Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids
ReviewThe retinoid X receptors and their ligands☆
Graphical abstract
Highlights
► Retinoid X receptor (RXR) α, β, and γ structural and molecular biology. ► RXR nuclear and cytoplasmic function and binding partners. ► RXR posttranscriptional processing. ► Impact of ligand on cofactor binding. ► Recently identified RXR ligands.
Introduction
The retinoid X receptor (RXR1) is an intriguing and essential member of the steroid/thyroid hormone superfamily of nuclear receptors (NRs) that predominately function as transcription factors with roles in development, cell differentiation, metabolism, and cell death. This review outlines the accomplishments made in understanding RXR biology from 2004 and also presents an overview of many of the RXR ligands (rexinoids) and their activities reported since 2000.
Briefly, the RXR subtypes or isotypes α–γ (NR2B1–3) (Table 1) are members of the orphan NR family of this NR superfamily because at their discovery natural ligands were unknown. The natural ligand of RXR remains controversial. Although 9-cis-retinoic acid (9-cis-RA in Fig. 1A) was first proposed to have this status, many groups have since been unable to detect endogenous 9-cis-RA in cells either in culture or in vivo unless its isomer, all-trans-retinoic acid (ATRA), had been present first or added [1], [2]. Compounding the uncertainty of its status as the natural ligand of RXR is the instability of the RA tetraene side chain that either in the presence of light or a mercaptan, such as reduced glutathione, can equilibrate to a mixture of double-bond isomers generally containing 80% ATRA, 8–10% 9-cis-RA, and other isomers. Polyunsaturated fatty acids (PUFAs) such as docosahexaenoic acid (DHA) and a saturated metabolite of chlorophyll, phytanic acid (Fig. 1A), were also identified as RXR ligands.
In the nucleus, RXR functions as a transcription factor by binding to specific six-base-pair sequences of DNA in the promoter regions of genes. In doing so, RXR functions as a dimer with either itself (homodimer) or another NR (heterodimer). Generally, binding by the ligand of the NR partner defines the promoter site (response element or RE) composed of two six base-pair sequences (half-sites) separated by a discrete number of bases to which the RXR–NR heterodimer binds [5′-PuG(G/T)TCA-(X)n-PuG(G/T)TCA-3′] [3]. As indicated by some of the REs listed in Table 2, these sequences can be repeated directly (DR), inverted (IR), everted (ER), palindromic (pal), or disordered depending on the dimer bound. Thus, RXR heterodimers with peroxisome proliferator-activated receptor (PPAR), retinoic acid receptor (RAR), vitamin D receptor (VDR), and thyroid hormone receptor (TR) consist of two directly repeated (DR) half-sites separated by one, two or five, three, and four bases (n), respectively, typically with RXR in the 5′-position. In the case of the RXR heterodimer with RAR bound to a DR-1 response element, RXR can occupy either the 5′ or 3′-position. The RXR homodimer preferentially recognizes two 5′-(A/G)GGTCA-3′ half-sites separated by one base (DR-1).
The status of RXR in cells remains controversial. In addition to forming heterodimers and homodimers in vitro, RXR homotetramers have also been detected. The cellular status of retinoic acid receptor (RAR) ligand-binding domain (LBD)–RXR LBD heterodimers and RXR LBD–RXR LBD homodimers was determined using fluorescence correlation spectroscopy (fluorescence fluctuation brightness analysis) [4]. CV-1 cells were transfected with constructs for yellow fluorescent protein (YFP)-RXR LBD and cyan fluorescent protein (CFP)-RAR LBD. Brightness intensities were then measured. Both YFP and CFP were identically bright after excitation at 905 nm, whereas only YFP fluoresced after excitation at 965 nm. These studies were used to demonstrate that in the transfected cells the labeled RXR existed either as a heterodimer with labeled RAR or as a monomer and not as a homodimer.
In Table 2 are listed those nuclear receptors that heterodimerize with the RXRs and have roles in regulating genes controlling metabolic signaling pathways, their typical REs and ligands. Among these are the peroxisome proliferator-activated receptor (PPAR) isotypes α, β/δ and γ, which also have roles in cell proliferation and differentiation.
The intracellular behavior of the RXRα–PPAR heterodimer in the presence or absence of a ligand was investigated using the combination of fluorescence recovery after photobleaching (FRAP), fluorescence correlation spectroscopy (FCS), and fluorescence resonance energy transfer (FRET) on transfected enhanced yellow fluorescent protein (eYFP)-PPARα–γ and eYFP-RXRα constructs [5]. Unlike the nuclear patterning exhibited by eYFP-ERα, the fluorescent flakes and foci produced after preliminary transfections of the eYFP-PPARα constructs were considered to be artifacts that were caused by protein over-expression. At lower expression levels, the eYFP-PPARα expression pattern became diffuse in the nuclei of living COS-7 cells. According to FRAP, the eYFP-PPAR proteins in the presence or absence of their ligands were highly mobile in cells that had the diffuse distribution patterns of fluorescence and were unaffected by co-expression of RXRα alone or with added 9-cis-RA. The diffusion pattern for eYFP-RXRα was similar to that of the apo-PPARs. The diffusion constants for the eYFP-PPARs were 4.8–5.5 μm2/s and decreased to 2.3–3.5 μm2/s after their ligand bound to suggest that the apo-PPAR were bound by cofactors in complexes on the order of “1–2 MDa”. The diffusion constant for the eYFP-RXRα was 4.6 μm2/sec. The authors estimated that about 3,600–120,000 fluorescent protein molecules were expressed per cell, whereas the actual number of PPAR target genes was < 1,000. Therefore, they speculated that many of the reported interactions of PPARs–RXRα on DNA would have been the consequence of transient or nonspecific interactions at sites resembling authentic PPREs. FRET indicated that PPAR–RXR dimerization occurred prior to ligand binding or DNA binding, however heterodimer binding to DNA was only observed to be stable in vivo after ligand had bound.
The RXRs also have major roles in regulating genes controlling cell proliferation and differentiation in the context of their heterodimers with the RARs, thyroid hormone receptors (TRs), and vitamin D receptor (VDR). Both non-denaturing nanoelectrospray ionization (nano-ESI) and high-mass matrix-assisted laser desorption ionization (MALDI) mass spectrometric methods were used to demonstrate that the RXR–RAR heterodimer bound to a DR-5 RARE in the presence of 9-cis-RA and that RXR was upstream (5′), in contrast to the DR-1 RARE in which RAR was upstream [6, and references therein]. Crosslinking was used to stabilize the complex for MALDI, but did not necessarily stabilize the bound ligand. RAR did not homodimerize in solution but was able to form such a homodimeric complex on the DR-5 in the presence of 9-cis-RA and excess DR-5 to indicate that RXR was not required for RAR to associate with its half-site. Limitations of these methods were also described by the authors. However, the subtypes of the mutant murine retinoid receptors RXRΔA/B and RARΔA/B used in the study were not identified.
Recently, RXR has been shown to have cytoplasmic functions that are distinct from its activity as a transcription factor.
RXR was reported to shuttle the orphan NR human TR3/mouse Nur77/rat NGFI-B from the nucleus to the cytoplasm, allowing TR3 to interact with mitochondrial Bcl-2 to reverse its anti-apoptotic function to one promoting apoptosis. This activity was first reported by Zhang and colleagues [7], [8] and confirmed by Wu and colleagues [9]. Stress induced by treatment of cancer cell lines with a cancer therapeutic agent or an adamantyl-substituted retinoid-related molecule induced TR3 relocalization in several cancer cell lines.
Using MGC80-3 human gastric cancer cells, Wu and colleagues investigated the role of RXRα in inducing TR3 nuclear export [9]. Apo-RXRα was unable to induce export, whereas 1.0 μM 9-cis-RA-treated RXRα effectively did so within 30 min. Deletion analysis indicated that the RXRα DBD contained a nuclear export signal (NES), as did TR3. However, the CRM1-dependent TR3 NES did not appear to be involved in export in this cell line. TR3 lacking its DNA-binding domain (DBD), hinge, and 90 N-terminal residues of the LBD still colocalized in mitochondria, as was shown by staining with the mitochondrial marker Hsp60, and induced apoptosis irrespective of 9-cis-RA treatment, whereas full-length TR3 resided in the nucleus and was unable to migrate out of the nucleus or to induce apoptosis in the absence of RXRα and 9-cis-RA. A mutant lacking the 106 N-terminal residues of the TR3 A/B domain underwent nuclear export to mitochondria in the presence of 9-cis-RA, whereas the C-terminal deletion of 25 residues from the TR3 LBD prevented TR3 interaction with RXRα and nuclear export induced by RXRα and 9-cis-RA.
Despite their lack of a nucleus, platelets expressed several NRs, including androgen receptor (AR), estrogen receptors (ERs), glucocorticoid receptor (GR), mineralcorticoid receptor (MR), PPARs, PXR, and RXRs α and β. Platelets are derived from the cytoplasm of megakaryocytes, which have nuclei that express mRNAs for these NRs and have enzymes for their translation [10]. In platelets these NRs were considered to function through nongenomic pathways. For example, platelet aggregation and thromboxane (TX) A2 release were inhibited by the RXR agonists 9-cis-RA and methoprene acid (Fig. 1B). Activated platelets released microparticles that were found to contain RXRα/β to suggest that RXRα/β had an extracellular role in modulating the results of platelet activation [11]. Treatment with RXR ligands 9-cis-RA and methoprene acid, but not ATRA, inhibited platelet aggregation induced by TXA2 mimetic U46619 and TXA2 release stimulated by adenosine diphosphate [12]. Inhibition occurred by ligand-bound RXR interacting with G-protein Gq to prevent the activation of the GTPase Rac.
Section snippets
RXR gene promoter
The human RXRα gene was found to have 10 exons but not a typical TATA transcription initiation site [13], [14]. Its promoter sequence has been considered to resemble that of a housekeeping gene because of the high G + C content in its 5′-untranslated region. In keeping with the high G + C content, 17 and 12 putative Sp1 sites were identified upstream and downstream, respectively, of the start site. Putative AP-1, AP-2, AP-4, GATA-1/2, N-Myc, v-myb, SRY, AML-1a, and imperfect DR-0, 3, 4, and 5 sites
Ligand binding alters the ligand-binding pocket conformation
Notable differences in complexes of the RXRα LBDs with the agonists 9-cis-RA, DHA (docasahexaenoic acid in Fig. 1A), and BMS649 (SR11237 in Fig. 1B) included their respective volumes (494-513, 528, and 472–480 Å3), ligand-binding pocket (LBP) volume occupied by ligand (71–74, 81, and 86–88%), and van der Waals/polar contacts (71/6, 89/6, and 89–92/6–7), which variously impacted their respective ligand-binding affinities to the RXRα LBD (2 nM, 50–100 μM, and 5–10 nM) [26], [27]. Overlap of these
RXR interaction partners
Protein partners of the RXRs include (i) transcription factors, including the NRs — CAR, EAR2, FXR, LXRs α and β, NGFI-B/Nur77/TR3, Nurr1, PPARs α, β/δ, and γ, PNR, PXR/SXR, RARs α–γ, RXRs α–γ, SHP, TRs α and β, and VDR; the circadian rhythm transcription factors Arntl/Bmal1, Clock, and nPAS2/MOP4; and others such as Bcl3, integrin β3-binding protein, MyoD, NFκB-1, NFκB-1B, Oct1/POU2F1, Oct2/POU2F2, PML, RelA, SMAD-2, SP1, and TATA-binding protein; (ii) transcriptional cofactors including CoAs
Dimerization
Increasing evidence indicates that RXR does not play a passive role as a heterodimeric partner but impacts the responses of its NR partner, regardless of its permissive, non-permissive, or conditionally permissive status.
Phosphorylation
Phosphorylation is the most extensively studied post-translational modification of RXRα. Rochette-Egly and colleagues reported that ATRA-induced activation of jun-terminal kinase (JNK) led to the phosphorylation of AF-1 domain residues (Ser61, Ser75, and Thr87) in mouse RXRα, which then allowed cooperation between the AF-1 and AF-2 domains in the RXRα–RARγ heterodimer leading to enhanced transactivation activity and was necessary for the antiproliferative effect of ATRA [104]. They also showed
Atherosclerosis
While cholesterol metabolism and its peripheral distribution are mediated by the liver, dysfunctional processing of cholesterol by macrophages leads to the development of hypercholesterolemia and cholesterol-containing atherosclerotic plaques and lesions (reviewed in Ref. [120]). PPARγ activated by 9- and 13-OH-octadienoic acids plays a role in macrophage differentiation, uptake of oxidized low-density lipoprotein (LDL), and the expression of LXRα, which, in turn, induces the expression of such
Background reading (reviews)
Since 2000, many excellent reviews describing RXR transcriptional function have appeared. In 2010, Lefebvre and colleagues provided a thought-provoking overview of research on the signaling mechanisms of the RXR isotypes (α–γ) with emphasis on the research issues that should be addressed and the unique role that the RXRs have in transcriptional signaling [13]. They stressed the need for RXR isotype-selective rexinoids for mechanistic studies and pharmacological use on the basis of differences
Conclusions and future directions
Despite many clinical trials, only two RXR agonists have been approved for drug use and then only for limited indications, namely topical and systemic treatment of CTCL using bexarotene (LGD1069) and topical treatment of Kaposi's sarcoma and systemic treatment of refractory chronic hand eczema using aliretinoin (9-cis-RA). Both drugs have significant adverse effects that may be due to their abilities to activate the RARs. Their effects on other nuclear receptors such as those that bind fatty
Acknowledgements
Research from the groups of Drs. Xiao-Kun Zhang and Marcia I. Dawson that is described in this review was partially supported by U.S. National Cancer Institute Grants , , , which is gratefully acknowledged. We also thank Ms. Laura Nelson for assistance with the manuscript.
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This article is part of a Special Issue entitled Advances in High Density Lipoprotein Formation and Metabolism: A Tribute to John F. Oram (1945–2010).