Overview Of The Nuclear Receptor Superfamily

Nuclear receptors are transcription factors characterized by two important properties: first, they are activated on the binding of specific ligands and second, they bind to specific response elements mainly located within the promoters of their target genes. Thus, in a simplified view, the effector function of nuclear receptors in a cell is to adapt gene expression according to signals received in the form of specific ligands. An official nomenclature for these receptors across species is now used, organized according to their phylogeny (Nuclear Receptors Nomenclature Committee, 1999; reviewed in Aranda and Pascual, 2001).

Nuclear receptors share a common structural organization and functional behavior. The poorly structured N-terminal domain encompasses, depending on the receptor, a very weak to strong ligand-independent transactiva-tion domain. The DNA-binding domain (DBD) is folded in two zinc fingers and is the hallmark of the nuclear receptor family. The hinge region links the DBD to the ligand-binding domain (LBD). The general fold of the LBD is structured by 12 a-helices and 3 b-sheets defining the ligand-binding pocket.

Most nuclear receptors function as dimers, either homodimers, such as the glucocorticoid receptor (GR) or the estrogen receptor (ER), but more often as heterodimers with RXR. The DNA response element of nuclear receptors comprises two motifs corresponding to or closely related to the hexamer AGGTCA. The organization of these two motifs in direct, inverted, or palindromic repeats and the spacing between the two hexamers determine the specificity of these response elements toward each receptor dimer.

The general scheme for transactivation via nuclear receptors is thought to occur in at least two steps. In absence of ligand, nuclear receptor dimers may bind a corepressor protein that inhibits their transactivation properties. In the presence of ligand, or due to an alternative pathway of activation such as phosphorylation, the corepressor is released and a coactivator is recruited, allowing interactions with the transcription initiation complex as well as local histone modifications, eventually triggering or enhancing transcription. These cofactors are shared by numerous transcription factors, among which are the nuclear receptors, and might play key roles in the integration and coordination of the response of multiple genes to a variety of signals (Nettles and Greene, 2005; Tsai and Fondell, 2004).

B. CLASSIFYING NUCLEAR RECEPTORS: AN INFORMATIVE MEAN FOR POSITIONING RXR-RELATED SIGNALING PATHWAYS

Analyses of the human genome identified 48 nuclear receptor genes, some of them generating more than 1 receptor isoform. Different classifications have been proposed according to different criteria.

1. Classification According to the Phylogenetic Tree

A classification according to the position along the phylogenetic tree provided a practical and significant tool for unifying the nomenclature of all nuclear receptors across species. This system is based on the evolution of the two well-conserved domains of nuclear receptors (the DBD and the LBD) and distinguishes six subfamilies (Nuclear Receptors Nomenclature Committee, 1999). Interestingly, besides their phylogenic relationship, some common functional properties may be found within each group. All receptors contained in subfamily I are forming heterodimers with RXR. The three RARs (RARa, RARb, and RARg), the thyroid hormone receptors (TRa and TRb), the vitamin D receptor (VDR), and the peroxisome proliferator-activated receptors (PPARa, PPARb, and PPARg) belong to this subfamily. Steroid receptors, which comprise the estrogen receptor, androgen receptor, progesterone receptor, mineralocorticoid receptor, and glucocorticoid receptor (ER, AR, PR, MR, and GR, respectively), mostly function as homodimers and are all found in subfamily III. Intriguingly, RXR that can function in both configurations (homodimer and heterodimer) is linked to yet another subfamily (subfamily II) together with HNF4.

This phylogeny-based classification can be in part superimposed to a structurally based classification, where nuclear receptors are ordered regarding the conservation of an amino acid located in the main dimer interface of the LBD and determinant for homodimer versus RXR-containing hetero-dimer formation (Brelivet et al., 2004). This ability to structurally distinguish the two classes, homodimers versus heterodimers, might become a very useful tool to further understand ligand interference, as will be discussed below, or some properties of orphan nuclear receptors. Intriguingly, RXR along this structural partitioning falls into the class of homodimers, consistent with its ability to function as a homodimer, while being the required partner for nuclear receptor heterodimers.

2. Classification According to the Ligand-Binding Properties

Ordering nuclear receptors according to their ligand-binding properties offers a functional classification in which they fall into three groups.

The orphan receptors possess the structural characteristics of nuclear receptors, including a sequence consistent with the presence of an LBD. However, no ligand has been identified so far for these receptors. Interestingly, the tight structure of the 12 helices in the Nurrl LBD has been shown to preclude the formation of a ligand-binding pocket (Wang et al., 2003b), suggesting that at least some of the orphan receptors function in a ligand-independent manner. Interestingly, orphan receptors are mainly grouped in the phylogenetic subfamilies IV, V, and VI (the latter containing only one nuclear receptor, GCNF1). As for now, the functions of many orphan receptors remain elusive.

On the opposite, the "classic" hormone receptors bind a narrow range of molecules with very high affinity. They comprise the steroid receptors (ER, AR, PR, MR, and GR), which mediate the corresponding endocrine functions. It also includes TRa and TRb, which bind triiodothyronine and VDR. Finally, the three RARs (RARa, RARb, and RARg) also belong to this class. While steroid receptors form homodimers, TRs, VDR, and RARs are functioning as RXR heterodimer.

The receptors of the "intermediary" class are metabolic sensors. This group comprises receptors binding to a broad range of molecules with, as a corollary, a relatively poor affinity. Rather than responding to hormones secreted by endocrine glands with tight feedback controls, these receptors can bind to molecules that are components of metabolic pathways as substrates, intermediates, or end-products. In this class are the PPARs, which are involved in many aspects of lipid metabolism, and more generally in energy metabolism. They can bind a wide variety of fatty acids, from dietary lipids to lipids derivatives such as eicosanoids. The liver X receptors (LXRa and LXRb) recognize cholesterol metabolites such as oxysterols. Together with the farnesoid X receptor (FXR), which binds bile acid derivatives, they are closely involved in cholesterol metabolism. The pregnane X receptor (PXR) is activated by many endobiotic and xenobiotic compounds, a property shared with its close relative, the constitutive androstane receptor (CAR). Their activities induce the expression of multiple genes from the

CYP family, forming a redundant network for the detoxification and excretion of potentially harmful molecules, including therapeutic drugs (Jacobs et al., 2005; Xie et al., 2000). In addition, they also contribute to the enter-ohepatic circulation of bile acids as well as bile acid detoxification (Cao et al., 2004; Kullak-Ublick et al., 2004). In summary, the receptors of this class are sensors of the metabolic status, respond both to incoming dietary signals and to metabolites, and orchestrate the metabolic adaptation at the cell, organ, and whole organism levels. Interestingly, all these receptors act as heterodimers with RXR.

RXR itself is difficult to assign to a distinct class following this criterium. While it behaves as a classic receptor with respect to 9-cis RA, the nature of its endogenous ligand(s) is still unclear (see Section IV.B).

3. Classification According to the Active Regulatory Role of RXR

Among the RXR heterodimer-forming receptors, a further functional distinction may be done between nonpermissive and permissive receptors, very important with respect to rexinoid signaling. In permissive heterodi-mers, the DNA-binding complex is active in the presence of an agonist for either RXR or the partner receptor (Mangelsdorf and Evans, 1995; Minucci and Ozato, 1996). PPAR:RXR, LXR:RXR, and FXR:RXR are the best characterized permissive heterodimers. The mechanism underlying such a property is not well known, especially regarding cofactor recruitment. In the context of PPAR:RXR, the AF2 domain of RXR is not required for permissiveness, suggesting that transcriptional activation by RXR agonists results from an indirect conformational change of the PPAR LBD, transmitted through the heterodimerization interface (Schulman et al., 1998). However, it might not be a general rule as the AF2 domain of RXR is required for the permissiveness of PPAR^:RXR bound to the Hmgcs2 promoter (Calleja et al., 2006). TR and VDR form nonpermissive complexes, in which the RXR ligand cannot trigger transcription, except in the context of the prolactin gene promoter where RXR may also be an active partner of TR (Castillo et al., 2004; Li et al., 2004). The fact that permissive receptors belong to the "sensor" receptors whereas TR and VDR are classic high-affinity hormonal receptors led to a detailed molecular analysis of the structural determinant for receptor permissiveness. One attractive hypothesis is that loss of this property in TR and VDR arose during evolution in parallel with the ability to recognize endocrine ligands with high affinity (Shulman et al., 2004). CAR:RXR heterodimers seem to be neither strictly permissive nor non-permissive for RXR signaling, as rexinoids have distinct effects depending on the context, particularly that of the response element (Tzameli et al., 2003). Finally, in this mode of action, RAR is not truly permissive since an RXR agonist is only active when the RAR agonist is previously bound to RAR (Germain et al., 2002), creating a so-called subordination mechanism.

Besides direct transcriptional activation, RXR may play an active modulating role by regulating the subcellular localization of its partners. This is convincingly described for VDR:RXR (Prufer and Barsony, 2002; Yasmin et al, 2005) and for Nurr/TR3/NGF1-B:RXR (Cao et al, 2004; Jacobs and Paulsen, 2005), but has not been reported for other RXR-dependent nuclear receptors.

We have emphasized in these classifications the particular place occupied by RXR heterodimers. We will now focus our attention on the RXR permissive complexes which are key metabolic regulators (Desvergne et al., 2006). These heterodimers are ideal targets in drug research on metabolic diseases, and understanding their main activities underlines the possible positive or negative therapeutic interference that rexinoids may provoke.

III. RXR IN PARTNERSHIP: THE PERMISSIVE HETERODIMERS AS METABOLIC SENSORS

A. AN OVERVIEW OF LXR:RXR PHYSIOLOGICAL ACTIVITIES

The endogenous activators of LXRs (NR1H3) are oxysterols and other derivatives of cholesterol metabolism. As such, they participate in cholesterol sensing and regulate important aspects of cholesterol and fatty acid metabolism (Tontonoz and Mangelsdorf, 2003).

Two isotypes, LXRa and LXRb, share 77% amino acid identity in their DBD and LBD, and are highly conserved between rodents and human. LXRa is highly expressed in the liver but is also found in kidney, intestine, adipose tissue, and macrophages, whereas LXRb is expressed ubiquitously. LXRs heterodimerize with RXR to bind to their DNA response element, formed of a direct repeat of two hexamers related to the sequence AGTTCA, separated by four nucleotides.

Mono-oxidized derivatives of cholesterol are potent LXR ligands. The most potent of these are 22(R)-hydroxycholesterol, 24(S)-hydroxycholesterol, and 24(S),25-epoxycholesterol, which activate both LXRa and LXRb (Janowski et al., 1996; Lehmann et al., 1997). Little is known about the sterol hydro-xylases that produce these metabolites, but it is assumed that oxysterol concentrations parallel those of cholesterol. Importantly, oxysterols are found at micromolar concentrations in tissues that express high levels of LXRa or LXRb.

LXR was initially characterized by its role in the positive regulation of the gene encoding cholesterol 7a-hydroxylase (CYP7A), the rate-limiting enzyme in the neutral bile acid biosynthetic pathway that diverts cholesterol into bile acids. The nature of LXR endogenous ligands further emphasized the importance of this receptor in cholesterol metabolism. This function has been confirmed by the phenotype of LXRa null mice, which display a severely impaired cholesterol and bile acid metabolism when fed with a cholesterol-enriched diet. Indeed, these mutant mice fail to induce CYP7A and consequently suffer from a dramatic accumulation of cholesteryl esters in the liver with no increase in bile acid production (Peet et al., 1998). However, it is now clear that the human CYP7A is not responsive to LXR and might even be repressed by LXRa activation (Chen et al., 2002; Goodwin et al., 2003). This difference between mouse and human is of interest as it might explain at least in part both the higher capability of mouse to face high-cholesterol diet and its increased resistance to the development of atherosclerosis.

As a consequence, the research focuses now more on other important LXR-mediated regulations that converge on the reverse cholesterol transport pathway. This pathway limits the exposure of peripheral cells to cholesterol excess and its modulation by LXR has been reported in both human and mouse, at least at three levels. First, LXR upregulates the expression of several genes encoding members of the ABC transporter family. ABCG5 and ABCG8, which are expressed almost exclusively in the liver and small intestine, favor the secretion of sterols from the liver epithelial cells to the bile duct and from the gut epithelial cells to the intestinal lumen (Berge et al., 2000). Activation of these two genes by LXR is considered as the main mechanism by which an LXR agonist in mice causes a total blockade of cholesterol absorption (Plosch et al., 2002; Repa et al., 2002). Another important target is the widespread ABCA1 transporter, which promotes efflux of intracellular and plasma membrane cholesterol to the nascent high-density lipoprotein (HDL) particles via interaction with ApoA1, thereby increasing HDL levels (Repa et al., 2000b). Second, LXR increases ApoE expression. Effluxed cholesterol from cell membrane can also be charged on HDL particles by ApoE, increasing their total capacity of accepting cholesterol. In addition, ApoE containing particles can interact with the scavenger receptor that increases the uptake of these particles in the liver. Third, LXR increases the expression of the cholesteryl ester transfer protein (CETP), which promotes cholesteryl ester transfer from VLDL to HDL and from HDL to low-density lipoprotein (LDL), a lipoprotein which is also efficiently taken up by the liver. By these means, LXR increases cholesterol clearance from the blood (reviewed in Tall et al., 2002). Some of these regulations are shared by LXRa and LXRb. If LXRft null mice do not have the dramatic phenotype described for LXRa, the double mutant mice are more strongly affected than the LXRa null mice (Repa and Mangelsdorf, 1999). However, no functional compensation by LXRb is seen in LXRa null mutant mice and a specific role for LXRb has not been clearly defined yet.

Activated LXR also acts on fatty acid synthesis mainly by mediating the insulin-induced expression of SREBP-1c (Chen et al., 2004), a transcription factor playing a major lipogenic role in hepatocytes (Repa et al., 2000a; Schultz et al., 2000). This lipogenic activity is also reinforced by the

LXR-dependent increase of fatty acid synthase (FAS) expression (Joseph et al., 2002). Together, these effects might explain the steatosis and the massive increase in VLDL and triglyceride blood levels observed in mice treated with pharmacological doses of an LXR ligand (Grefhorst et al., 2002).

In summary, LXR is a major transcription factor, which acts as a sensor of cholesterol levels via its interaction with oxysterols and, in turn, drives the disposal of the excess of cholesterol. It also acts at the level of individual cells, by increasing the ABC transporter molecules responsible for cholesterol efflux, and at the level of the organism by decreasing the cholesterol uptake from the diet. In mouse liver, it also increases the conversion of cholesterol into bile acids. These positive activities on cholesterol metabolism are, however, minored by the strong lipogenic and hypertriglyceridemia effects of LXR agonists.

B. AN OVERVIEW OF FXR:RXR

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