteristics of a NR coactivator, but revealed the presence of a NR-interaction module containing the NR box motif LXXLL, which was also present in two putative TIFs/mediators/coregulators, RIP140 and Trip3 (Ref. 42 and references therein). Importantly, we demonstrated that this NR box motif was necessary and sufficient for ligand-dependent direct interaction between TIF1{alpha} or RIP140 and a cognate LBD surface that required the integrity of the AF2 AD core (see above) (42). The presence of such NR box motifs in a number of putative NR coregulators, as well as their implication in NR-coregulator binding, was confirmed in two subsequent reports (43, 44). The TIF2-NR interaction domain, as well as those of its orthologues SRC-1 and p/CIP (44), the two other members of the p160 family, was found to comprise three NR binding modules, each containing the NR box motif LXXLL (Ref. 45 and references therein). Furthermore, TIF-2 [also known as GR-interacting protein-1 (GRIP-1) or nuclear receptor coactivator (NcoA)-2], SRC-1 (NcoA-1), and p/CIP (also known as NcoA-3, AIB-1, ACTR, RAC, or TRAM-1), were shown to contain an activation domain (AD-1), which interacted with cAMP binding protein-binding protein (CBP)/p300 through a LLXXLXXXL motif (44), which was distinct from the LXXLL NR box motif (Refs. 44, 45, 46, 47, 48 and references therein).

The observation that CBP/p300, and to a lesser extent the p160 coactivators, possessed an intrinsic histone acetyltransferase activity (HAT), and the belief that histone acetylation played an important role in the "opening" of the chromatin during transcription activation, provided the first indication that coactivators like those of the p160 family could mediate the NR AF-2 ligand-induced transcription activity through modification of the chromatin environment of target genes, making it accessible to the transcription machinery (Refs. 44, 45, 46, 47, 48 and references therein). Furthermore, our finding of a ligand-dependent interaction between the ER LBD and components of the SWI/SNF ATP-dependent nucleosome remodeling complex (49) made it likely that additional chromatin remodeling complexes interacting with NR-liganded LBDs could be involved in mediating their AF-2 activity (Ref. 50 and references therein).

On the other hand, it had been found that some NRs (particularly RARs and TRs) actively repressed transcription, when in the unliganded apoform (Refs. 24 , 33 , 46 , and 48 and references therein). Approaches similar to those used for the characterization of coactivators led to the identification of the corepressors N-CoR (nuclear receptor corepressor) (51) and SMRT (silencing mediator of RA and TR) (52) that bind through a LXX[I/H]IXXX[IL] Cor-NR box to ligand-free apo-LBDs (e.g. RARs and TRs) or antagonist-bound NRs (Refs. 24 , 33 , 46 , 48 , and 53 and references therein). Interestingly, deletion of the AF-2 AD core enhanced the binding of N-CoR (N-CoR1) or SMRT (N-CoR2) to TR and RAR apo-LBDs, and prevented their agonistic ligand-induced release (48, 51, 52). Corepressors were shown to reside in or recruit high molecular weight complexes that have intrinsic histone deacetylase activities (HDAC) (Ref. 48 and references therein). Thus, because acetylated histones are known to be mostly associated with silent "condensed" regions of the genome, active transcriptional repression by some NRs appeared to be mediated by the binding of corepressor complexes to their unliganded LBD, which renders their target genes transcriptionally unavailable through chromatin "closure."

How AF-2 could be related to the recruitment of RNA polymerase II holoenzyme was unveiled when its mediator complex component SMCC was characterized in 1996–1999 as the thyroid hormone (TRAP complex) or vitamin D (DRIP complex) receptor-interacting complexes that enhanced activation of transcription by TR and vitamin D receptor (VDR) cell-free systems. The SMCC subunit that interacted with the agonist-bound NR LBD was identified as DRIP205 (identical to TRAP220), and shown to contain a functional LXXLL NR box motif (for review see Refs. 24 , 48 , and 50).

Finally, direct or indirect interactions between NRs and components of the basal transcription machinery (e.g. TBP, TAF subunits of TFIID, and TFIIH) were also revealed in studies in vitro, and reported to enhance the transcriptional activity of NRs, at least in transient transfection experiments (Refs. 5 , 24 , and 46 and references therein; and see below).

Not much has been yet learned concerning the coactivators that may possibly selectively mediate the effect of the ligand-independent AF-1 (see Ref. 54 and references therein), although analysis of the ability of ER AF-1 and AF-2 to homosynergize and to heterosynergize with one another and with AFs of other activating domains (28), as well as a comparison of their transcriptional interference/squelching properties (38), suggested that they could interact with different TIFs/coactivators (38). However, it has been shown that TIF-2, using distinct binding surfaces, can bind simultaneously to isolated ER{alpha} AF-1 and AF-2, which may, at least in part, account for the synergistic activation of transcription by the two AFs (54).

Ultimately, the multiple functions of NRs (homo- and heterodimerization, ligand binding, ligand-induced switch from an inactive or repressive condition to a transcriptionally active state) must be understood in terms of structural features that mediate these functions. The initial crystallographic studies of the RXR{alpha} unliganded apo-LBD (55) and the RAR{gamma} all-trans RA-liganded holo-LBD (56), performed in our Institute in collaboration with Dino Moras’ group, allowed us to propose in 1995 a canonical structure for NR LBDs (57) that forms a single protein domain. This fold was described as a three-layer antiparallel {alpha}-helical sandwich with 12 {alpha}-helices (H1 to H12) and one ß-turn. An alignment of LBD sequences of all NRs suggested that they all share a similar fold, and this fold has indeed been found in all NR LBDs whose crystal structures have been as yet described (Refs. 24 , 33 , 53 , 57 , and 58 and references therein). These initial and additional crystallographic and molecular genetics studies (58, 59, 60, 61) revealed the structures of the RAR/RXR heterodimerization interface (60) and of the ligand binding pockets (LBP) of RAR{gamma} (56, 59) and RXR{alpha} (61), as well as the basis for the selectivity of RAR{alpha}, ß, and {gamma} for the binding of isotype-selective synthetic retinoids, which was shown to only reside in three divergent residues in their LBPs, whereas, in contrast, modeling the LBP of RXRß and RXR{gamma} isotypes did not reveal any difference with the residues forming the RXR{alpha} LBP.

Importantly, making the assumption [subsequently shown to be correct (61)] that the structure of the RXR{alpha} holo-LBD would be similar to that of the RAR{gamma} holo-LBD, the initial comparison of the RXR{alpha} apo-LBD and RAR{gamma}-holo LBD suggested that a transconformation of NR LBDs was induced by binding of the ligand, thus generating the cognate surface required for efficient interaction with the transcriptional coactivators that mediate the AF-2 activation function to the chromatin template and/or the transcription machinery. We proposed that this was achieved through a mouse trap mechanism that most notably involves a major repositioning of the transactivation helix H12 that corresponds to the core of the AF-2 activation domain (AF-2AD) (33, 56, 57, 58). The validity of our proposal was supported by subsequent structural studies performed by others with complexes formed between holo-LBDs and coactivator peptides containing NR box LXXLL motifs, which showed that these latter make contact with helices 3, 4, and 12 through binding to a hydrophobic groove on the receptor surface (Refs. 48 and 53 and references therein).

The observations that helix 12 (AF-2 AD core) was required for agonist-induced release of corepressors from apo-LBDs, and also that corepressors bound more tightly to LBDs lacking the AF-2 AD core (see above), initially suggested to us that the conformational changes, that allow coactivators to bind, could concomitantly destroy the corepressor-interacting surface present in TR and RAR apo-LBDs, making the binding of these coregulators mutually exclusive (33, 57). This was subsequently supported by others who performed mutational studies aimed at mapping LBD surfaces that interact with coactivators and corepressors, as well as structural studies of complexes between agonist-bound LBD and corepressor peptides containing CoR-NR box motifs. It was indeed shown that the corepressor peptide does bind in the same hydrophobic groove as the coactivator, but with H12 displaced from the active position (Refs. 48 and 53 and references therein).

RXRs AS PROMISCUOUS HETERODIMERIZATION PARTNERS FOR DNA BINDING, AND SUBORDINATION OF THEIR TRANSCRIPTIONAL ACTIVITY

The observation that purified RARs did not bind efficiently to RAREs led us to purify and to clone the nuclear accessory factor that enhanced the binding of RARs to RAREs in vitro. Surprisingly, we discovered that this protein was RXRß (62). Concomitantly Yu et al. (63), in a search for proteins that could interact with RARs, also identified RXRß as the factor that stimulates the binding of RARs to RAREs. Both approaches led to the demonstration that, through the formation of heterodimers, RXRs ({alpha}, ß, and {gamma}) stimulated not only the binding of RARs to RAREs, but also of TRs to thyroid hormone response elements (TREs) and VDR to vitamin D response elements (VDREs) (reviewed in Ref. 32). Shortly afterward, these seminal observations were confirmed by several groups that showed that cloned RXRs could substitute for nuclear extracts in enhancing binding of RAR, TR, VDR, and perosixome proliferator-activated receptors (PPARs) to their cognate response elements (REs) (reviewed in Ref. 33). Subsequently, the formation of heterodimers between RXRs and LXR, farnesoid X receptor (FXR), pregnane X receptor (PXR), NGFIB, and Nurr1 was also reported (see Refs. 24 , 26 , and 33 for references).

The finding that heterodimers between RXRs and a number of NRs were possibly the functional units transducing the effect of their cognate ligands, raised the question as to whether, in such heterodimers, the RXR partner was a silent or a transcriptionally active partner. Although, using RAR/RXR heterodimers, the results of initial in vitro ligand binding and transactivation transfection experiments were conflicting (reviewed in Ref. 33), studies much closer to physiological conditions performed with cultured embryonal carcinoma cells (F9 and P19 cells) exposed to suboptimal concentrations of synthetic RAR-selective retinoids and RXR-selective rexinoids resulted in synergistic activation of expression of a number of endogenous RA-responsive genes, whose expression was known to require both RAR{gamma} and RXR{alpha} (Refs. 65, 66, 67, 68 and references therein; see below). Importantly, the expression of these genes was also efficiently activated by retinoids on their own at higher concentrations but could not be activated by rexinoids on their own, even at higher concentrations. This silencing of RXR activity in the absence of a RAR ligand was referred to RXR subordination or silencing by apo-RAR (Refs. 24 , 25 , and 33 and references therein). Similar RXR subordinations were also reported for RXR/apo-TR and RXR/apo-VDR heterodimers, but not for RXR/apo-PPARs and RXR heterodimerized with other apo-NR partners (see above).

A molecular mechanism accounting for RXR subordination and permissivity in heterodimers has been recently proposed (69). In short, RXR in heterodimers with apo-RAR can bind its ligand and recruit coactivators in vitro. However, in the usual cellular environment, corepressors do not dissociate from the apo-RAR, which inhibits coactivator binding, as bindings of coactivators and corepressors are mutually exclusive. Thus, RXR subordination can be overcome in heterodimers that bind corepressor weakly or in cells with high coactivator and low corepressor contents. The study of Germain et al. (69), which showed how RXR could be a transcriptionally active partner within RAR/RXR heterodimers, also demonstrated that the synergy between RXR ligands and RAR ligands results from increased interaction efficiency of two NR boxes, present in a single p160 coactivator molecule, with both holo-RAR and holo-RXR of the heterodimer. That transcriptionally active RXRs can be effectively instrumental to RAR/RXR-mediated signaling in vivo has been strongly supported by our genetic studies, which have shown that the ligand-dependent activation functions (AF-2) of RXR{alpha} and RXRß are important for retinoid signaling during mouse development (Refs. 70 and 71 and references therein; and our unpublished results; see below). Interestingly, we also found that the RXRß AF-2 is required in RXRß/LXRß heterodimers that control cholesterol efflux in testis Sertoli cells (72).

Assuming that RXR AF-2 activity requires the binding of an agonistic ligand, raises the question of the possible identity of this ligand. In vitro, RXRs are activated by 9-cis RA, which binds to RXR and RAR LBDs with high affinity (Ref. 33 and references therein). However, 9-cis RA has been difficult to detect in vivo, which suggested that it might not be the endogenous ligand activating the ligand-dependent AF-2 of RXRs (Ref. 73 and references therein). In any event, assuming that 9-cis RA might be the ligand activating RXRs in permissive heterodimers (e.g. RXR/PPARs, RXR/LXR, RXR/FXR, see above) would create a problem of promiscuity, as it would result in concomitant activation of the RAR/RXR-mediated retinoid signaling pathway. For instance, this would not permit, in the same cell, the simultaneous occurrence of 9-cis RA-induced transcriptional activation events mediated by permissive NR/RXR heterodimers, with transcriptional repression events mediated by RAR/RXR heterodimers. In this respect, we have recently found that the RXRß AF-2 activity of RXRß/LXRß heterodimers was required for the control of cholesterol efflux in testis Sertoli cells, and concluded that it was unlikely to be induced by a retinoid (72). Docosahexaenoic acid, a long-chain polyunsaturated fatty acid that is abundant in brain and binds RXR with a low efficiency, has been found to activate RXR in cell-based assays (73). These observations raise the interesting possibility that the RXR subunit of heterodimers could be bound to low affinity ligands to maximally "sensitize" (through synergistic effects) the transcriptional activity of the heterodimer to discrete variations in the concentration of the cognate ligand of its heterodimeric partner. For instance low affinity metabolites may sensitize RXR/PPAR, RXR/LXR and RXR/FXR heterodimers which are all involved in regulating energy and nutritional homeostasis (Ref. 24 and references therein).

Finally, the question as to whether, under physiological conditions, RXR homodimers could be transcriptionally active on their own through binding to RXR response element remains open, even though it has recently been shown that RXR homodimers bound to PPAR response elements are able to functionally substitute for PPAR-RXR heterodimers and to regulate complex metabolic pathways in the mouse. This functional substitution indeed required the administration of supraphysiological amounts of 9-cis RA to the mice (74).

F9 EMBRYONAL CARCINOMA CELLS AS A CELL AUTONOMOUS MODEL TO STUDY THE FUNCTIONAL SELECTIVITY OF RARs AND RXRs IN RETINOID SIGNALING

The high degree of conservation of the various RAR and RXR isoforms across vertebrate evolution, as well as their selective spatiotemporal expression patterns in developing embryos and adult tissues (Refs. 32 , 33 , and 71 and references therein), initially suggested to us that each isoform may perform unique functions, thus accounting for the highly pleiotropic effects of RA throughout the life of vertebrates (32). The results of subsequent studies in vitro led to the further proposal that these highly pleiotropic effects could reflect highly combinatorial molecular mechanisms in which the multiplicity of the actors (the heterodimers and their coregulators) differentially transduce retinoid signals to selectively orchestrate the expression of numerous sets of RA target genes (33). A genetic dissection of the functions of RAR and RXRs in vivo was obviously required as a first step to evaluate the physiological relevance of this proposal.

We first chose the RA-responsive F9 murine embryonal carcinoma (EC) cell line as a simple cell-autonomous model system for analyzing RA signaling under in vitro conditions that mimics, at least to some extent, physiological process occurring during early embryogenesis (reviewed in Ref. 68). Indeed, upon RA treatment, and depending on the culture conditions, the F9 EC cell line differentiates into cells resembling either one of the three distinct extra-embryonic endoderms [primitive, parietal (in the additional presence of cAMP) and visceral (culture in suspension)]. These differentiations are accompanied by a decrease in proliferation rate (antiproliferative response), triggering of apoptosis, and RA-induced variations in expression of subsets of responsive genes (see Ref. 68 and references therein). Thus, F9 cells represent an interesting model system to study the molecular mechanisms underlying complex biological events induced by retinoids. Moreover, as they respond to cAMP, these cells also constitute a good system to study how, at the molecular level, retinoid signaling cross talks with other signaling pathways, such as the protein kinase A (PKA) pathway.

Combining genetic approaches (targeted mutagenesis of RARs and RXRs through homologous recombination, followed by reexpression of wild-type or mutant receptors in "rescued" lines), and a pharmacological strategy using RAR isotype-specific retinoids and pan-RXR-selective synthetic rexinoids, we showed (Ref. 68 and references therein) that 1) RAR{gamma}/RXR{alpha} heterodimers were required for growth arrest, visceral endodermal differentiation, and primitive endodermal differentiation, whereas RAR{alpha}/RXR{alpha} heterodimers were further required for parietal endodermal differentiation in the presence of cAMP. In all cases, RAR and RXR could act synergistically, but RXR activity was always subordinated to that of its RAR partner; 2) the induced expression of RA-target genes was mediated by RXR{gamma}/RXR{alpha} heterodimers in which the RXR activity was also subordinated to that of its partner; 3) AF-1 and AF-2 of RAR{gamma}, RAR{alpha} and RXR{alpha} acted synergistically and selectively to control the physiological and molecular responses of F9 cells to RA; 4) in some cases, gene knockouts generated artifactual functional redundancies between RAR or RXR isotypes, which did not exist under wild-type conditions; for instance expression of RARß2, which is selectively induced by a RAR{gamma}, but not a RARß selective agonist in wild-type F9 cells, was efficiently activated by a RARß selective agonist in RAR{gamma}-null mutant cells, clearly demonstrating that gene knockouts can artifactually create situations of redundancy of gene function, that do not exist in wild-type conditions (75); 5) upon interaction with TFIIH, a general transcription factor (GTF), RAR{alpha}, and RAR{gamma} were constitutively phosphorylated in a ligand-independent manner in their AF-1 domain by the cdk7 subunit of TFIIH (Refs. 68 and 76 and references therein). Incidentally, this was the first example of activation of a transactivator through binding to and phosphorylation by a GTF.

Using the rescue strategy in RAR{gamma}- and RAR{alpha}-null F9 mutant cells, we also demonstrated that phosphorylation of RAR{gamma} AF-1 was required for primitive endodermal differentiation, whereas phosphorylation of the same AF-1 domain in RAR{alpha} or RXR{alpha} was not mandatory for endodermal differentiation (76, 77). Furthermore, phosphorylation of the AF-1 domain of RAR{gamma} was required for induction of RA-responsive genes, but in a differential promoter-dependent fashion (68, 77). Interestingly, phosphorylation by PKA at a conserved site within the LBD of RAR{alpha} and RAR{gamma} was required in RAR{alpha}, but not in RAR{gamma} for parietal endodermal differentiation (77), thus demonstrating the existence of cross talk between RA and PKA pathways for parietal differentiation through phosphorylation of RAR{alpha} at its PKA site. The observation that the AF-1 domain of RAR{alpha} and RAR{gamma} could also be phosphorylated by MAPK (Refs. 68 and 76 and references therein) raised the question of whether the MAPK cascade might also cross talk with the retinoid signaling pathway, as first reported by us in the case of the estrogen signaling pathway (78). In any event, these F9 cell studies clearly supported the conclusion that through binding of cognate ligands and phosphorylation of their activation domains, retinoid receptors are sophisticated transducers, integrating signals belonging to several signaling pathways.

Finally, our studies on F9 cells also revealed that degradation of RAR{gamma}/RXR{alpha} heterodimers by the ubiquitin-proteasome system was dependent on phosphorylation of the RAR{gamma} partner, supporting the proposal that transcriptional activation and activator degradation are closely coupled events, and leading to a model in which this degradation may participate in the regulation of duration and magnitude of retinoid action (see references in Ref. 68).

FUNCTIONS OF RARs AND RXRs IN VIVO: LESSONS FROM A GENETIC DISSECTION OF THE RETINOID SIGNALING PATHWAY IN THE MOUSE

The above F9 studies supported our initial assumption that distinct RAR/RXR heterodimers could be differentially involved in cellular and molecular events induced by RA, and also that their activation functions AF-1 and AF-2 could be differentially required in these RA-induced events. Moreover, they revealed that phosphorylation of the AF-1 and AF-2 domains could be instrumental for cross talks between retinoid and other signaling pathways. It was, however, clear that genetic approaches in vertebrates (the mouse) will be required to determine, at the organismal, cellular, and molecular levels, the function of the multiple retinoid receptors under truly physiological conditions, from the conception to the death of the animal.

Genetic Evidence that RARs Transduce Retinoid Signals in Vivo
Through homologous recombination in ES cells (see Refs. 71 , 79 , and 80 and references therein), we engineered germline knockout mutations in the mouse for the three RAR isotypes ({alpha}, ß, and {gamma}), the three RXR isotypes ({alpha}, ß, and {gamma}), as well as for the main eight RAR isoforms (RAR{alpha}1, RAR{alpha}2, RARß1/ß3, RARß2/ß4, RAR{gamma}1, RAR{gamma}2). None of these single RAR mutations were lethal, and thorough examination of these mouse mutants revealed that RARs transduced retinoid signals in vivo, as RAR ({alpha}, ß, and {gamma}) null mutants, and some mutant isoforms (RARß2/ß4 and RAR{gamma}1), displayed several aspects of the postnatal vitamin A deficiency (VAD) syndrome, and congenital abnormalities corresponding to atavistic traits, strongly supporting the view that modulation of RA signaling has been instrumental during vertebrate evolution to modify skull shapes and functions (71). As altogether, RAR single mutant abnormalities were confined to only a small subset of the tissues normally expressing these receptors during embryogenesis and postnatally, we assumed that RARs could be functionally redundant. To test this possibility, mutants lacking a pair of isotypes (RAR{alpha}ß, RAR{alpha}{gamma}, and RARß{gamma} double mutants), or two or more isoforms belonging to distinct isotypes, were generated.

All of these compound null mutants, with the exception of RARß2/RAR{gamma}2, died in utero or at birth, because of severe developmental defects that, altogether, included the complete spectrum of malformations corresponding to the classical fetal VAD-induced syndrome described 52 yr ago by Warkany’s group (Refs. 71 , 79 , and 80 and references therein). This demonstrated that liganded RARs play crucial roles at many distinct stages of early embryogenesis and organogenesis. These analyses also provided the first compelling evidence that RA was actually the active metabolite of vitamin A during embryonic development, which was subsequently confirmed by the demonstration that RA synthesized by the retinaldehyde deshydrogenases acts as an indispensable developmental hormone (see references in Ref. 71).

Signaling through RXRs Is Indispensable for Embryonic Patterning and Shaping and Organogenesis
All RXR{alpha} null mutants died around embryonic d 14.5, and displayed an hypoplasia of the myocardium that was previously observed in VAD fetuses and in some RAR double null fetuses, which suggested that RXR{alpha} was involved in the transduction of a RA signal. RXR{alpha} mutants also displayed additional abnormalities belonging to the fetal VAD syndrome, and also found in RAR double null mutants. That all the congenital defects exhibited by RXR{alpha} null fetuses were also observed in RAR single or double null mutants (References 70 , 71 , and 79 and references therein) provided the first genetic evidence of a convergence between RAR and RXR signaling pathways, and also gave the first clue that RXR{alpha}/RAR heterodimers are the functional units that transduce RA signals in vivo.

RXRs Can Be Transcriptionally Active, and Both AF-1 and AF-2 Activation Functions of RXR Are Differentially Involved in Development
Whether RXRs are transcriptionally active within RAR/RXR heterodimers has been a controversial issue (see above). To determine the roles played by RXR{alpha} AF-1 and AF-2 activities in vivo, we engineered mouse mutants that expressed truncated RXR{alpha} proteins lacking either 1) most of the terminal A/B region that includes AF-1 (RXR{alpha}af1° mutant), or 2) the AF-2 core (RXR{alpha}af2° mutant), or both AF-1 and AF-2 activities (RXR{alpha}af° mutant) (references in Refs. 70 and 71 ; and our unpublished data). Both RXR{alpha}af2° and RXR{alpha}af°) died at birth. In impaired genetic backgrounds that eliminated functional redundancies between RXRs, both RXR{alpha} activation functions were required for normal eye development, whereas they were dispensable for myocardial growth, indicating that their requirement depends on the nature of the RA-controlled event. Furthermore, RXR{alpha}af1°/RARnull ({alpha}, ß, or {gamma}), as well as RXRaf2°/RARnull ({alpha}, ß, or {gamma}) compound mutants died in utero, and exhibited a large array of malformations that nearly recapitulated the full spectrum of the fetal VAD syndrome, showing that both RXR AF-1 and AF-2 can be instrumental to the activity of RAR/RXR heterodimers in vivo. Compound mutants in which a null mutation of a given RAR isotype ({alpha}, ß, or {gamma}) was associated with either 1) a RXR{alpha} null mutation or 2) a RXR{alpha}af1° mutation, or 3) a RXR{alpha}af2° mutation or 4) a RXR{alpha}af° mutation, altogether also recapitulated the abnormalities exhibited by RAR double mutants (Refs. 70 , 71 , and 79 and references therein). This synergism between RAR and RXR loss-of-function mutations definitely supported the conclusion that RXR{alpha}/RAR heterodimers are the functional units that transduce RA signals during embryonic development. Importantly, the abnormalities exhibited by these various RXR{alpha}/RAR compound mutants also identified the heterodimers that actually transduce the RA signal in a given developmental process.

What do functional redundancies within RAR and RXR families actually mean?
Altogether, our mouse genetic studies support the conclusion that many of the molecular mechanisms underlying the transduction of the RA signal by retinoid receptors, as they have been deduced from in vitro studies using cellular and acellular systems, are also instrumental to RA signaling under truly physiological conditions, i.e. at the organismal level. RAR signals appear to be transduced by RXR/RAR heterodimeric functional units that are selectively involved in given developmental processes. Furthermore, depending on the developmental event under consideration, the RXR partner can be transcriptionally active, which raises the question of the possible existence of physiological RXR ligands, and what these ligands could be (see above). All of this supports our initial proposal (64) that the highly pleiotropic effects of RA reflect the combinatorial mechanisms through which multiple RXR/RAR heterodimers differentially transduce retinoid signals to selectively regulate the expression of numerous sets of RA target genes that control the shaping and axial patterning of the early embryo, and subsequently multiple aspects of organogenesis.

However, our genetic study of the physiological role of RAR and RXR has also revealed an extensive functional redundancy within the members of each family, although in all cases each of these members appears to individually exert at least one specific physiological function. Even though functional redundancy may not be surprising within family members sharing a common ancestor, the striking interspecies conservation of a given RAR{alpha}, ß, or {gamma} (32) isoform raises the question whether this redundancy is physiologically relevant or artifactually generated when a given RAR or RXR is knocked out, as it has been shown to be the case in F9 cells (see above). In fact, redundancy is frequent within members of the RAR or RXR families, a given defect being frequently or exclusively seen in compound RAR or RXR double null mutants (although it is lacking or weakly penetrant in single mutants), whereas in striking contrast redundancy is much less frequent in the case of given compound RAR/RXR heterodimer knockouts, in which this defect is generated in a fully penetrant manner. As discussed (Ref. 70 and references therein), assuming that RAR/RXR heterodimers are indeed the functional transducing units, the easiest way to interpret these observations is to postulate that redundancy can occur only when one of the two partners of the physiological heterodimer is ablated. In other words, the activity of a given heterodimer that is selectively involved in the control of a given event may still be above a physiological threshold level when either one of the two partners (RAR or RXR) is ablated, but not when both are missing. Thus, the selective involvement of a given RAR or RXR isotype might be revealed only under conditions where the threshold level is not reached, which would account for the observation that the role of the RXR{alpha} AF-1 and AF-2 functions cannot be fully revealed unless the activity of the heterodimer is altered in some way (e.g. additional mutation of the RAR partner, additional knockout of potentially redundant RXR isotypes, decreased availability of intracellular RA). As the actual intracellular concentrations of RA might be lower in the wild than in animal facilities, it is therefore possible that the selective functions of the various RAR and RXR isoforms would be revealed only under conditions of limited RA supply. In any event, the results of our genetic studies strongly support the view that the functional redundancies observed between retinoid receptors in the mouse are actually artifactually generated upon impairment of one of the partner of physiological relevant heterodimers.

Somatic Spatiotemporally Controlled vs. Germline Mutations to Genetically Dissect NR Signaling Pathways
Clearly, the generation of germline mutations have provided many valuable insights into the functions of RARs and RXRs, as well as of other members of the NR superfamily (for an early review, see Ref. 79). However, this strategy has intrinsic limitations. First, the effect of a germ line mutation may be functionally compensated during development, thus precluding the appearance of a defect postnatally. On the other hand, the mutation could be lethal in utero (e.g. RXR{alpha}) or postnatally, thus preventing analysis of the functions of the gene at later developmental or postnatal stages. The mutation could also arrest the development of a given organ at an early stage, thus precluding further analysis of the gene functions at later stage. Moreover, introducing mutations in germline makes it very difficult to distinguish cell-autonomous from non-cell-autonomous functions of a gene belonging to families, such as RARs and RXRs, that are involved in highly pleiotropic signaling pathways. In many instances, these limitations may actually prevent the determination of the function of a given gene product in a defined cell-type/tissue at a given time of the animal life. This is obviously the case for RARs and RXRs.

To overcome these limitations, we have designed a strategy for targeted spatiotemporally controlled somatic mutagenesis in the mouse, which is based on the engineering of "floxed" genes and cell type-specific expression of a transgene expressing a conditional tamoxifen-inducible Cre recombinase (Cre-ERT2) generated by fusing Cre with a mutated LBD of ER{alpha}, which binds tamoxifen, but not estrogens (Ref. 81 and references therein). This approach has been used for generating selectively in epidermal keratinocytes of the mouse, somatic null mutations of RXR{alpha}, and of both RAR{alpha} and RAR{gamma}, whose germ line mutations are lethal in utero. This allowed us to genetically dissect the retinoid signaling in the epidermis (81, 82, 83). Using the same approach to dissect the function of RXR in adipocytes of the adult mouse, we have shown that RXR{alpha}/PPAR{gamma} heterodimers are instrumental to preadipocyte differentiation, adipogenesis, lipogenesis, and survival of mature adipocytes (64, 84). There is no doubt that the combined use of transgenic mouse lines expressing the tamoxifen-inducible chimeric Cre-ERT2 recombinase in specific cell types/tissues (the so-called Cre-Zoo), together with mouse lines harboring "floxed" NRs will provide invaluable mouse models to further investigate the mechanisms that underlie signaling through NRs.



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ACKNOWLEDGMENTS

Research is not a lonely adventure: I am particularly thankful to the numerous students and post-docs who worked with me over the last 20 yr for their enthusiastic contributions and devotion to Science. I also thank my colleagues at the Institut de Génétique et de Biologie Moléculaire et Cellulaire and Institut Clinique de la Souris for fruitful collaborations, and our engineers, technicians and administrative staff for their efficient help.

FOOTNOTES

I am grateful to the French research organizations, the Institut National de la Santé et de la Recherche Médicale and the Centre National de la Recherche Scientifique, as well as to the Université Louis Pasteur and the Hopitaux Universitaires in Strasbourg, for their financial support. I am thankful to Bristol-Myers Squibb who generously built the Institut de Génétique et de Biologie Moléculaire et Cellulaire for us.

Abbreviations: AD, Activation domain; AF, activation function; CBP, cAMP binding protein-binding protein; DBD, DNA binding domain; ER, estrogen receptor; FXR, farnesoid X receptor; GRIP-1, GR-interacting protein-1; GR, glucocorticoid receptor; LBD, ligand binding domain; LBP, ligand binding pocket; MMTV, mouse mammary tumor virus; N-CoR, nuclear receptor corepressor; NR, nuclear receptor; PKA, protein kinase A; PPAR, perosixome proliferator-activated receptor; RA, retinoic acid; RAR, RA receptor; RARE, retinoic acid response element; SRC, steroid receptor coactivator; TIF, transcriptional intermediary factor; TR, thyroid hormone receptor; VAD, vitamin A deficiency; VDR, vitamin D receptor.

Received for publication March 16, 2005. Accepted for publication March 28, 2005.

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